Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of Pyk2 and FAK in B cell migration, adhesion, and spreading Tse, Kathy Wan-Kei 2010

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2010_fall_tse_kathywankei.pdf [ 6.08MB ]
Metadata
JSON: 24-1.0069997.json
JSON-LD: 24-1.0069997-ld.json
RDF/XML (Pretty): 24-1.0069997-rdf.xml
RDF/JSON: 24-1.0069997-rdf.json
Turtle: 24-1.0069997-turtle.txt
N-Triples: 24-1.0069997-rdf-ntriples.txt
Original Record: 24-1.0069997-source.json
Full Text
24-1.0069997-fulltext.txt
Citation
24-1.0069997.ris

Full Text

 THE ROLE OF PYK2 AND FAK IN B CELL MIGRATION, ADHESION, AND SPREADING   by  Kathy Wan-Kei Tse  B.Sc., University of British Columbia, 2004   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies  (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   May 2010     Kathy Wan-Kei Tse, 2010    ii Abstract The ability of the B cell receptor (BCR) to stimulate integrin-mediated adhesion, and induce cytoskeletal reorganization and cell spreading enhances the ability of B cells to bind and respond to antigens (Ag).  The proper localization and trafficking of B cells in the secondary lymphoid organs are also critical for B cells to encounter Ags and to be activated.  Proline- rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that have been shown to regulate cell adhesion, morphology, and migration. However, their functions in B cells are not clear.  The overall hypothesis of this thesis was that Pyk2 and FAK are downstream targets of BCR, integrin, and chemokine receptor signaling, and that they are involved in B cell morphological regulation, migration, and adhesion.  I showed that the BCR and integrins collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, modifications that increase the kinase activity of Pyk2 and FAK.  Activation of the Rap1 GTPase is critical for BCR-induced integrin activation and for BCR-induced reorganization of the actin cytoskeleton and I showed that inhibition of Pyk2 and FAK function by either gene knockdown or the use of chemical inhibitors impaired B cell spreading.  Marginal zone (MZ) B cells are innate-like B cells that are responsible for T cell-independent responses to microbial pathogens. The proper localization of MZ B cells is dependent on integrated migration and retention signals provided by the stromal cells in the spleen. Because MZ B cells are not found in Pyk2-/- mice, I hypothesized that Pyk2 and FAK are involved in MZ B cell retention in the spleen.  I showed that Pyk2 and FAK are required for MZ B cell migration and that Pyk2 is required for integrin-dependent adhesion in response to chemoattractant stimulation.  Moreover, I found that FAK is involved in chemokine-induced Akt phosphorylation in MZ B cells.  In summary, Pyk2 and FAK are downstream targets of the Rap GTPases and play a key role in regulating B cell morphology, migration, and adhesion.   iii Table of contents Abstract .................................................................................................................................... ii Table of contents .................................................................................................................... iii List of tables............................................................................................................................ vi List of figures ......................................................................................................................... vii List of abbreviations .............................................................................................................. ix Acknowledgements .............................................................................................................. xiii Dedication ............................................................................................................................. xiv Co-authorship statement ...................................................................................................... xv 1. Introduction ........................................................................................................ 1 1.1 B cell development and humoral immunity ......................................................... 1 1.1.1 Follicular B-2 cells ............................................................................................... 3 1.1.2 Marginal zone B cells ........................................................................................... 4 1.1.3 B-1 cells ................................................................................................................ 7 1.1.4 Regulatory B cells ................................................................................................ 8 1.1.5 B cells and disease ................................................................................................ 9 1.2 B cell activation ................................................................................................. 15 1.2.1 B cell antigen acquisition ................................................................................... 15 1.2.2 B cell receptor signaling ..................................................................................... 19 1.3 Integrins and B cell adhesion ............................................................................. 22 1.3.1 Inside-out signaling and integrin activation ....................................................... 23 1.3.2 Outside-in integrin signaling .............................................................................. 26 1.4 B cells migration ................................................................................................ 27 1.4.1 Lymphocyte migration across the endothelium ................................................. 28 1.4.2 Chemoattractants important for B cell migration ............................................... 28 1.4.3 Chemoattractant receptor signaling .................................................................... 29 1.5 Cytoskeleton remodeling ................................................................................... 33 1.6 Pyk2 and FAK.................................................................................................... 35 1.6.1 The structures of Pyk2 and FAK ........................................................................ 35 1.6.2 The regulation of Pyk2 and FAK by tyrosine phosphorylation ......................... 37 1.6.3 The role of FAK in cell migration ...................................................................... 38 1.6.4 Functions of Pyk2 and FAK in haemaopoetic lineage cells ............................... 38 1.6.5 Functions of Pyk2 and FAK in macrophages ..................................................... 39 1.6.6 Activation and function of Pyk2 and FAK in T cells ......................................... 39 1.6.7 Activation and function of Pyk2 and FAK in B cells ........................................ 40 1.7 The Rap GTPases ............................................................................................... 42 1.8 Objective and aims ............................................................................................. 46 1.9 References .......................................................................................................... 48   iv 2. B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading ........................................................................................................... 70 2.1 Introduction ........................................................................................................ 70 2.2 Results ................................................................................................................ 73 2.2.1 Expression and localization of Pyk2 and FAK in B cells .................................. 73 2.2.2 Adhesion to ECM enhances BCR-induced tyrosine phosphorylation of Pyk2 and FAK activation ................................................................................................... 77 2.2.3 BCR/integrin-induced B cell spreading involves Pyk2 and FAK ...................... 80 2.2.4 BCR/integrin-induced tyrosine phosphorylation of Pyk2 and FAK depends on activation of the Rap GTPases ........................................................................... 83 2.2.5 Rap activation is important for integrin-induced phosphorylation of Pyk2 and FAK .................................................................................................................... 87 2.2.6 Rap activation is important for BCR-induced phosphorylation of Pyk2, but not for BCR-induced phosphorylation of FAK ........................................................ 89 2.2.7 The role of Rap activation in the phosphorylation of Pyk2 and FAK corresponds to a requirement for actin dynamics ................................................................... 91 2.2.8 B cell spreading requires Pyk2/FAK kinase activity ......................................... 94 2.3 Discussion .......................................................................................................... 98 2.4 Materials and methods ..................................................................................... 102 2.4.1 Antibodies and inhibitors ................................................................................. 102 2.4.2 Cells .................................................................................................................. 102 2.4.3 Expression of Pyk2 and FAK ........................................................................... 103 2.4.4 Immunofluorescence ........................................................................................ 103 2.4.5 Phosphorylation of Pyk2, FAK, ERK, Akt, and Paxillin ................................. 103 2.4.6 Short Hairpin RNA (shRNA)-mediated knockdown of Pyk2 and FAK expression in A20 cells .................................................................................... 104 2.4.7 Cell spreading ................................................................................................... 104 2.4.8 Rap activation ................................................................................................... 104 2.5 References ........................................................................................................ 105 3. Small molecule inhibitors of proline-rich tyrosine kinase 2 and focal adhesion kinase modulate marginal zone B cell responses to the chemoattractants sphingosine-1-phosphate and CXCL13 ......................... 110 3.1 Introduction ...................................................................................................... 110 3.2 Results .............................................................................................................. 114 3.2.1 S1P and CXCL13 induce Pyk2 and FAK tyrosine phosphorylation in B cells 114 3.2.2 The small molecule inhibitors PF-719 and PF-228 block tyrosine phosphorylation of Pyk2 and FAK ................................................................... 116 3.2.3 PF-719 and PF-228 inhibit chemoattractant-induced migration of splenic B-2 and MZ B cells ................................................................................................. 119 3.2.4 PF-719 reduces B-2 and MZ B cell adhesion to ICAM-1 ................................ 124 3.2.5 The FAK inhibitor PF-228 inhibits chemoattractant- and anti-Ig-induced Akt phosphorylation ................................................................................................ 127 3.3 Discussion ........................................................................................................ 130 3.4 Materials and methods ..................................................................................... 136   v 3.4.1 Animals, cells, and reagents ............................................................................. 136 3.4.2 Western blotting ............................................................................................... 136 3.4.3 Cell viability assay ........................................................................................... 137 3.4.4 Chemotaxis and chemokinesis assays .............................................................. 137 3.4.5 Adhesion assay ................................................................................................. 137 3.4.6 Phospho-flow analysis ...................................................................................... 138 3.5 References ........................................................................................................ 139 4. Concluding chapter ........................................................................................ 146 4.1 Summary and overview ................................................................................... 146 4.2 Discussion and future directions ...................................................................... 149 4.2.1 The in vivo functions of Pyk2 in MZ B cells ................................................... 149 4.2.2 The role of Pyk2 and FAK in B cell immune synapse formation, polarization, and proliferation ............................................................................................... 151 4.2.3 Pathways upstream of Pyk2 and FAK in B cells ............................................. 152 4.2.4 Potential signaling pathways downstream of Pyk2 and FAK in B cells .......... 155 4.2.5 Role of Pyk2 and FAK in tumor dissemination and development ................... 159 4.3 Conclusion ....................................................................................................... 160 4.4 References ........................................................................................................ 161 A. Appendix:  The expression of Pyk2 and FAK in different mouse and human cell lines ........................................................................................................... 166 A.1 Rationale ........................................................................................................... 166 A.2 Experimental procedure ................................................................................... 167 A.3 Results .............................................................................................................. 168 A.4 Conclusions ...................................................................................................... 169 A.5 References ........................................................................................................ 170 B. Appendix:  The role of Src family kinases in Pyk2 tyrosine phosphorylation .......................................................................................................................... 171 B.1 Rationale ........................................................................................................... 171 B.2 Experimental procedure ................................................................................... 172 B.3 Results .............................................................................................................. 173 B.4 Conclusions ...................................................................................................... 175 B.5 References ........................................................................................................ 176 C. Appendix:  UBC research ethics board’s certificates of approval............. 177     vi List of tables Table 1.1 Human mature B cell malignancies................................................................... 13 Table 1.2  Integrins expressed on B cells. .......................................................................... 22 Table 3.1 Statistical analysis of the effects of the Pyk2 inhibitor (PF-719) and FAK inhibitor (PF-228) on CXCL13- and S1P-induced chemotaxis and chemokinesis. .................................................................................................. 122 Table 3.2 Statistical analysis of the effects of the Pyk2 and FAK inhibitors on B cell adhesion to ICAM-1 ........................................................................................ 126     vii List of figures Figure 1.1  B cell development. ............................................................................................. 3 Figure 1.2 A schematic view of the anatomy of the spleen and MZ B cell trafficking. ....... 6 Figure 1.3     Mechanisms by which B cells encounter Ags. .................................................. 16 Figure 1.4     BCR signaling. .................................................................................................. 20 Figure 1.5 Integrin signaling pathways. ............................................................................. 25 Figure 1.6 Chemokine receptor signaling events leading to cell polarization and migration.  ........................................................................................................................... 32 Figure 1.7 The effectors of Rho family GTPases that regulate cytoskeleton dynamics. .... 34 Figure 1.8 The structures of Pyk2 and FAK. ...................................................................... 36 Figure 1.9 Rap effectors and Rap signaling pathways. ...................................................... 43 Figure 2.1 Expression and localization of Pyk2 and FAK in B cells. ................................ 75 Figure 2.2 Adhesion of B cells to ECM selectively enhances BCR-induced tyrosine phosphorylation of Pyk2 and FAK. ................................................................... 78 Figure 2.3 A20 cell spreading involves both Pyk2 and FAK. ............................................ 81 Figure 2.4 BCR/integrin-induced tyrosine phosphorylation of Pyk2 and FAK depends on activation of the Rap GTPases. ......................................................................... 85 Figure 2.5 Rap activation is important for integrin-induced phosphorylation of Pyk2 and FAK. .................................................................................................................. 88 Figure 2.6 Rap activation is important for BCR-induced tyrosine phosphorylation of Pyk2 but not FAK. ...................................................................................................... 90 Figure 2.7 Rap-dependent phosphorylation of Pyk2 and FAK requires actin dynamics. .. 92 Figure 2.8 An inhibitor of Pyk2/FAK activity blocks B cell spreading. ............................ 96 Figure 3.1 S1P and CXCL13 induce FAK tyrosine phosphorylation in B cells. ............. 115 Figure 3.2 Inhibition of Pyk2 and FAK tyrosine phosphorylation by the small molecule inhibitors PF-719 and PF-228. ........................................................................ 117 Figure 3.3 PF-719 and PF-228 inhibit chemoattractant-induced migration of splenic B-2 and MZ B cells. ............................................................................................... 120 Figure 3.4 PF-719 reduces B-2 and MZ B cell adhesion to ICAM-1 ............................... 125 Figure 3.5 PF-228 inhibits chemoattractant- and anti-Ig-induced Akt phosphorylation .. 128 Figure 4.1 Potential downstream targets of Pyk2 and FAK in B cells ............................. 155 Figure A.1 Expression of Pyk2 and FAK in mouse and human B cell lines. .................... 168   viii Figure B.1 The effect of SFK inhibitor on Pyk2 tyrosine phosphorylation in response to PMA stimulation and integrin activation ........................................................ 173      ix List of abbreviations Ab Antibody Ag Antigen APC Antigen presenting cell BAFF B cell activating factor BCAP B cell adaptor for phosphoinositide 3-kinase BCR B cell receptor BLNK B cell linker BM Bone marrow Btk Bruton’s tyrosine kinase CCR Chemokine (C-C motif) receptor CLL Chronic lymphocytic leukemia CLP Common lymphoid progenitors CR1/CD35; CR2/CD21 Complement receptor 1 and 2 cSMAC Central supramolecular activation cluster CXCL CXC chemokine ligand DAG Diacylglyercol DCs Dendritic cells DL1 Delta-like 1 Dock2 Dedicator of cytokinesis 2 EAE Experimental autoimmune encephalitis ECM Extracellular matrix ERK Ras/Raf/extracellular signal-regulated kinase ERM Ezrin radixin moesin proteins FAK Focal adhesion kinase FAT Focal adhesion targeting FDCs Follicular dendritic cells FERM Protein 4.1, ezrin, radixin and moesin homology FRCs Fibroblastic reticular cells    x GAPs GTPase activating proteins GC Germinal center GDP Guanosine diphosphoate GEFs Guanine nucleotide exchange factors GPCRs G-protein-coupled receptors Grb Growth-factor receptor-bound protein GTP Guanosine triphosphate HEV High endothelial venules HSC Hematopoietic stem cell ICAM Intercellular adhesion molecule IFNγ Interferon gamma Ig Immunoglobulin IL Interleukin IP3 Inositol (1,4,5)-trisphosphate ITAMs Immunoreceptor tyrosine-based activation motifs Itk IL-2-inducible T cell kinase JAM Junctional adhesion molecule LAD-I Leukocyte adhesion deficiency I LFA-1 Lymphocyte function-associated antigen-1 LIMK LIM domain kinase MAdCAM-1 Mucosal vascular addressin cell adhesion molecule-1 MALT Mucosal associated lymphoid tissue MAPK Mitogen-activated protein kinase mDia Mammalian diaphanous MHC II Major histocompatibility class II MLC Myosin light chain MMP Matrix metalloproteinase MZ Marginal zone NFAT Nuclear factor of activated T cells NFκB Nuclear factor kappa B NK Natural killer   xi NOD Non-obese diabetic PAK p21-activated kinase PAMPs Pathogen-associated molecular patterns PI3K Phosphoinositide-3-OH kinase PIP2 Phosphatidylinositol-(3,4)-bisphosphate PIP3 Phosphatidylinositol-(3,4,5)-trisphosphate PKC Protein kinase C PLCγ Phospholipase C gamma PRRs Pattern recognition receptors PSGL-1 P-selectin glycoprotein ligand 1 pSMAC Peripheral supramolecular activation complex PTP Protein tyrosine phosphatases Pyk2 Proline rich tyrosine kinase RA Rheumatoid arthritis RAG Recombinase activating genes RANKL Receptor activator of NF-kappaB ligand RGS Regulator of G protein signaling RIAM Rap1-GTP-interacting adapter molecule ROCK Rho-associated coiled coil containing protein kinase S1P Sphingosine 1-phosphate SCF Stem cell factor SCID Severe combined immunodeficiencies SCS Subcapsular sinus SEM Scanning electron microscopy SFK Src family kinase SH2 Src homology 2 SHM Somatic hypermutation SKAP Src-kinase-associated phosphoprotein SLE Systemic lupus erythematosus SLOs Secondary lymphoid organs SLP-76 SH2 domain-containing leukocyte phosphoprotein-76   xii SOCS Suppressor of cytokine signaling T1 Transitional 1 TCR T cell receptor TGF-β Transforming growth factor-beta TIAM T lymphoma invasion and metastasis-inducing protein TLR Toll-like receptors TNF-α Tumor necrosis factor alpha VCAM-1 Vascular cell-adhesion molecule-1 VLA-4 Very late antigen-4 WASP Wiskott-Aldrich Syndrome protein WAVE WASP family Verprolin-homologous protein Zap-70 Zeta-chain associated protein kinase of 70 kDa    xiii Acknowledgements I would like to thank Dr. Michael Gold for his guidance, insight, support, and for being a fantastic and understanding supervisor.  I would also like to thank the members of my supervisory committee, Dr. Ninan Abraham, Dr. Pauline Johnson and Dr. Catharine Pallen for their intellectual contribution to this work.  I would especially like to thank my collaborators Dr. Leonard Buckbinder and Pfizer for their collaboration and for supplying the Pyk2 and FAK inhibitors.  Special thanks to Dr. Linda Matsuuchi for helpful insight, scientific guidance, and encouragement.  I also wish to acknowledge the past and present members of the Gold lab for useful discussions and technical support.  I would also like to thank the Wesbrook Animal Unit and the UBC FACS Facility for very helpful technical support, as well as the members of the Abraham lab, the Johnson lab, the Matsuuchi lab, the Roskelley lab, the Teh lab, and the Underhill lab for useful discussions and technical support. Financial assistance from the Michael Smith Foundation for Health Research and the Natural Sciences and Engineering Research Council of Canada was greatly appreciated.   xiv Dedication    I would like to dedicate this thesis to my parents and Woofy.      xv Co-authorship statement My participation in the work presented in Chapter 2: • I performed all experiments except for: o Figure 2.4: Some of the experimental repeats were performed by Doris Vong o Figure 2.6A: Some of the experimental repeats were performed by Rosaline Lee o Figure 2.7A, B, & D: Some of the experimental repeats were performed by May Dang-Lawson o Figure 2.7C&E: Some of the experimental repeats were performed by Anica Bulic • I analyzed all the data • PF-431396 was supplied by Dr. Leonard Buckbinder • I designed the research program and prepared the manuscript with Dr. Michael Gold My participation in the work presented in Chapter 3: • I performed and analyzed all experiments and data except for: o Figure 3.5: Performed by Dr. Kevin Lin.         I analyzed the data with Dr. Kevin Lin • PF-431396, PF-573228, and PF-3430719 were supplied by Dr. Leonard Buckbinder • I designed the research program and prepared the manuscript with Dr. Michael Gold My participation in the work presented in Appendices: • I performed all experiments except for: o Figure A.1: Some of the lysates were prepared by Anica Bulic and May Dang- Lawson • I analyzed all the data • I designed the research program with Dr. Michael Gold     1 1. Introduction 1.1 B cell development and humoral immunity B cells are an essential component of the adaptive immune system.  The primary function of B cells is to make antibodies (Abs) against pathogens.  They are characterized by the clonally diverse expression of antigen (Ag) receptors termed the B cell receptor (BCR). B cells also express Toll-like receptors (TLRs) and undergo activation and proliferation when they encounter structurally conserved molecules derived from pathogens.  In mice and humans, B cell progenitors are generated primarily in the fetal liver and adult bone marrow (BM), undergoing an extensive rearrangement of their Ig genes to generate a diverse repertoire of BCRs.  Besides being the source of protective Abs, B cells also regulate many other functions essential for immune homeostasis.  It is now appreciated that B cells can initiate T cell activation by presenting Ag via their MHC II molecules, producing immunomodulatory cytokines, and facilitating the transport of Ags (1). B cell progenitors develop in the BM before they migrate into the blood to reach the spleen for the final steps in their maturation process (Figure 1.1) (1, 2).  Like all hematopoietic cells, B cells are derived from hematopoietic stem cells (HSC), which have the capacity for self-renewal.   HSCs undergo multiple steps of differentiation and eventually give rise to common lymphoid progenitors (CLPs), which can give rise to B cells, T cells, and NK cells but not myeloid-lineage cells (3-5).  Common lymphoid progenitors, defined as Lin-Sca-1loc-kitloCD127+ cells, have traditionally been considered to be precursor cells that give rise to T and B cells but recently revised models of lymphopoiesis suggest that CLPs represent early B-lineage specific progenitors, and that the branchpoint between B and T cell development occurs at an earlier stage of development (6, 7).  Rumfelt et al. showed that Ig H-chain rearrangement is initiated in CLPs and continues as these cells mature into pre-pro-B cells (Lin-CD45R+CD43+AA4.1+CD19-Ly6C-) and then pro-B cells (CD45R+CD19+CD43+AA4.1+) (6).    2 The presence of various secreted factors (CXCL12, FLT3 ligand, IL-7, SCF, RANKL) in the BM and the expression of specific transcription factors (PU.1, Ikaros, E2A, Bcl11a, EBF and Pax-5) are critical for the development of B cell progenitors (Figure 1.1) (5, 8, 9). Pre-pro-B cells and pro-B cells acquire survival, differentiation, and proliferation signals from BM stromal cells (10).  IL-7 is a key cytokine that drives early B cell lymphopoiesis, promoting V to DJ rearrangement and transmiting survival and proliferation signals (11). Pre-B cells, which have undergone functional Ig H-chain rearrangement, express the pre- BCR (Ig H-chain and surrogate L-chain consists of the heterodimer of λ5 and VpreB) (12). The interaction of the pre-BCR with its putative ligands serves as a proliferative stimulus (13).  Although the ligand for the pre-BCR is not known, one current model suggests that ligand-independent oligomerization of the pre-BCR provides a survival and proliferation signal (14).  Finally, IgM+ immature B cells are produced from pre-B cells following productive Ig L-chain gene rearrangement (15).  These newly produced IgM+ cells then migrate from the BM to the spleen where they undergo further maturation. Immature B cells exiting the BM express cell surface IgM as well as CD21, CD22 and the B-lineage precursor marker AA4.1 (CD93) on their surface.  Immature B cells are also referred to as “transitional” (T1 and T2) B cells (16-18).   Immature B cells enter the spleen as T1 B cells (AA4.1+IgD-IgM+CD21loCD23-).  T1 B cells develop into T2 B cells (AA4.1+IgDhiIgMhiCD21intCD23+), a process that depends on tonic BCR signaling, survival signals derived from binding of the cytokine BAFF to its receptor, and activation of the non- canonical NF-κB pathway.  Ultimately, T2 B cells give rise to two different subsets of long- lived mature B cells, follicular B cells (or B-2 cells) and marginal zone (MZ) B cells.  A third subset of mature B cells, with self-renewing ability, called B-1 cells are derived from a fetal liver B cell progenitor and are enriched in the peritoneal and pleural cavities.   3  Figure 1.1  B cell development. B cell progenitors develop in the BM from the CLP.  Immature B cells then migrate to the spleen and differentiate further into follicular B-2 cells and MZ B cells.  Upon Ag encounter, these mature B cells undergo differentiation into Ab-secreting plasma cells.  Activated B-2 cells form germinal centers and differentiate into plasma cells or memory B cells.  Other minor B cell subsets (B-1a, B-1b, and B10) are also illustrated.  CLP, common lymphoid progenitor; SHM, somatic hypermutation; CSR, class switch recombination. Adapted from (1).  1.1.1 Follicular B-2 cells The majority of mature B cells are recirculating B-2 cells (IgMloIgDhiCD21intCD23+) that home to B cell follicles in the secondary lymphoid organs.  These B cell follicles are always adjacent to T cell zones. This arrangement allows activated follicular B cells and activated T helper cells to migrate towards each other and interact at the interface between these two areas.  B-2 B cells encounter T cell-dependent foreign Ags that are bound to follicular dendritic cells (FDC) within the lymphoid follicles.  These activated B cells can   4 then proliferate and differentiate into either short-lived plasmablasts or initiate a germinal center (GC) reaction (1). Ag activation of mature B cells leads to the transient generation of short-lived plasmablasts that secrete germ line-encoded Abs.  However, in a secondary immune response, other B cells can initiate a GC reaction, which is characterized by clonal expansion, class switch recombination at the IgH locus, somatic hypermutation of the VH genes, and selection for increased affinity of the BCR for the Ag (affinity maturation) (1).  GC-derived memory B cells with enhanced affinities for the specific Ag persist for a long time and can rapidly expand and differentiate into plasma cells upon secondary challenge.  Some of the B cells produced during the GC reaction will become long-lived plasma cells, which will migrate to the BM, survive for prolonged period without the need for self-replenishment or turnover, and continuously secrete Abs (19, 20).  1.1.2 Marginal zone B cells MZ B cells (IgMhiIgDloCD21hiCD23-) are non-circulating B cells that reside in the region surrounding the marginal sinuses of the lymphoid follicles in the spleen (21-23). Because of their special location they scan the blood on its way from the arterial sinuses to the venous sinuses, making them the first population of cells encountered by blood-borne Ags.  MZ B cells are distinguished from conventional follicular B cells by their rapid response to T-independent type II Ags (mainly microbial Ags with many repeating subunits) and their ability to make natural Abs (23-26).  These Abs are produced spontaneously without any apparent exposure to the Ag (27).  Natural Abs are predominantly IgM and can also be made by B-1a cells (see below) in the absence of obvious stimulation by exogenous Ags.   The Ig repertoire of MZ B cells is skewed towards the recognition of self-Ags and microbial Ags.  Accordingly, natural Abs often recognize epitopes on encapsulated Gram- positive bacteria, pathogenic viruses, apoptotic cells, and oxidized low-density lipoproteins, thereby providing immediate protection against infection in addition to preventing inflammation by facilitating the clearance of oxidized lipids, oxidized proteins, and apoptotic cells (27-29).  MZ B cell deficiency results in a reduction in T-independent immune   5 responses and natural Ab titers (30-32), resulting in an inability to clear microbial infections. In addition, the high level of CD21 (also known as complement receptor type 2) on MZ B cells can facilitate the transport of immune complexes from the circulation to the splenic follicles, where Ags can be captured by follicular DCs and facilitate the initiation of an adaptive immune response by circulating B-2 cells (33).  Thus, MZ B cells contribute to the host defense by providing “natural memory” and by bridging the innate and adaptive immune responses. The initial MZ B cell fate decision depends on the strength of BCR signaling as well as inductive signals from Notch2, the receptor for BAFF, and the canonical NFκB pathway, as well as signals involved in cell migration and adhesion (Figure 1.2) (23).  The precise order and the mechanism by which these signals are integrated to drive MZ B cell commitment and differentiation is unclear.  The current model suggests that the commitment of MZ B cell precursors to the MZ B cell fate occurs in either the red pulp or the MZ and is driven by weak BCR signals, signals from the BAFF receptor, and signaling induced by the Notch2 ligand delta-like 1 (DL1), which is highly expressed in the fenestrated venules of the red pulp.  Signaling by integrins and chemoattractant receptors then direct the proper positioning and trafficking of MZ B cells in the spleen.    6  Figure 1.2 A schematic view of the anatomy of the spleen and MZ B cell trafficking. Developing MZ B cell precursors migrate to the fenestrated venules in the red pulp where Notch2 on their surface can bind the Notch ligand DL1.  MZ B cell localization at the MZ depends on chemotatic signals from S1P.  MZ B cells can also migrate into the B cell follicles in response to CXCL13.  DL1, delta-like 1; S1P, sphingosine-1-phosphate.  Adapted and modified from (23).  The signals from integrins and chemoattractant receptors are important for the localization and function of MZ B cells.  MZ B cells express the αLβ2 (LFA-1) and α4β1 (VLA-4) integrins, which allow them to adhere strongly to marginal sinus stromal cells that express ICAM-1 and VCAM-1, the ligands for LFA-1 and VLA-4, respectively (34).  The lipid chemoattractant sphingosine-1-phosphate (S1P) is also necessary for MZ B cell positioning in the MZ area, overcoming signals from CXCL13 that induce the migration of B cells to the lymphoid follicles (35).  The shuttling of MZ B cells between the MZ area and B cell follicles is the result of oscillating responses to the chemotatic signals from S1P and CXCL13 (33).  This shuttling allows MZ B cells that have acquired blood-borne Ags in the MZ to deliver these Ags to FDCs (33).   7 Signals that regulate MZ B cell migration and adhesion have a direct effect on the homing, localization, and the immune function of MZ B cells.  Consistent with this idea, mutations in genes encoding components of integrin or chemokine signaling pathways often result in the loss of MZ B cells.  MZ B cells are either missing or greatly reduced in number in mice deficient for Pyk2, (a kinase involved in both adhesion and migration), Lsc (a RhoGEF involved in chemokine signaling), Dock2 (an activator of the Rac GTPase), Rap1b (an important regulator for B cell migration and adhesion), or RapL (a downstream effector of Rap involved in integrin activation).   This suggests that signals from both chemoattractant receptors and integrins may be important for the maturation and survival of MZ B cells, as well as their retention in the spleen (31, 32, 36-38).  1.1.3 B-1 cells  B-1 cells (CD45R/B220loIgMhiIgDloCD23-CD43+) are distinguished from B-2 cells based on their anatomical localization, cell surface phenotype, self-renewal capacity, BCR signaling, and contribution to natural IgM Abs present in the serum (39, 40).  B-1 cells are normally found in the peritoneal and pleural cavities, Peyer’s patches, tonsils, and spleen (they represent ~5% of splenic B cells) but are absent from lymph nodes.  B-1 cells are further subdivided into the B-1a (CD5+) and B-1b (CD5-) subsets.  B-1a cells make natural Ab and provide innate protection against bacterial infections in naïve hosts.  In contrast, B-1b cells function independently as the primary source of long-term adaptive Ab responses to polysaccharides and other T cell-independent type II Ags during infection (41, 42).  The B-1 cell progenitor has a fetal origin and appears to be distinct from the progenitors that develop into B-2 and MZ B cell populations (43, 44).  Two models have been proposed to account for the origin of B-1 cells.  The lineage model proposes the existence of distinct progenitors for B-1 and B-2 cells.  By contrast, the selection model proposes that B-1 and B-2 cells are derived from a common progenitor and that Ag selection at the sIgM+ transitional 1 B cell stage determines whether a B cell will commit to the B-1 or B-2 lineage (39, 40).    8  Unlike B-2 and MZ B cells, B-1 cells are hypo-responsive to BCR cross-linking and share some similarities with anergic cells (45).  The CD5 molecule on B-1a cells inhibits signaling through the BCR by recruiting the SHP-1 protein tyrosine phosphatase to the BCR complex (46, 47).  As B-1b cells do not express CD5, the mechanism by which BCR signaling is inhibited in these cells is not clear.  A recent study suggests that the mechanism of BCR signaling inhibition is due to the elevated levels of activated Lyn tyrosine kinase in B-1 cells (48).  Although BCR signaling is downregulated in B-1 cells, microbial components such as CpG DNA are potent activators of these cells.  Importantly, both MZ and B-1 cell numbers are increased in several murine models of autoimmune disorders and in human patients with lupus or rheumatoid arthritis (49-53), suggesting a clinical relevance of these innate-liked B cells in autoimmunity.  This may be related to the fact that both of these cell populations have Ig repertoires that are skewed towards microbial Ags that crossreact with self-Ags.  This molecular mimicry may have evolved as an attempt by microbes to escape immune recognition.  1.1.4 Regulatory B cells Recently, regulatory B cell subsets that oppose autoimmune responses and inflammation have been identified.  Diverse subsets of B cells with B-1a (CD5+), MZ (CD21+CD23-), and MZ precursor (CD1d+CD21+CD23+) phenotypes have regulatory activities (54-56), in particular suppressing the activation and differentiation of CD4+ T cells, CD8+ T cells, NKT cells, and other immune cells (57).  These B cells exert their regulatory role mainly through the production of IL-10 and other cytokines such as IL-4, IL-6, IFN-γ, and TGF-β (55, 58, 59). Another rare subset of CD1dhiCD5+CD19hi regulatory B cells termed B10 cells has recently been identified.  B10 cells share cell surface markers with B-1a, MZ, and MZ precursor B cells and are found within the spleen of naïve wild-type mice at frequencies of 1- 2% (60).  B10 cells inhibit T cell-mediated inflammatory responses through the production of IL-10 (60, 61).  Signals from the BCR, CD40, and the TLRs are involved in the initiation of IL-10 production by these regulatory B cells (55, 62).   9 Similar to regulatory T (Treg) cells, regulatory B cells suppress inflammatory responses and prevent autoimmunity.  The loss of regulatory B cells can exacerbate disease symptoms in experimental autoimmune encephalitis (EAE), chronic colitis, contact hypersensitivity (CHS), collagen induced arthritis (CIA), and non-obese diabetic (NOD) mouse models (54, 60, 63-67).  Conversely, adoptive transfer of pre-activated regulatory B cells can reduce inflammation and reduce the onset or severity of autoimmune disease in these disease models (58, 60, 61, 65-68).  Thus in addition to producing Abs and acting as antigen presenting cells (APCs), B cells act as important regulatory cells that control autoimmunity and inflammation.  1.1.5 B cells and disease B cells undergo strictly regulated developmental process in which both central and peripheral tolerance mechanisms enable the removal of potentially harmful and auto-reactive B cells from the immune system (69, 70).  Under normal situations, self-reactive B cells undergo apoptosis or become anergic when they encounter multivalent self-Ag during development or when mature B cells are activated without CD40 co-stimulatory signal provided by T cells.  Hence, defects in immunological tolerance can lead to autoimmunity (64, 71).  Other B cell processes such as gene rearrangement, receptor editing, Ig class switching, and proliferation can potentially introduce unwanted mutations that result a block in B cell development or malignant transformation.  Therefore, immune diseases such as immunodeficiency, leukemia/lymphoma, and autoimmunity can arise from altered B cell development or a failure to induce tolerance (71).  Previous research on autoimmunity had focused largely on T cells, but now studies are increasingly directed towards understanding the mechanisms by which B cells participate in the induction and maintenance of autoimmunity (64, 71).  Given their ability to produce self-reactive Abs, secrete inflammatory cytokines, participate in Ag presentation, and facilitate T cell activation, B cells are now regarded as important therapeutic targets (1, 64, 71).    10 1.1.5.1 B cell deficiency  B cell deficiencies can result from mutations in signaling molecules that are important for B cell development or contribute to the tonic BCR signaling that promotes the survival and differentiation of B cell progenitors.  Mutations in the recombinase activating genes (RAG) often leads to severe combined immunodeficiencies (SCID) as both T and B cells are unable to develop in these individuals.  X-linked agammaglobulinemia (XLA) is the most common abnormality of B cell development (72, 73).  This is caused by mutation in a single gene known as Bruton’s tyrosine kinase (Btk), which is a signaling molecule downstream of the pre-BCR and the BCR.  Loss-of-function mutations in Btk arrest B cell development at the pre-B cell stage and result in a severe deficit in Ab production.  Mutations in the gene encoding CD19, a co-receptor for the BCR, results in common variable immune deficiency (CVID) in which patients have normal numbers of mature B cell number but often exhibit inefficient B cell activation, low serum Ig titers, and reductions in memory B cell number (74).  Thus, understanding the signaling mechanisms involved in B cell development is important for identifying the cause of these immunodeficiency diseases.  1.1.5.2 B cells in autoimmunity  B cells play a central role in the induction and pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and type1 diabetes. The mechanisms by which they contribute to the initiation of these diseases or amplify on- going inflammatory processes include loss of tolerance or hyperactivity leading to auto-Ab production, presentation of self Ags to T cells, and increased cytokine production (75, 76). In particular, auto-Ab production can lead to activation of the complement system resulting, in exacerbation of inflammatory diseases. In SLE and RA patients, the serum concentration of BAFF and anti-nuclear Abs are elevated (77-80).  BAFF is a member of the TNF family of cytokines that promotes B cell survival and activation.  In SLE and RA patients, ectopic GCs containing activated B cells develop within the tissues.  At these sites, the B cells can efficiently present Ags and trigger auto-reactive T cell activation (71, 76, 78).  Therefore, B cells can contribute to the   11 pathogenesis of RA and SLE by Ag presentation, auto-Ab production, and inflammatory cytokine production.  B cell depletion using anti-CD20 monoclonal Abs (Rituximab) or other approaches has become an effective therapy for these autoimmune diseases (81, 82). The accumulation of B cells in ectopic GCs and inflamed tissues is dependent on chemokine-induced migration.  Chemokines contribute to homeostatic migration as well as the entry of immune cells into acute and chronic inflammatory sites.  It has been suggested that the chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4 and CXCR5 are important for the formation of ectopic GCs and the accumulation of immune cells at these sites (83).  In particular, CXCL12 supports local immune cell proliferation, cytokine production, and the expression of ICOS-L on B cells, a co-stimulatory molecule that promotes T cell activation (84).  Moreover, elevated levels of B-1 cells have been documented in patients with autoimmune disorders such as RA and Sjogren’s syndrome (50, 85).  Cell surface expression of CXCR5, the receptor for the B cell chemoattractant CXCL13, is significantly higher on CD5+B-1 cells than on B-2 cells (86).  Studies in mouse models of SLE suggest that the increased expression of CXCL13 by myeloid DCs in the target organs may also be involved in the recruitment of auto-Ab-producing B cells and the activation of self-reactive T cells during disease onset (87-89).  Furthermore, in vivo evidence for the pathogenic relevance of the CXCL13–B cell axis has been recently provided in collagen- and Ag-induced arthritis, in which CXCL13 neutralization with Abs or genetic ablation of CXCR5 interferes with the formation of ectopic GCs in the synovia, thereby improving disease outcome (90, 91).  Collectively, these findings suggest that chemokine- induced B cell migration plays a key role in the pathogenesis of autoimmune disorders. B cells have been also implicated in the development of type 1 diabetes, which is an autoimmune disease characterized by the destruction of insulin-producing islet cells in the pancreas.  Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes and have been widely used to study the pathogenesis of this disease.  B cell deficient-NOD mice do not develop diabetes (92, 93), supporting the idea that B cells are involved in the disease initiation.  Accordingly, depletion of B cells with anti-CD20 Ab delays the onset of the disease (94, 95).  Interestingly, a recent study has shown that the MZ B cell population is expanded in NOD mice (96), suggesting that MZ B cells are involved in the disease   12 progression.  Although MZ B cells are non-circulating B cells located in the spleen, TLR signaling causes MZ B cells to alter their chemoattractant receptor expression, allowing them to leave the MZ area and migrate to the pancreatic lymph node (97).  These activated MZ B cells can then present auto-Ag to auto-reactive T cells (96), which can subsequently migrate to the pancreas and destroy the insulin-producing islet cells.  Together, these findings highlight an important role for MZ B cell migration in the pathogenesis of type 1 diabetes and suggest that the ability of MZ B cells to migrate to other lymphoid tissues may be a key factor in the initiation of many autoimmune diseases.  1.1.5.3 B cells in cancer  B cells undergoing Ig gene rearrangement during development or somatic hypermutation during the GC reaction have a high probability of acquiring unwanted mutations.  Mutations that lead to the loss of growth control can result in the development of B cell leukemias and lymphomas (Table 1.1).  Infection by hepatitis C virus and Helicobacter pylori can also initiate the development of splenic marginal zone lymphomas (SMZL) and mucosa-associated lymphoid tissue (MALT) type MZ lymphomas, respectively (98).   13  Malignancy Putative cell of origin Frequency among all lymphomas (%) Mantle-cell lymphoma Pre-GC B cell, CD5+ mantle- zone B cell 5 Chronic lymphocytic leukemia (CLL) Ag-experienced B cell (pre-or post-GC) 7 Burkitt’s lymphoma GC B cell 2 Follicular lymphoma GC B cell 20 Marginal-zone lymphoma – nodal, extranodal (MALT) and splenic  Marginal zone B cell, subset of naïve B cells that have partially differentiated into marginal zone B cells 7 Diffuse large B cell lymphoma (DLBCL) GC B cell or post-GC B cell 30-40 Lymphoplasmacytic lymphoma (LPL) Post-GC B cell 1 Multiple myeloma Plasma cell 10 Hodgkin lymphoma (classical type) Defective GC B cell 10 Hodgkin lymphoma (nodular lymphocyte pre-dominant type) GC B cell 0.5 B cell prolymphocytic leukemia  Memory B cell <1 Hairy cell leukemia Memory B cell <1  Table 1.1 Human mature B cell malignancies. List of mature B cell malignancies, their putative origins, and their frequencies of incidence in Europe and North America.  GC, germinal center.  Adapted from (98, 99).   Chemokine-mediated migration, and subsequent interactions of malignant B cells with stromal cells or other cell types in the tumor microenvironment, are important for the initiation and maintenance of lymphoma and leukemia (100).  Lymphomas normally promote the formation of a complex surrounding microenvironment that favors cell growth, the inhibition of apoptosis, and angiogenesis.  Macrophages, DCs, and regulatory T cells residing within the tumor can produce immunosuppressive cytokines such as IL-10 and TGF-β, as well as factors such as BAFF and CXCL12 that support the survival of the lymphoma cells (100-102).  Chemokines and their receptors are important for the initiation and maintenance of many hematopoietic malignancies.  For examples, B-CLL cells express high levels of CXCR4, which allows them to home to the BM (102).  CXCL12 can also upregulate MMP-9 expression in B-CLL cells (103), promoting cell invasion and tranendothelial migration. Other chemokine receptors such as CCR7 and CXCR5 that are expressed by B-CLL cells are   14 also important for the migration and accumulation of leukemic cells in lymphoid tissues (104, 105).  Therefore, preventing the homing and interaction of these tumor cells with their microenvironment can potentially deprive them of growth and survival signals, a potential strategy for controlling B cell-derived cancers.    15 1.2 B cell activation Ag-induced signaling by the BCR initiates an activation program that leads to B cell proliferation and subsequent differentiation into Ab-producing cells.  BCR clustering by Ags initiates multiple signaling pathways that control gene expression, cell survival, proliferation, and morphology (106-108).  Thus, B cell recognition of Ag is necessary for B cell activation and function.  1.2.1 B cell antigen acquisition B cells need to find their cognate Ag in order to initiate a humoral immune response. The sites of first encounter between B cells and Ag have only recently been visualized with the advent of intravital two-photon microscopy, which allows the direct visualization of the spatial and temporal organization of the B cell response to Ag in intact lymphoid organs in real-time (109, 110).  This work has revealed that B cells come into contact with Ags in multiple ways (see Figure 1.3).   16   Figure 1.3   Mechanisms by which B cells encounter Ags. A, B cells can encounter small Ags from the lymphatic fluid.  B cells can also recognize Ag that is presented on the surface of FDCs and SCS macrophages in the form of immune complexes. B, B cells entering the lymph node can recognize Ags presented on the surface of recently migrated DCs that are in close proximity to the HEVs. C, The conduit network in the lymph node is composed of a collagen core and is lined with FRCs.  DCs can extend short protrusions into the conduits and sample lymphatic fluid.  Ags, antigens; FRC, fibroblastic reticular cells; FDC, follicular dendritic cell, DC, dendritic cells; SCS, subcapsular sinus; HEV, high endothelial venules.  Adapted and modified from (111).   17 Subcapsular sinus (SCS) macrophages in the lymph nodes serve an important role in the early detection of blood-borne Ags by B cells (112-114).  As shown by intravital two- photon microscopy, SCS macrophages often send cytoplasmic protrusion into the subcapusular sinus to sample the lymph and in doing so become coated with Ag complexes (Figure 1.3) (110, 114).  Large particulate Ags such as vesicular stromatitus virus (113), inert beads coated with Ags (112), and immune complexes can be trapped by SCS macrophages via their complement receptors and potentially via their Fc and mannose receptors (110, 114, 115).  Ag-Ab complexes are initially trapped in the floor of the SCS but rarely stay there for more than a day (110, 112-114).  B cells that reside underneath the SCS macrophages can sample Ags acquired by SCS macrophages in the subcapsular region.  Therefore, SCS macrophages are important for B cells to encounter particulate Ags, allowing this interaction to occur within the first few hours of an immune response.  SCS macrophages express high levels of adhesion molecules VCAM-1 and ICAM-1 (113), which may facilitate their interactions with B cells and promote the formation of an immunological synapse between the SCS macrophage and the B cell (116). B cells can also relay Ags from SCS macrophages to follicular DCs (FDCs) within the lymphoid follicles.  B cells can capture immune complexes via complement receptors, which then localize to the uropod of the B cells as it migrates into the lymphoid follicle and then trasnsfers the Ag to the FDC (114).  MZ B cells in the spleen may also shuttle Ags into lymphoid follicles via a similar mechanism (33, 117). FDCs, and the prolonged display of these Ags by FDCs may ensure that rare cognate B cells can encounter Ags and become activated (110).  A recent study by Suzuki et al. showed that Ag acquisition by B cells from FDCs could be detected more than one week after immunization (118).  Similar to SCS macrophages, immune complexes are maintained on FDCs by Fc receptors and complement receptors (119-122).  FDCs also express the integrin ligands ICAM-1, VACM-1 and MAdCAM-1, allowing B cells to adhere to them via integrins.  FDCs also produce the chemokine CXCL13, and this attracts B cells to the follicles where they can deposit and capture Ags (118, 123, 124).  Therefore, FDCs serve as important sites of B cell Ag capture by maintaining Ags for a prolonged period so that even rare B cells traveling from distant sites have the chance to encounter Ag.   18 B cells can also encounter Ags in the T cell zone, where Ags are transported by DCs from the periphery to the lymph nodes (125).  CD11c+ splenic DCs pulsed with hen egg lysozyme have been shown to migrate to the lymph node and activate newly arrived B cells in the vicinity of HEVs, which that are located in the outer T cell zone and inter-follicular regions (125).  It appears that certain DCs do not degrade Ags but can maintain the Ag on its surface (or protected in endosomes), possibly bound to FcγRIIB (126). Mature recirulating B cells enter lymph nodes via HEVs and migrate to the follicle where they spend about 24 hr before re-entering the circulation (127).  Not all lymph-borne Ags require binding by SCS macrophages to enter the lymph node follicles.  Pape et al. found that small protein molecules (less than 70 kDa) rapidly enter the B cell follicles in draining lymph nodes in an SCS macrophage-independent manner (128).  These small Ags enter the follicles via small gaps (0.1-1 µm wide) in the floor of the lymph node sinus or through lymphoid conduits (Figure 1.3).  The conduit system is an interconnected network of collagen fibers ensheathed by fibroblastic reticular cells (FRCs) (129).  These networks are found throughout most lymphoid organs and serve as an important “highway” for small molecules to reach the interstitial space of the lymph nodes (130).  In support of this model, Roozendaal et al. tracked the distribution of fluorescently-labeled turkey egg lysozyme in real time by two-photon microscopy and found that it first traveled into the follicles via conduits before reaching follicular B cells (131).  Conduits not only deliver Ag to FDCs but also provide a rich source of CXCL13 secreted by FRCs (131).  Therefore, B cells can be attracted to the conduit network by chemokines and acquire small protein Ags that are released from the conduits. In summary, during the first hours after the arrival of immune complexes in a lymph node, B cells have the opportunity to encounter Ags displayed by SCS macrophages.  At later times, FDC-bound Ags may be the main form of Ag encountered by B cells as FDCs can retain Ags on their surface for a longer time.  Importantly, efficient Ag encounter and trafficking relies on chemotatic and adhesive signals provided by the specialized stromal cells in the lymphoid organs.  Once B cells bind Ag via their BCR, intracellular signaling by the BCR initiates B cell activation.    19 1.2.2 B cell receptor signaling The BCR complex consists of the two Ig H-chains and two L-chains that are non- covalently associated with the Igα and Igβ subunits, which have the ITAM domains that are critical for signal transduction (132).  BCR oligomerization induced by the binding of multivalent Ags, by Ag arrays displayed on the surface of other cells, or experimentally with anti-Ig Abs initiates BCR signaling.  The initial clustering of the BCR allows Src family kinases (SFKs) such as Lyn to phosphorylate the tyrosine residues in the ITAM motifs of the Igα and Igβ cytoplasmic domains (133).  The Syk tyrosine kinase as well as the BLNK and BCAP adaptor proteins can then be recruited to the phosphorylated BCR (132, 134).  This promotes the recruitment and activation of PLCγ and PI3K, and subsequently the production of key second messengers (Figure 1.4). PLCγ activation results in the production of IP3 and DAG, second messengers that each activates downstream signaling pathways.  The binding of IP3 to its receptor on the endoplasmic reticulum (ER) results in the release of Ca2+ from the ER and causes a Ca2+ flux in the cells.  Calmodulin bound with Ca2+ activates the phosphatase calcineurin, which dephosphorylates the transcription factor NFAT, allowing it to migrate into the nucleus and promote transcription (135).  DAG activates RasGRP and PKCβ, which will then activate the Ras/Raf/ERK pathway and the transcription factor NFκB, respectively (136).  The translocation of NF-κB and NFAT into the nucleus results in activation of genes involved in cell proliferation, survival, and differentiation.  Moreover, DAG can activate guanine nucleotide exchange factor (GEF) proteins such as RasGRP2 (CalDAG-GEF1), which activates the Rap GTPases, key regulators of integrin activation and cytoskeletal rearrangement (137, 138).   20   Figure 1.4   BCR signaling. BCR aggregation allows Syk and SFKs recruitment and activation.  SLP65/BLNK recruits PLCγ2, which generates DAG and IP3, leading to activation of transcription factor NFAT.  SLP65/BLNK also recruits Grb2 and Vav, which in turn lead to activation of ERK and Rac, respectively.  The Ras/ERK pathway is activated primarily by RasGRP, a Ras-GEF, in B cells.  DAG also leads to the activation of PKC and the subsequent activation of NFκB pathway and other signaling molecules such as Rap1.  Btk, Bruton’s tyrosine kinase; DAG, diacylglyceral; ERK, extracellular-signal-regulated kinase; Grb2, growth-factor receptor-bound protein2; IKK, inhibitor of nuclear factor-κB (IκB) kinase; InsP3, inositol-1,4,5- trisphosphate; NFAT, nuclear factor of activated T cells, NFκB, nuclear factor-κB; PKCβ, protein kinase C-β; PLCγ2, phospholipase C-γ2; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; SOS, son of sevenless homologue; RasGRP, Ras guanyl releasing protein; GEF, guanine nucleotide exchange factor. Adapted and modified from (139).     21 BCR activation also promotes cell survival via the PI3K/Akt pathway.  BCR signaling induces phosphorylation of the BCAP adaptor proteins and this provides a binding site for the SH2 domain of the p85 subunit of PI3K.  The Syk tyrosine kinase can also phosphorylate the YXXM motif on CD19, providing binding sites for PI3K, Vav, and Lyn (140-142).  Thus, BCAP and CD19 cooperatively facilitate the membrane localization of PI3K (143, 144).  This allows PI3K to generate the membrane lipid PIP3, which recruits PH domain-containing proteins such as Akt.  Mice with a targeted gene disruption of p85α, a regulatory subunit of PI3K, exhibit impaired B cell development at the pro-B cell stage, reduced numbers of mature B cells, and reduced B cell proliferative responses (145). Accordingly, deficiency in both BCAP and CD19 results in a complete abrogation of BCR- induced Akt activation and severe defects in the generation of immature and mature B cells (146).   Therefore, BCAP and CD19 have an important role in BCR-mediated PI3K activation and B cell development.  Furthermore, the production of PIP3 also facilitates the phosphorylation of Vav by Syk.  Vav can then promote activation of the Rac GTPase, which in turn activates the p38 and Jnk1/2 MAP kinase pathways, which are important for cell survival (132). The activation of B cells by APCs involves integrins and the formation of an immunological synapse.  During the early stage of membrane-bound Ag recognition, a B cell spreads on the surface of the APC and then contracts, collecting the surface bound Ags into a central aggregate (134).  The interface formed between the B cell and APC is termed an immunological synapse (147), which is characterized by the central accumulation of BCR and Ags (central supramolecular activation complex, c-SMAC), surrounding by a ring of integrins (peripheral supramolecular activation complex, p-SMAC) (134, 148).  Intracellular signaling molecules such as Syk, PLCγ, and Vav are co-localized with the initial BCR- containing microclusters (149).  After maximal spreading (approximately 3 min), these BCR microclusters start to concentrate at the c-SMAC.  At a later time point, CD19, Syk, PLCγ, PI3K, and Vav dissociate from the BCR clusters in the c-SMAC due to BCR dephosphorylation.  Finally, the BCRs in the c-SMAC are internalized with the captured Ags and trafficked to endocytic compartments where the bound Ag can be processed into peptides and bound to MHC II proteins for presentation to T cells (132, 150).   22 1.3 Integrins and B cell adhesion The major function of integrins is to mediate adhesion and to link the extracellular matrix (ECM) to the cytoskeleton.  Integrins are a group of heterodimeric transmembrane receptors that consist of an α- and a β-subunit.  In mammals, there are 18 α-subunits and 8 β- subunits, and together they can give rise to 24 different αβ-pairs (151-153).  Lymphocytes mainly express the αLβ2 integrin (LFA-1), α4β1 integrin (VLA-4), and α4β7 integrin (151). B cell integrin ligands are predominantly ECM proteins (e.g. fibronectin and collagen) and cell surface molecules (e.g. ICAM, VCAM-1 and MAdCAM-1) (Table 1.2).  In B cells, integrin-dependent adhesion is involved in development, survival, activation, migration, spreading, and immunological synapse formation (1, 154-158).  The importance of integrin function is also demonstrated in patients with type I lymphocyte adhesion deficiency (LAD) who lack β2 integrins, resulting in defects in adhesion for multiple cell types, recurrent bacterial infections, and impaired wound healing ability (153, 159).  Integrin Common ligands αLβ2 (LFA-1) ICAM-1, 2, 3, 5, JAM-A α4β1 (VLA-4) VCAM-1, Fibronectin, JAM-B α4β7 (LPAM-1) MAdCAM-1, Fibronectin α1β1 (VLA-1) Collagen, Laminin α2β1 (VLA-2)  Collagen, Laminin  Table 1.2  Integrins expressed on B cells. List of integrins normally found on B cells and their major ligands.  LFA, lymphocyte function-associated; ICAM, intercellular adhesion molecule; VLA, very late antigen; VCAM, vascular cell adhesion molecule; LPAM, lymphocyte Peyer’s patch adhesion molecule; MAdCAM, mucosal addressin cell adhesion molecule; JAM, junctional adhesion molecule.  Adapted from (160, 161).   The adhesive ability of integrins is determined by their affinity (the strength of the receptor binding to their ligands) and their avidity (the extent to which the receptors cluster on the cell surface). This change in adhesion ability involves structural changes in the integrins as well as releasing the integrins from the cytoskeleton such that their lateral mobility in the membrane is increased (153).  Integrin are involved in two types of signaling:   23 “inside-out” signaling, which is the process by which integrins are activated, and “outside-in” signaling where ligand-bound integrins initiate intracellular signaling (Figure 1.5) (153).  1.3.1 Inside-out signaling and integrin activation In a circulating or resting lymphocyte, integrins remain in a low affinity and avidity state.  Signals from the BCR or chemoattractant receptors can trigger a conformational change that leads to activation of the integrin, i.e. conversion to a state of high affinity/high avidity for their ligands (151).  Moreover, mechanical forces supplied by the blood flow can stabilize the adhesive interactions of integrins with their ligands (162).  It is thought that the coupling of integrins to the actin cytoskelelton by the adaptor protein talin allows mechanical tension to alter the interaction of the integrin cytoplasmic domains and promote conversion of the extracellular domain to a state of high affinity for ligand. BCR-induced integrin activation via “inside-out” signaling requires the activation of Lyn, Syk, PI3K, Btk, Vav, Rac2, PLCγ, Ca2+ release, and the activation of PKC and the Rap1 GTPase (157, 163, 164).  Recent studies on integrin signaling using genetic knockouts have suggested that Rap GTPase is the master regulator of integrin activation in B cells (see section 1.7 for more details on Rap).  Expression of a constitutively active mutant form of Rap (Rap1V12) in B cells results in increased integrin affinity (163).  Conversely, B cells lacking Rap1b exhibit defects in integrin-dependent adhesion.  Second messengers such as DAG can lead to Rap activation via RasGRP2 (CalDAG-GEF) in neutrophils and platelets (159) but it is not known if this GEF is responsible for Rap activation in lymphocytes.  Other studies have shown that Rap can be activated via protein kinase D (PKD) (153, 165).  In this model, DAG production can activate PKC, which can phosphorylate PKD1.  PKD1 can associate with Rap via its PH domain, leading to Rap activation and relocalization of Rap1 to the integrin cytoplasmic tails (166).  Activated Rap can recruit the adaptor protein RapL and the Mst1 kinase to the α-chain of the integrin (38, 167, 168).  This Rap/RapL/Mst1/integrin complex association is required for integrin activation as Mst1 kinase activity is not detected in RapL-deficient cells and receptor-induced integrin activation is attenuated when RapL or Mst1 expression is lost (38, 169).  In T cells, Rap can also be activated via the adaptor   24 proteins RIAM and ADAP, which can associate with Skap55 (170-172).  The formation of a Rap1/RIAM/ADAP/Skap55 complex leads to recruitment of talin to the cytoplasmic domain of the integrin β tail chain and subsequently to the unfolding and activation of the integrin. In Skap-H deficient B cells, LFA-1 and VLA-4-mediated adhesion is attenuated (173), suggesting that Rap and Skap-H might function in the same integrin activation pathway in B cells. Kindlin-3 has recently been identified as a haematopoietic-specific activator of integrins and also as a binding partner for the β1 and β3 integrins (174).  Kindlin-3-deficient mice die shortly after birth due to severe bleeding from platelet dysfunction, a consequence of defective inside-out signaling and integrin activation.  Kindlin is thought to function as a co-activator of integrins with talin because co-expression of the talin and kindlin-2 FERM domains has a synergistic effect on integrin activation (175).  The binding of kindlin-3 to β2 is necessary for integrin activation in neutrophils (176), and it is likely that kindlin-3 might be involved in the activation of the LFA-1 integrin in B cells.    25  Figure 1.5 Integrin signaling pathways. The figure outlines the key signaling events of integrin activation via “inside-out” signaling (top) and “outside-in” integrin signaling (bottom).  The mechanisms are described in the main text.  RIAM, Rap1- GTP-interacting adapter molecule; ADAP, adhesion and degranulation promoting adapter protein; SFK, Src family kinases. Adapted from (153).   26 1.3.2 Outside-in integrin signaling Integrins have no intrinsic enzymatic activity, but their binding to ligands can initiate “outside-in” signaling via direct association of integrin tails with kinases, phosphatases, and adaptor proteins (151, 177).  In lymphocytes, antibody-induced clustering of β2 integrins result in formation of a focal area of polymerized actin (referred to as an actin cloud) that contains many signaling molecules (178).  Activation of SFKs is the key initial step in “outside-in” signaling (179).  SFKs and Syk can bind directly to integrin tails and are crucial for proximal integrin signaling as either SFK or Syk/Zap70 deficiency results in severe defects in cell adhesion and spreading (180).  How integrins activate SFK is not clear, but it has been suggested that an ITAM-containing adaptor protein (e.g. DAP12 and FcγR) may be involved in the initial activation of Syk, followed by SFK activation (181).  Activated SFK and Syk/Zap70 can phosphorylate Vav, which can activate Rho GTPases, leading to actin cytoskeletal reorganization.  SFK can also activate the non-receptor tyrosine kinases FAK and Pyk2, leading to phosphorylation of the Cbl adaptor protein on sites that promote the translocation of PI3K to the membrane (182, 183).  Other signaling molecules such as SLP- 76, ADAP, cytohesin-1, JAB-1, and PKC are also downstream targets of “outside-in” integrin signaling in T cells and myeloid cells.   These signaling pathways control a variety of cellular responses such as proliferation, cytokine secretion, and production of reactive oxygen species (153). Integrin activation by the BCR also plays an important role in immunological synapse formation when B cells bind Ags on the surface of APCs (147, 158, 184).  This integrin- mediated attachment prolongs the interaction between the B cell and the APC and allows the concentration of Ags to the center of the synapse, thereby lowering the threshold for B cell activation (158).  Signaling molecules such as Rap GTPase, Dock8 (a Rac and Rho GEF), and Rac2, which are important for integrin activation and actin cytoskeletal rearrangement, are involved in B cell immunological synapse formation (157, 158, 185).  Blocking the function of Rap or Dock8 results in an inability to cluster LFA-1 into the p-SMAC (185, 186), suggesting that BCR signaling controls the distribution of integrins on the plasma membrane.    27 1.4 B cells migration Cell migration is characterized by the establishment of a polarized cell morphology and the asymmetrical redistribution of signaling molecules.  The leading edge sends out pseudopods, where chemokine receptors are concentrated and actin is actively polymerized (187-191).  This is followed by contraction of the mid-body by actomyosin-dependent forces, allowing the rear of the cell to move forward.  At the trailing edge, an actin-supported appendage, the uropod, is usually formed.  The uropod contains the Golgi apparatus and is enriched in ERM family proteins (actin binding proteins) and adhesion molecules such as ICAMs, PSGL-1, CD43 and CD44 (192).  Although the function of the uropod is largely unknown, it may initiate the myosin-based contraction required for propelling the rigid nucleus through narrow gaps when the cell undergoes transmigration through constricted spaces such as those found in endothelial monolayers (193).  In addition, Lammermann et al. has shown that lymphocytes can migrate within lymph nodes in an integrin-independent fashion (194).  They have shown that lymphocytes devoid of β1, β2, β7, and αv integrins are still able to migrate effectively within lymph nodes.  Thus it appears that lymphocytes migrate within peripheral lymphoid tissue by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge (191, 194). Lymphocytes also exhibit amoeboid motility (191, 195), which mimics the amoeba Dictyostelium discoideum.  Amoeboid movement is special because it allows cells to rapidly sense and integrate signals from the extracellular environement.  This is important for lymphocytes when they migrate in the lymphoid organs and scan for Ags on APCs. Amoeboid movement lacks the traditional focal adhesions and stress fibres that are normally found in slower moving cells such as fibroblasts and endothelial cells.  Although lymphocytes lack focal adhesions, Shulman et al. have shown that T cells form “focal dots” of high affinity LFA-1 when crawling over endothelial cells (196).  These focal dots are often localized at the tips of adhesive filopodia, ensure resistance to detachment by shear forces, and allow migration of the lymphocyte across the endothelial cell layer (196).    28 1.4.1 Lymphocyte migration across the endothelium Lymphocytes emigrate from blood to lymphoid organs or tissue via a multi-step process that consists of tethering and rolling, chemoattractant-mediated activation, firm adhesion, and transendothelial migration (197, 198).  First, L-selectin expressed on lymphocytes allows them to tether and roll before making firm adhesion to the endothelium. Chemokines presented on the endothelium bind to the rolling cells and rapidly trigger integrin activation, allowing the rolling cells to mediate firm adhesion to the endothelium (198).  The shear forces caused by the blood flow also stabilize integrin-mediated attachment.  After arrest, lymphocytes can slowly crawl along the luminal surface of the endothelium to arrive at a site suitable for tranendothelial migration.  The route of transmigration can either be paracellular (through intercellular junctions) or transcellular (through the endothelial cell body).  Passage through the endothelium requires active participation of both the migrating cells and the endothelial cells.  E-selectin, VCAM-1, and ICAM-1 on the apical surface of the endothelial cell and the CD31, CD99, Jam-A, and ICAM-2 at the inter-endothelial junctions promote adhesive interactions required for transendothelial migration (160).  1.4.2 Chemoattractants important for B cell migration Chemoattractant-induced B cell migration is critical for B cell development, routine immune surveillance, and humoral immune responses.  During B cell development, CXCL12, which is secreted by the BM stromal cells, binds to its receptor CXCR4 to retain developing B cells in the BM and provide survival signals (199, 200).  As B cells mature, they become less responsive to CXCR4 but show increased responsiveness to secondary lymphoid organs-associated chemokines such as CCL19, CCL21, and CXCL13.  After leaving the BM, developing B cells arrive the spleen and accumulate at the T-B cell border (198).  As they receive appropriate survival and differentiation signals (201), they increase CXCR5 expression and migrate to the follicles in response to CXCL13 (198).    29 Importantly, the ability of the immune system to respond to the presence of foreign Ags depends on chemokine networks to recruit specific leukocytes to the right place and to activate these leukocytes at the right time (190, 202, 203).  Naïve lymphocytes are continuously motile within different lymphoid tissues in the body (109, 204, 205). Recirculating B cells enter secondary lymphoid organs (expect the spleen) through HEVs. For lymphocytes entering peripheral lymph nodes, signals from CCR7 and CXCR4 activate integrins and facilitate the migration of the cell across the endothelial layer and into the lymphoid tissue (206, 207).  B cells then migrate into the lymphoid follicles in response to CXCL13 and scan for their cognate Ags.  Once they bind Ags, B cells transiently increase their CCR7 level.  This allows them to move to the interface between the B cell follicles and the T cell area, where they can receive T cell help and differentiate into Ab-secreting plasma cells.  Some of the long-lived plasma cells will then leave the secondary lymphoid organ and home to the BM in response to CXCL12.  In contrast, B cells that do not encounter their Ags begin re-expressing S1P receptors and gradually lose responsiveness to CXCL13.  This frees them to move back to the follicle edge.  They can then enter the lymphatic vessels via the cortical sinusoid of the lymph nodes, re-enter the circulation by chemotaxing towards S1P in the blood (S1P levels are low within lymph nodes), and continue to traffic to other secondary lymphoid organs (208, 209).  Although S1PR3 is the primary receptor that mediates B cell chemotaxis towards S1P (210), S1PR1 is needed to facilitate passage of the cells through the lymphatic endothelium (211).  S1PR1-mediated integrin activation may be involved in this process.  1.4.3 Chemoattractant receptor signaling Chemoattractants stimulate cell migration via binding to their cognate G protein coupled receptors (GPCR) and causing conformational changes that trigger intracellular signaling pathways involved in cell movement.  GPCRs are transmembrane receptors with seven helical membrane spanning regions connected by extramembranous loops (212). These receptors propagate their signals by interacting with heterotrimeric (α, β, and γ) G proteins. The Gα subunit interacts directly with the Gβ subunit, which in turn forms a tight complex with the Gγ subunit.  Chemokine receptor triggers the exchange of GTP for GDP on Gα,   30 which then allows GTP-bound Gα and Gβγ to dissociate and independently activate downstream effectors.  There are 23 α, 5 β, and 10 γ subunits (203).  The different Gα subunits have been grouped into Gαi, Gαs, Gαq/11, and Gα12/13 subfamilies, and they are coupled to different signaling pathways.  Almost all hematopoietic cells use the Gαi protein to propagate signals from chemokine receptors.  Consistent with this, most chemokine- induced signaling events are suppressed by pertussis toxin treatment, which causes ADP ribosylation of Gαi subfamily members and inhibits the exchange of GTP for GDP (190, 213).  In contrast, Gα12/13 subfamily members are coupled to the receptors for lipid chemoattractants such as lysophosphatidic acid and S1P (203).  The activity of trimeric G proteins is negatively regulated by regulator of G protein signaling (RGS) proteins, which enhance the rate of GTP hydrolysis by Gα (203).  This may help cells decipher rapidly changing agonist profiles.  Chemokine receptor signaling is also regulated by G protein receptor kinases, arrestins, and PDZ domain containing proteins (e.g. postsynaptic density protein-95) that act as scaffolds for effector molecules (190, 214). Chemoattractants bind to their cognate receptors and initiate the activation of multiple signaling pathways leading to cell migration and integrin activation (Figure 1.6).  GTP-bound Gαi and Gαs can bind directly to the catalytic domain of Src and change the conformation of Src, leading to increased accessibility of the active site for substrates (215, 216). Alternatively, Gα12/13 subfamily members can interact directly with Btk, Pyk2, and Lsc (p115RhoGEF) (217-219).  The Gβγ subunits play an important role in cell migration as it leads to the activation of the PLCβ and PI3K pathways.  PLCβ catalyzes the hydrolysis of PIP2, thereby generating the second messengers IP3 and DAG.  These second messengers can activate the Rap GTPases (via DAG-regulated Rap-GEFs), which can then mediate rapid integrin activation, cytoskeletal remodeling, and cell polarization (151, 220-222).  The activation of PI3K results in the activation of Rac, which controls cell spreading and migration (223-225).  PI3K also activates Akt, which regulates cell survival (225).  In B cells, p110δ is the dominant isoform of the PI3K catalytic subunit and is crucial for B cell chemotaxis (30, 226).  The lack of a functional p110δ subunit (e.g. in p110δ knockout mice or mice in which the p110δ gene has been replaced with a catalytically inactive mutant form), as well as treatment with a pharmacological inhibitor of p110δ, impairs directional   31 migration along a chemokine gradient but not random cell motility in B cells (30, 226-229). How PI3K regulates B cell chemotaxis is not entirely clear, but it might play a role in directing the localized polymerization of actin via PIP3-dependent recruitment of PH domain- containing proteins such as Vav (an upstream activator of the Rac GTPase) that regulate actin assembly and dynamics. The activation of several small GTPases including Rac, Cdc42, RhoA, and Rap1 is important for the establishment of cell polarity and actin cytoskeletal remodeling during cell migration.  In general, Rac is important for the formation of lamellipodia, while Cdc42 is required for stabilization of the leading edge and directed motility.  RhoA is important for cell retraction and the release of the uropod from substrate whereas Rap1 induces integrin activation and cell polarization (190).  In lymphocytes, chemokine stimulation induces a very rapid increase in F-actin formation at the leading edge, which is mediated by Dock2, a Rac2GEF that activates Rac (37, 235).  Besides increasing integrin affinity, Rap-GTP contributes to cell polarization via the assembly of Par3/Par6/Cdc42-GTP complex and PKC- ζ activation (236, 237).  RhoA localizes both at the leading edge and in the uropod of migrating cells.  Activated RhoA can control the activities of myosin, phospholipase D (PLD), and phosphatidylinositol-4-phosphate 5-kinase 1γ (PIP5K1γ), which can then regulate cell body contraction, integrin activation, and polarization during migration (238, 239).   32   Figure 1.6 Chemokine receptor signaling events leading to cell polarization and migration. A chemoattractant binding to its GPCR can activate different signaling pathways that control cell migration and polarization.  Activation of Rac leads to an increase in intracellular F-actin.  The assembly of a Par3- Par6-Cdc42-GTP complex helps define the position of the leading edge.  Activation of the Mst1 kinase via Rap1 is important for integrin activation.  The actin assembly factor mDia1 is important during the chemotaxis of T cells but not B cells.  Finally, activation of RhoA is important for uropod retraction.  Two different isoforms of PI3K have been shown to be important for immune cells.  The p110γ isoform is important for chemokine-induced migration in neutrophils, macrophages, and T cells (230-232), while the p110δ isoform is important for B cell development, activation, and migration (30, 226, 233, 234).  Adapted from (190).    33 1.5 Cytoskeleton remodeling Lymphocytes that are circulating in the bloodstream, migrating through the tissue, and interacting with APCs undergo shape changes that involve massive cytoskeletal rearrangement.  This process requires dynamic actin polymerization and depolymerization (192, 240, 241).  The Rho family GTPases RhoA, Rac and Cdc42 are the major players involved in these processes (242).  Actin nucleation is catalyzed by various assembly factors such as Arp2/3 and formins (243, 244).  So far, Arp2/3 and the formins have been identified as central participants in lymphocyte morphology.  The Arp2/3 complex promotes actin polymerization that creates branched structures.  Wiskott Aldrich syndrome protein (WASP), which is activated by Cdc42-GTP, can link receptor signals to actin polymerization by triggering Arp2/3 activity (Figure 1.7).  The WASP family verprolin homologous protein (WAVE/Scar) acts downstream of Rac-GTP to activate the Arp2/3 complex (245).  There are three isoforms of WAVE proteins, and lymphocytes only express WAVE2, which has been shown to regulate lamellipodial F-actin formation in a PI3K- and ERK- dependent pathway in fibroblasts (246, 247).  In contrast to the Arp2/3 complex, formin proteins, which are activated by Rho GTPases, promote the nucleation and elongation of non-branched F-actin. However, formin might not be necessary for B cell migration as B cells lacking mDia, an isoform of formin, migrate normally (248, 249). Cdc42 and Rac can also activate another actin modulating protein beside the Arp2/3 complex (242).  This module involves the p21-activated kinase (PAK) family kinases, which phosphorylate and activate LIM domain kinase (LIMK), a kinase that in turn phosphorylates and inhibits the enzymatic activity of cofilin.  Cofilin promotes the severing of actin filaments, which is a prerequisite for reorganization of the actin cytoskeleton.  LIMK can also be activated by Rho-associated coiled coil containing protein kinase (ROCK), an effector that is downstream of RhoA.  ROCK also increases the phosphorylation of myosin light chain (MLC) by phosphorylating and inhibiting myosin light chain phosphatase (MLCP), and also by directly phosphorylating MLC.  Phosphorylation of MLC leads to its increased association with actin filaments and facilitates cell contraction.  Conversely, PAK counteracts ROCK function by inhibiting myosin light chain kinase (MLCK), thereby   34 reducing phosphorylation of MLC.  Thus, the Rho family GTPases are central regulators of actin cytoskeleton dynamics.    Figure 1.7 The effectors of Rho family GTPases that regulate cytoskeleton dynamics. Regulation of the cytoskeleton dynamics downstream of the Rho family GTPases (Cdc42, Rac, and RhoA) is mediated by several effector proteins.  Activation of Cdc42 and Rac can facilitate actin cytoskeleton reorganization by promoting actin polymerization and severing.  Also, activation of RhoA can facilitate cell contraction during cell movement.  Adapted and modified from (242).    35 1.6 Pyk2 and FAK Focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2/RAFTK) are two related non-receptor tyrosine kinases that are critical regulators of cell migration, proliferation, and survival (250).  Both Pyk2 and FAK can be activated following integrin- mediated adhesion, but Pyk2 is also activated by stimuli that increase intracellular calcium. FAK is expressed in almost all tissues, whereas Pyk2 is expressed mainly in the central nervous system, epithelial cells, and in cells derived from hematopoietic lineages (251).  In hematopoietic cells an alternative shorter RNA spliced isoform of Pyk2, named Pyk2-H, is the predominant form of Pyk2 (252).  Given the difference in the sequence at the C-termini of Pyk2 and Pyk2-H, it has been suggested that these two isoforms of Pyk2 can interact with different sets of signaling molecules (252).  Moreover, although Pyk2 and FAK share a similar structural sequence, they can interact with different proteins with their unique sequences, conferring these kinases with the ability to function in a variety of cellular processes (250).  Notably, increased expression and activation of Pyk2 and FAK has been found in a number of metastatic cancers (253-258).  Their correlation with invasive phenotype in cancer cells together with their contribution to cell adhesion, motility, and cell cycle regulation have positioned Pyk2 and FAK as potential targets for disease modulation, particularly in cancer cell metastasis (259, 260).  1.6.1 The structures of Pyk2 and FAK Pyk2 shares significant homology with FAK, with 60% amino acid identity in the central catalytic domain and 40% identity at C and N termini.  Both proteins are composed of an N-terminal protein 4.1, ezrin, radixin and moesin homology (FERM) domain, a central kinase domain, proline-rich regions (PRRs), and a C-terminal focal-adhesion targeting (FAT) domain (Figure 1.8).  The FERM domain plays an important role in Pyk2 and FAK activation as well as in propagating downstream signaling events.  The FERM domain of FAK can bind to receptors such as epidermal growth factor receptor, GPCRs, and integrins and recruit other signaling molecules to these receptors (261).  Beside cell surface receptors, actin- and membrane-associated adaptor proteins such as ezrin can bind to the FERM domain   36 of FAK and lead to FAK activation in an integrin-independent manner (262).   Moreover, an auto-regulatory role for the FERM domain has also been described (263-265).  In the inhibited state, the FERM domain of FAK binds directly to the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from phosphorylation (265).  The FERM domain in Pyk2 also seems to have an auto-inhibitory role but this is less characterized (266).  Recent findings show that Ca2+/calmodulin binding to the FERM domain of Pyk2 releases the Pyk2 kinase domain from auto-inhibition by promoting the formation of a Pyk2 dimer (267).  Furthermore, the FERM domain of Pyk2 and FAK are important for their nuclear localization, where they can promote cell proliferation and growth (268, 269).  This recent finding challenges the conventional idea that Pyk2 and FAK are solely cytoplasmic signaling molecules.  Figure 1.8 The structures of Pyk2 and FAK. Pyk2 and FAK are composed of an N-terminal FERM domain, a central kinase domain, PRR, and a C- terminal FAT domain.  Pyk2 tyrosines 402, 579, 580 and 881 correspond to FAK tyrosines 397, 576, 577 and 925, respectively.  FERM, protein 4.1, ezrin, radixin and moesin homology; PRR, proline-rich region; SH2, Src-homology-2; FAT, focal-adhesion targeting; Grb2, growth-factor-receptor-bound-2.  Adapted from (270).    37 Both Pyk2 and FAK do not contain SH2 or SH3 domains but contain several sites for binding SH2/SH3-containing signaling proteins (250).  The C-terminal domains of Pyk2 and FAK contain multiple PRRs that function as binding sites for SH3-containing proteins.  For instance, these PRRs mediate the binding of the adaptor protein p130Cas to Pyk2 and FAK, which is important in promoting cell migration through the coordinated activation of Rac at membrane extensions (271, 272).  The PRR domain of FAK can also interact with other proteins such as the GTPase regulator associated with FAK (GRAF) and the Arf-GTPase activating protein (ASAP1), both of which are involved in the regulation of cytoskeletal dynamics and focal contact assembly.  The C-terminal domain of Pyk2 and FAK also includes the FAT region.  However, only the FAT domain of FAK contains the sequence for focal adhesion localization.  The FAT domain of FAK promotes the co-localization of FAK with integrins at focal contacts via indirect association with integrin-associated proteins such as paxillin and talin (273).  Despite the similar structure of Pyk2 and FAK, the differences between the two kinases allow them to couple unique signaling pathways to different cell surface receptors.  1.6.2 The regulation of Pyk2 and FAK by tyrosine phosphorylation Pyk2 and FAK have multiple tyrosine residues (Y402, Y579, Y580, and Y881 for Pyk2 and Y397, Y407, Y576, Y577, Y861, and Y925 for FAK) that are important for their kinase activities and for binding to other signaling molecules.  The mechanism of Pyk2 activation and its interaction with SFKs have been extensively characterized.  In response to receptor signaling, Pyk2 autophosphorylation occurs at Y402. This is thought to occur via trans-phosphorylation in a SFK-independent manner.  SFKs can then bind via their SH2 domain to the phosphorylated Y402, resulting in SFK activation (274).  The activated SFK then phosphorylates Pyk2 at Y579 and Y580, which enhances Pyk2 activity towards substrates such as paxillin (250).  FAK undergoes similar phosphorylation events at analogous sites.  However, it has been shown that Y397 on FAK can also be phosphorylated in a SFK-dependent manner following an initial autophosphorylation event (i.e. SFKs amplify the phosphorylation at this site) (275, 276).  Phosphorylation of FAK at Y397 and Pyk2 at Y402 allows the binding of various SH2 domain-containing proteins such as SFKs,   38 PLCγ, SOCS, Grb7, the Shc adaptor protein, p120RasGAP and PI3K (270), although these interactions appear to be cell type-dependent.  In addition, the enzymatic activities of Pyk2 and FAK are modulated by protein tyrosine phosphatases (PTPs) (277-279).  For example, PTPα can function as an upstream regulator of Pyk2 and FAK phosphorylation by removing the inhibitory tyrosine residue on Src, thereby allowing maximal Src catalytic activity (278- 280).  1.6.3 The role of FAK in cell migration FAK has been studied mostly in adherent cells, where it is crucial for efficient cell migration.  FAK has been shown to promote integrin activation, leading edge formation, focal adhesion turnover, and trailing edge retraction (270, 281, 282).  During cell migration, FAK can facilitate actin polymerization and modulate Rho/Rac activities, resulting in the formation and stabilization of leading edge.  Upon receptor activation, FAK recruits SFK at focal adhesion sites to form a FAK-SFK signaling complex.  This signaling complex can then phosphorylate other focal adhesion signaling proteins including paxillin, p130Cas, and PAK-interacting exchange factor (PIX), which activates Rac GTPases (283-285). Concurrently, FAK can also inhibit Rho activity at the leading edge by activating p190RhoGAP (286), thereby facilitating lamellipodia formation and cell polarization.  Also, FAK can bind directly to and activate the Apr2/3 complex, thereby stimulating leading edge protrusion (287).  Moreover, the binding of FAK to talin can recruit talin to focal adhesions and thereby promote integrin activation (288).  At the trailing edge, FAK promotes RhoA activation via its association with RhoGEFs, resulting in increased contractility and focal adhesion disassembly (289, 290).  Thus, FAK regulates multiple pathways required for efficient cell movement.  1.6.4 Functions of Pyk2 and FAK in haemaopoetic lineage cells Mice deficient in either FAK or Pyk2 have been generated, but they have different phenotypes, suggesting that these two kinases have different and non-compensatory functions.  FAK-/- mice exhibit embryonic lethality (291).  Fibroblasts isolated from these   39 mice show increased immature focal contact formation, impaired focal contact turnover, and impaired integrin-dependent migration.  In contrast, Pyk2-/- mice are viable and fertile. Although they have no obvious impairment in development or behavior (32), they do have defects in cells of the immune system.  Macrophages and B cells isolated from the Pyk2-/- mice are unable to migrate in response to chemokines and these mice lack MZ B cells.  Thus, Pyk2 and FAK may participate in distinct signaling mechanisms and functions in hematopoietic lineage cells.  1.6.5 Functions of Pyk2 and FAK in macrophages Both Pyk2-/- and FAK-/- macrophages display morphological alterations and have reduced migratory ability (292, 293).  Okigaki et al. reported that while wild type macrophages that polarize form lamellopodia at one cell pole and move the cell body in the direction established by the leading edge, Pyk2-/- macrophages extend multiple lamellopodia in different directions and show a reduced ability to follow the leading edge (292). Additionally, in response to localized CXCL12 stimulation, F-actin is concentrated at the edge of the cell facing the chemokine gradient in wild type macrophages whereas in Pyk2-/- macrophages, F-actin accumulates at multiple sites along the cell periphery.  FAK-/- macrophages also exhibit a generalized defect in chemotaxis, random motility, and invasion through matrigel (3D cell-matrix tissue culture system) (293).  FAK-/- macrophages make numerous short-lived protrusions all around the cell periphery and exhibit reduced kinetics of adhesion formation and disassembly.  Thus, Pyk2 and FAK both appear to be involved in morphological regulation and directional movement in macrophages.  1.6.6 Activation and function of Pyk2 and FAK in T cells Pyk2 and FAK have been implicated in morphological regulation, polarization, survival, integrin functions, and migration in T cells (294-300).  Both Pyk2 and FAK are downstream targets of signaling by the T cell receptor (TCR), integrins, and chemokine receptors in T cells (294, 297, 299, 300).  CXCL12 stimulation results in Pyk2 phosphorylation and T cell migration.  It has been suggested that the adaptor protein Lck-   40 interacting adaptor protein (Lad) can co-localize Pyk2 with Lck and the Gβ subunit of heterotrimeric G proteins (301).  This leads to the association of Pyk2, the Zap-70 tyrosine kinase and the Vav RacGEF, thereby facilitating actin cytoskeleton remodeling (299, 301, 302). Pyk2 is also involved in immunological synapse formation in T cells.  Pyk2 is rapidly translocated to the T cell-APC contact area upon T cell recognition of Ag presented on APCs (303).  Pyk2 phosphorylation and translocation to the synapse requires Lck and Itk (a Tec family kinase) kinase activities (303, 304).  Pyk2 phosphorylation and activation during immune synapse formation requires integrin activation, the microtubular cytoskeleton, and Ca2+ signaling (267, 305).  Other proteins involved in Pyk2 activation in lymphocytes include Lck, Zap-70, PKC, PI3K, Jak2, and the Rap GTPases (163, 299, 302, 306, 307).  An intact cytoskeleton, or cytoskeletal dynamics, is thought to be required for Pyk2 activation in T cells as treating cells with drugs that disrupt the actin cytoskeleton abolishes anti-CD3- induced Pyk2 phosphorylation (308).  However, a contradictory result presented by Collins et al. showed that Ca2+ influx, Zap-70 activation, actin cytoskeleton rearrangement, and PI3K function were not required for TCR-induced Pyk2 tyrosine phosphorylation (309).  These authors suggested that the use of more stringent lysis buffers in their experiments might account for the different observation compared to other studies.  1.6.7 Activation and function of Pyk2 and FAK in B cells In B cells, signaling by integrins, the BCR, and chemoattractant receptors result in Pyk2 and FAK tyrosine phosphorylation (163, 310-313).  We have shown that Rap activation is crucial for BCR-, integrin-, and chemoattractant-induced Pyk2 phosphorylation in B cells but the functions of Pyk2 and FAK in B cells are not well known (163, 314).  As mentioned above, MZ B cells are lacking in Pyk2-/- mice, suggesting that Pyk2 plays an essential role in the development and localization of this specific subset of B cells (32).  Recently, immunochistochememical analysis of normal lymphoid tissue showed that FAK is highly expressed in GC and MZ B cells (315).  However, it is not known how Pyk2 contributes to MZ B cell development and whether FAK plays a role in MZ B cell functions.   41 Recent studies have suggested that sustained adhesion of progenitor B cells is associated with chemokine-induced activation of FAK (316).  CXCL12 induces prolonged phosphorylation of FAK in BM progenitor B cells while FAK is only transiently phosphorylated in mature B cells.  This prolonged adhesion mediated by FAK may play an important role in the retention of developing B cells within the BM microenvironments, where they receive survival and differentiation signals (1).  The transient phosphorylation of FAK in mature B cells is regulated by the suppressor of cytokine signaling 3 (SOCS3), which targets FAK to the ubiquitin-proteasome pathway upon CXCL12 stimulation (317). Accordingly, SOCS3 expression is low in progenitor B cells and gradually increases as B cells mature (317).  Notably, SOCS3 expression can also inhibit CXCL12-induced Pyk2 phosphorylation and migration in mature T cells (318).  This suggests that chemokine- induced Pyk2 and FAK phosphorylation is differentially regulated in mature and immature lymphocytes.    42 1.7 The Rap GTPases There are five members of the Rap GTPase family, Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C, each encoded by a separate gene.  The Rap GTPases, referred to collectively as Rap, have been implicated in cell adhesion, junction formation, polarity, and secretion (Figure 1.9) (319, 320).  Like other GTPases, Rap is activated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP to GTP.  This induces a conformational change that allows downstream effector proteins to bind to Rap-GTP and trigger signaling that can stimulate a variety of cellular events (321).  Conversely, Rap GTPase activating proteins (GAPs) terminate the signal output through Rap by enhancing its intrinsic rate of GTP hydrolysis (222, 321-324).  Several Rap GEFs have been identified, including C3G, Epac1, Epac2, RasGRP2, RasGRP3 (which also can activate Ras), PDZ- GEF1, PDZ-GEF2, and PLCε (322, 324).  Also, a number of Rap GAPs have been identified, including RapGAPI, RapGAPII, the Spa-1 family of GAPs (Spa-1, Spa-1-like, and E6TP1), and SPAR2 (322, 325).  Given the large number of Rap-specific GEFs and GAPs, it is not clear which GEFs and GAPs are important for regulating Rap function in lymphocytes. However, in B cells Rap1 is activated via the PLC-dependent production of DAG (326), suggesting that one of the RasGRP family members is the primary Rap GEF in B cells. The list of Rap effectors is rapidly expanding and contains proteins both with and without catalytic activity.  Most Rap effectors are involved in cell adhesion and modulation of the actin cytoskeleton.  This includes the adaptor proteins RapL, RIAM, AF-6, and Krev interaction trapped 1 (Krit1); the RacGEFs Tiam1 and Vav2; and the RhoGAPs RA-RhoGAP and Arap3 (322, 324).  Other Rap1-binding proteins have been identified such as PKD1and the scaffold protein IQGAP1 (166, 327), which are important for determining the subcelluar localization of the activated form of Rap1.   43   Figure 1.9 Rap effectors and Rap signaling pathways. Rap can activate integrin via targeting RapL and talin to the integrin tail.  Rap can also facilitate actin cytoskeleton remodeling by binding to and activating GEFs for Rac and Cdc42.  Finally, Rap can lead to cell polarization by controlling the localization of Cdc42-GTP and the Par3/Par6 polarity complex.  GEF, guanine exchange factor.  Adapted and modified from (321).    44 Rap can be activated by a variety of receptors, including receptors for Ags, growth factors, cytokines, chemokines, and cell-adhesion molecules (320, 321, 328).  Recent studies using genetic approaches indicate that Rap1 plays an important role in B cell development, adhesion, migration, and BCR repertoire selection (163, 314, 329).  Rap1b is the dominant isoform of Rap1 expressed in B cells (31).  This is supported by the finding that B cells develop normally in Rap1a-deficient mice while B cells deficient of Rap1b have numerous developmental and functional defects (31, 330, 331).  Rap1b has a critical role in mature B cell trafficking to lymph nodes and in MZ B cell development (31, 331).  MZ B cell number is reduced in Rap1b-/- mice, and the remaining MZ B cells exhibit defects in S1P-induced migration and adhesion to ICAM-1 (331).  This is also supported by our previous findings that overexpression of Rap1GAP or the dominant negative form of Rap1 (Rap1N17) in B cells inhibit their migration, adhesion, actin polymerization, synapse formation, spreading, and Pyk2 phosphorylation (163, 186, 314, 332).  Moreover, conditional overexpression of a dominant negative from of Rap1 (Rap1A17) in B-lineage cells results in early B cell developmental defect (333).  These mutated B cells are unable to proliferate in response to IL-7 and this results in progressive cell death (333).  Furthermore, mice deficient in SPA-1 (a Rap1GAP protein) show an Ag-dependent increase in B-1a cells producing anti-dsDNA Ab as well as lupus-like nephritis (329).  Overall, these findings highlight the essential roles of Rap in B cell development, trafficking, and in particular the development and proliferation of innate-like B cell subsets (MZ and B-1a cells) that contribute to both anti-microbial immunity and autoimmunity. Rap1 has been implicated in receptor-triggered signaling pathways that activate integrins, induce lymphocyte polarization, and mediate lymphocyte motility (334).  The major role of Rap in integrin receptor activation is to form an “integrin activation complex” that consists of Rap1 and several other effectors and adaptors such as RIAM, PKD, and RapL (also see section 1.3).  Rap effector, regulator of adhesion and cell polarization enriched in lymphoid tissues (RapL), has been identified as the key effector of Rap for integrin activation (335).  In RapL-deficient mice, both B and T lymphocytes exhibited poor adhesion and homed poorly to lymphoid tissues.  Also, there is a reduction in MZ B cell numbers in RapL- deficient mice (38), suggesting an important function of integrin activity in MZ B cell development.  Besides increasing integrin affinity, Rap-GTP contributes to T cell   45 polarization via the assembly of Par3/Par6/Cdc42-GTP complex and PKC-ζ activation (236, 237).  Moreover, a number of Rap effectors can regulate the balance of Rac and Rho GTPase activities, thereby allowing Rap to control the dynamics of actin cytoskeleton.    46 1.8 Objective and aims The overall goal of this thesis was to gain new insights into the regulation and function of Pyk2 and FAK in B cells.  As Pyk2 and FAK are implicated in integrin functions and the regulation of cell migration and cell morphology in many cell types, I hypothesized that these two kinases were involved in B cell morphological changes.  Specifically, I explored the role of Pyk2 and FAK in B cell spreading on integrin ligands.  Based on the previous findings from our lab that adhesion to ECM enhances BCR-induced Pyk2 phosphorylation (163), I tested whether FAK is also regulated in the same manner in B cells. Also, I examined the role of the Rap GTPases in Pyk2 and FAK tyrosine phosphorylation at specific regulatory sites (Y402, Y579, and Y580 for Pyk2; Y397, Y576, and Y577 for FAK). I examined the function of Rap in Pyk2 and FAK phosphorylation directly downstream from the BCR and integrins.  Finally, I investigated how Rap may regulate Pyk2 and FAK via actin cytoskeleton dynamics. Since Pyk2 is required for MZ B cell development, I hypothesized that Pyk2 promotes the retention of mature MZ B cells in the spleen by regulating the chemotatic and adhesive signals.  Recently, it has been shown that FAK is highly expressed in MZ B cells (315).  Therefore, my second objective was to study the role of Pyk2 and FAK in MZ B cells. Specifically, I focused on cellular processes mediated by integrins and by the chemoattractants CXCL13 and S1P, which are important for MZ B cell retention in the spleen (34, 35).  Initially, I characterized the activation of Pyk2 and FAK in response to CXCL13 and S1P.  I then examined the role of Pyk2 and FAK in MZ and follicular B-2 cell migration and adhesion.  Finally, since chemoattractants also stimulate PI3K/Akt activation, which promotes directional movement and survival, and Pyk2 and FAK can potentially interact with the regulatory subunit of PI3K, I investigated whether Pyk2 and FAK are involved in Akt phosphorylation in B cells. Overall, the work in this thesis showed that Pyk2 and FAK are important regulators of B cell morphology and migration.  In particular, I showed that Pyk2 is an important signaling molecule that acts downstream of the integrins and chemoattractant receptors and that it regulates MZ B cell migration and adhesion.  Together, these findings highlight the regulation of Pyk2 and FAK as well as their functions in B cell trafficking and morphological   47 regulation.  My results suggest that regulating Pyk2 and FAK activities may be a good approach for controlling B cell-mediated diseases as well as the dissemination of malignant B cells.   48 1.9 References  1. LeBien, T. W., and T. F. Tedder. 2008. B lymphocytes: how they develop and function. Blood 112:1570-1580. 2. Nutt, S. L., and B. L. Kee. 2007. The transcriptional regulation of B cell lineage commitment. Immunity 26:715-725. 3. Adolfsson, J., O. J. Borge, D. Bryder, K. Theilgaard-Monch, I. Astrand-Grundstrom, E. Sitnicka, Y. Sasaki, and S. E. Jacobsen. 2001. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15:659-669. 4. Adolfsson, J., R. Mansson, N. Buza-Vidas, A. Hultquist, K. Liuba, C. T. Jensen, D. Bryder, L. Yang, O. J. Borge, L. A. Thoren, K. Anderson, E. Sitnicka, Y. Sasaki, M. Sigvardsson, and S. E. Jacobsen. 2005. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121:295-306. 5. Nagasawa, T. 2006. Microenvironmental niches in the bone marrow required for B- cell development. Nat Rev Immunol 6:107-116. 6. Rumfelt, L. L., Y. Zhou, B. M. Rowley, S. A. Shinton, and R. R. Hardy. 2006. Lineage specification and plasticity in CD19- early B cell precursors. J Exp Med 203:675-687. 7. Igarashi, H., S. C. Gregory, T. Yokota, N. Sakaguchi, and P. W. Kincade. 2002. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 17:117-130. 8. Singh, H., K. L. Medina, and J. M. Pongubala. 2005. Contingent gene regulatory networks and B cell fate specification. Proc Natl Acad Sci U S A 102:4949-4953. 9. Li, Y. S., R. Wasserman, K. Hayakawa, and R. R. Hardy. 1996. Identification of the earliest B lineage stage in mouse bone marrow. Immunity 5:527-535. 10. Tokoyoda, K., T. Egawa, T. Sugiyama, B. I. Choi, and T. Nagasawa. 2004. Cellular niches controlling B lymphocyte behavior within bone marrow during development. Immunity 20:707-718. 11. Milne, C. D., and C. J. Paige. 2006. IL-7: a key regulator of B lymphopoiesis. Semin Immunol 18:20-30. 12. Sakaguchi, N., and F. Melchers. 1986. Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. Nature 324:579-582. 13. Melchers, F. 2005. The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire. Nat Rev Immunol 5:578-584. 14. Bankovich, A. J., S. Raunser, Z. S. Juo, T. Walz, M. M. Davis, and K. C. Garcia. 2007. Structural insight into pre-B cell receptor function. Science 316:291-294. 15. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, and K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J Exp Med 173:1213-1225. 16. Chung, J. B., M. Silverman, and J. G. Monroe. 2003. Transitional B cells: step by step towards immune competence. Trends Immunol 24:343-349. 17. Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton, and R. R. Hardy. 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals   49 multiple selection points during peripheral B cell maturation. J Immunol 167:6834- 6840. 18. Allman, D., and S. Pillai. 2008. Peripheral B cell subsets. Curr Opin Immunol 20:149-157. 19. Radbruch, A., G. Muehlinghaus, E. O. Luger, A. Inamine, K. G. Smith, T. Dorner, and F. Hiepe. 2006. Competence and competition: the challenge of becoming a long- lived plasma cell. Nat Rev Immunol 6:741-750. 20. McHeyzer-Williams, L. J., and M. G. McHeyzer-Williams. 2005. Antigen-specific memory B cell development. Annu Rev Immunol 23:487-513. 21. Pillai, S., A. Cariappa, and S. T. Moran. 2005. Marginal zone B cells. Annu Rev Immunol 23:161-196. 22. Martin, F., and J. F. Kearney. 2002. Marginal-zone B cells. Nat Rev Immunol 2:323- 335. 23. Pillai, S., and A. Cariappa. 2009. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 9:767-777. 24. Martin, F., A. M. Oliver, and J. F. Kearney. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14:617-629. 25. Balazs, M., F. Martin, T. Zhou, and J. Kearney. 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17:341-352. 26. Martin, F., and J. F. Kearney. 2000. B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a "natural immune memory". Immunol Rev 175:70-79. 27. Fleming, S. D. 2006. Natural antibodies, autoantibodies and complement activation in tissue injury. Autoimmunity 39:379-386. 28. Ochsenbein, A. F., T. Fehr, C. Lutz, M. Suter, F. Brombacher, H. Hengartner, and R. M. Zinkernagel. 1999. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286:2156-2159. 29. Binder, C. J., M. Y. Chou, L. Fogelstrand, K. Hartvigsen, P. X. Shaw, A. Boullier, and J. L. Witztum. 2008. Natural antibodies in murine atherosclerosis. Curr Drug Targets 9:190-195. 30. Clayton, E., G. Bardi, S. E. Bell, D. Chantry, C. P. Downes, A. Gray, L. A. Humphries, D. Rawlings, H. Reynolds, E. Vigorito, and M. Turner. 2002. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation. J Exp Med 196:753-763. 31. Chen, Y., M. Yu, A. Podd, R. Wen, M. Chrzanowska-Wodnicka, G. C. White, and D. Wang. 2008. A critical role of Rap1b in B-cell trafficking and marginal zone B-cell development. Blood 111:4627-4636. 32. Guinamard, R., M. Okigaki, J. Schlessinger, and J. V. Ravetch. 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 1:31-36. 33. Cinamon, G., M. A. Zachariah, O. M. Lam, F. W. Foss, Jr., and J. G. Cyster. 2008. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat Immunol 9:54-62.   50 34. Lu, T. T., and J. G. Cyster. 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297:409-412. 35. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, and J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat Immunol 5:713-720. 36. Girkontaite, I., K. Missy, V. Sakk, A. Harenberg, K. Tedford, T. Potzel, K. Pfeffer, and K. D. Fischer. 2001. Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses. Nat Immunol 2:855-862. 37. Fukui, Y., O. Hashimoto, T. Sanui, T. Oono, H. Koga, M. Abe, A. Inayoshi, M. Noda, M. Oike, T. Shirai, and T. Sasazuki. 2001. Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412:826-831. 38. Katagiri, K., N. Ohnishi, K. Kabashima, T. Iyoda, N. Takeda, Y. Shinkai, K. Inaba, and T. Kinashi. 2004. Crucial functions of the Rap1 effector molecule RAPL in lymphocyte and dendritic cell trafficking. Nat Immunol 5:1045-1051. 39. Berland, R., and H. H. Wortis. 2002. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol 20:253-300. 40. Montecino-Rodriguez, E., and K. Dorshkind. 2006. New perspectives in B-1 B cell development and function. Trends Immunol 27:428-433. 41. Haas, K. M., J. C. Poe, D. A. Steeber, and T. F. Tedder. 2005. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. Immunity 23:7-18. 42. Alugupalli, K. R. 2008. A distinct role for B1b lymphocytes in T cell-independent immunity. Curr Top Microbiol Immunol 319:105-130. 43. Dorshkind, K., and E. Montecino-Rodriguez. 2007. Fetal B-cell lymphopoiesis and the emergence of B-1-cell potential. Nat Rev Immunol 7:213-219. 44. Montecino-Rodriguez, E., H. Leathers, and K. Dorshkind. 2006. Identification of a B- 1 B cell-specified progenitor. Nat Immunol 7:293-301. 45. Hippen, K. L., L. E. Tze, and T. W. Behrens. 2000. CD5 maintains tolerance in anergic B cells. J Exp Med 191:883-890. 46. Sen, G., G. Bikah, C. Venkataraman, and S. Bondada. 1999. Negative regulation of antigen receptor-mediated signaling by constitutive association of CD5 with the SHP- 1 protein tyrosine phosphatase in B-1 B cells. Eur J Immunol 29:3319-3328. 47. Bondada, S., G. Bikah, D. A. Robertson, and G. Sen. 2000. Role of CD5 in growth regulation of B-1 cells. Curr Top Microbiol Immunol 252:141-149. 48. Dasu, T., V. Sindhava, S. H. Clarke, and S. Bondada. 2009. CD19 signaling is impaired in murine peritoneal and splenic B-1 B lymphocytes. Mol Immunol 46:2655-2665. 49. Burastero, S. E., P. Casali, R. L. Wilder, and A. L. Notkins. 1988. Monoreactive high affinity and polyreactive low affinity rheumatoid factors are produced by CD5+ B cells from patients with rheumatoid arthritis. J Exp Med 168:1979-1992. 50. Dauphinee, M., Z. Tovar, and N. Talal. 1988. B cells expressing CD5 are increased in Sjogren's syndrome. Arthritis Rheum 31:642-647. 51. Hayakawa, K., R. R. Hardy, D. R. Parks, and L. A. Herzenberg. 1983. The "Ly-1 B" cell subpopulation in normal immunodefective, and autoimmune mice. J Exp Med 157:202-218.   51 52. Mohan, C., L. Morel, P. Yang, and E. K. Wakeland. 1998. Accumulation of splenic B1a cells with potent antigen-presenting capability in NZM2410 lupus-prone mice. Arthritis Rheum 41:1652-1662. 53. Sidman, C. L., L. D. Shultz, R. R. Hardy, K. Hayakawa, and L. A. Herzenberg. 1986. Production of immunoglobulin isotypes by Ly-1+ B cells in viable motheaten and normal mice. Science 232:1423-1425. 54. Evans, J. G., K. A. Chavez-Rueda, A. Eddaoudi, A. Meyer-Bahlburg, D. J. Rawlings, M. R. Ehrenstein, and C. Mauri. 2007. Novel suppressive function of transitional 2 B cells in experimental arthritis. J Immunol 178:7868-7878. 55. Bouaziz, J. D., K. Yanaba, and T. F. Tedder. 2008. Regulatory B cells as inhibitors of immune responses and inflammation. Immunol Rev 224:201-214. 56. Spencer, N. F., and R. A. Daynes. 1997. IL-12 directly stimulates expression of IL-10 by CD5+ B cells and IL-6 by both CD5+ and CD5- B cells: possible involvement in age-associated cytokine dysregulation. Int Immunol 9:745-754. 57. Mauri, C., and M. R. Ehrenstein. 2008. The 'short' history of regulatory B cells. Trends Immunol 29:34-40. 58. Tian, J., D. Zekzer, L. Hanssen, Y. Lu, A. Olcott, and D. L. Kaufman. 2001. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J Immunol 167:1081-1089. 59. Harris, D. P., L. Haynes, P. C. Sayles, D. K. Duso, S. M. Eaton, N. M. Lepak, L. L. Johnson, S. L. Swain, and F. E. Lund. 2000. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol 1:475-482. 60. Yanaba, K., J. D. Bouaziz, K. M. Haas, J. C. Poe, M. Fujimoto, and T. F. Tedder. 2008. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity 28:639-650. 61. Matsushita, T., K. Yanaba, J. D. Bouaziz, M. Fujimoto, and T. F. Tedder. 2008. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest 118:3420-3430. 62. Yanaba, K., J. D. Bouaziz, T. Matsushita, T. Tsubata, and T. F. Tedder. 2009. The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals. J Immunol 182:7459-7472. 63. Wolf, S. D., B. N. Dittel, F. Hardardottir, and C. A. Janeway, Jr. 1996. Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice. J Exp Med 184:2271-2278. 64. Mizoguchi, A., E. Mizoguchi, R. N. Smith, F. I. Preffer, and A. K. Bhan. 1997. Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice. J Exp Med 186:1749-1756. 65. Fillatreau, S., C. H. Sweenie, M. J. McGeachy, D. Gray, and S. M. Anderton. 2002. B cells regulate autoimmunity by provision of IL-10. Nat Immunol 3:944-950. 66. Mizoguchi, A., E. Mizoguchi, H. Takedatsu, R. S. Blumberg, and A. K. Bhan. 2002. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 16:219-230. 67. Mauri, C., D. Gray, N. Mushtaq, and M. Londei. 2003. Prevention of arthritis by interleukin 10-producing B cells. J Exp Med 197:489-501.   52 68. Hussain, S., and T. L. Delovitch. 2007. Intravenous transfusion of BCR-activated B cells protects NOD mice from type 1 diabetes in an IL-10-dependent manner. J Immunol 179:7225-7232. 69. Manjarrez-Orduno, N., T. D. Quach, and I. Sanz. 2009. B cells and immunological tolerance. J Invest Dermatol 129:278-288. 70. Stadanlick, J. E., and M. P. Cancro. 2008. BAFF and the plasticity of peripheral B cell tolerance. Curr Opin Immunol 20:158-161. 71. Martin, F., and A. C. Chan. 2006. B cell immunobiology in disease: evolving concepts from the clinic. Annu Rev Immunol 24:467-496. 72. Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow, and et al. 1993. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein- tyrosine kinases. Nature 361:226-233. 73. Tsukada, S., D. C. Saffran, D. J. Rawlings, O. Parolini, R. C. Allen, I. Klisak, R. S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, and et al. 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72:279-290. 74. Bacchelli, C., S. Buckridge, A. J. Thrasher, and H. B. Gaspar. 2007. Translational mini-review series on immunodeficiency: molecular defects in common variable immunodeficiency. Clin Exp Immunol 149:401-409. 75. Shlomchik, M. J. 2008. Sites and stages of autoreactive B cell activation and regulation. Immunity 28:18-28. 76. Shlomchik, M. J. 2009. Activating systemic autoimmunity: B's, T's, and tolls. Curr Opin Immunol 21:626-633. 77. Pers, J. O., C. Daridon, V. Devauchelle, S. Jousse, A. Saraux, C. Jamin, and P. Youinou. 2005. BAFF overexpression is associated with autoantibody production in autoimmune diseases. Ann N Y Acad Sci 1050:34-39. 78. Yanaba, K., J. D. Bouaziz, T. Matsushita, C. M. Magro, E. W. St Clair, and T. F. Tedder. 2008. B-lymphocyte contributions to human autoimmune disease. Immunol Rev 223:284-299. 79. Moore, P. A., O. Belvedere, A. Orr, K. Pieri, D. W. LaFleur, P. Feng, D. Soppet, M. Charters, R. Gentz, D. Parmelee, Y. Li, O. Galperina, J. Giri, V. Roschke, B. Nardelli, J. Carrell, S. Sosnovtseva, W. Greenfield, S. M. Ruben, H. S. Olsen, J. Fikes, and D. M. Hilbert. 1999. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260-263. 80. Tangye, S. G., V. L. Bryant, A. K. Cuss, and K. L. Good. 2006. BAFF, APRIL and human B cell disorders. Semin Immunol 18:305-317. 81. Dorner, T., A. Radbruch, and G. R. Burmester. 2009. B-cell-directed therapies for autoimmune disease. Nat Rev Rheumatol 5:433-441. 82. Levesque, M. C. 2009. Translational Mini-Review Series on B Cell-Directed Therapies: Recent advances in B cell-directed biological therapies for autoimmune disorders. Clin Exp Immunol 157:198-208. 83. Nanki, T., K. Takada, Y. Komano, T. Morio, H. Kanegane, A. Nakajima, P. E. Lipsky, and N. Miyasaka. 2009. Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis. Arthritis Res Ther 11:R149.   53 84. Hutloff, A., A. M. Dittrich, K. C. Beier, B. Eljaschewitsch, R. Kraft, I. Anagnostopoulos, and R. A. Kroczek. 1999. ICOS is an inducible T-cell co- stimulator structurally and functionally related to CD28. Nature 397:263-266. 85. Plater-Zyberk, C., R. N. Maini, K. Lam, T. D. Kennedy, and G. Janossy. 1985. A rheumatoid arthritis B cell subset expresses a phenotype similar to that in chronic lymphocytic leukemia. Arthritis Rheum 28:971-976. 86. Ansel, K. M., R. B. Harris, and J. G. Cyster. 2002. CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. Immunity 16:67-76. 87. Ishikawa, S., and K. Matsushima. 2007. Aberrant B1 cell trafficking in a murine model for lupus. Front Biosci 12:1790-1803. 88. Sato, T., S. Ishikawa, K. Akadegawa, T. Ito, H. Yurino, M. Kitabatake, H. Yoneyama, and K. Matsushima. 2004. Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus. Eur J Immunol 34:3346-3358. 89. Ishikawa, S., T. Sato, M. Abe, S. Nagai, N. Onai, H. Yoneyama, Y. Zhang, T. Suzuki, S. Hashimoto, T. Shirai, M. Lipp, and K. Matsushima. 2001. Aberrant high expression of B lymphocyte chemokine (BLC/CXCL13) by C11b+CD11c+ dendritic cells in murine lupus and preferential chemotaxis of B1 cells towards BLC. J Exp Med 193:1393-1402. 90. Zheng, B., Z. Ozen, X. Zhang, S. De Silva, E. Marinova, L. Guo, D. Wansley, D. P. Huston, M. R. West, and S. Han. 2005. CXCL13 neutralization reduces the severity of collagen-induced arthritis. Arthritis Rheum 52:620-626. 91. Wengner, A. M., U. E. Hopken, P. K. Petrow, S. Hartmann, U. Schurigt, R. Brauer, and M. Lipp. 2007. CXCR5- and CCR7-dependent lymphoid neogenesis in a murine model of chronic antigen-induced arthritis. Arthritis Rheum 56:3271-3283. 92. Yang, M., B. Charlton, and A. M. Gautam. 1997. Development of insulitis and diabetes in B cell-deficient NOD mice. J Autoimmun 10:257-260. 93. Wong, F. S., L. Wen, M. Tang, M. Ramanathan, I. Visintin, J. Daugherty, L. G. Hannum, C. A. Janeway, Jr., and M. J. Shlomchik. 2004. Investigation of the role of B-cells in type 1 diabetes in the NOD mouse. Diabetes 53:2581-2587. 94. Fiorina, P., A. Vergani, S. Dada, M. Jurewicz, M. Wong, K. Law, E. Wu, Z. Tian, R. Abdi, I. Guleria, S. Rodig, K. Dunussi-Joannopoulos, J. Bluestone, and M. H. Sayegh. 2008. Targeting CD22 reprograms B-cells and reverses autoimmune diabetes. Diabetes 57:3013-3024. 95. Hu, C. Y., D. Rodriguez-Pinto, W. Du, A. Ahuja, O. Henegariu, F. S. Wong, M. J. Shlomchik, and L. Wen. 2007. Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice. J Clin Invest 117:3857-3867. 96. Marino, E., M. Batten, J. Groom, S. Walters, D. Liuwantara, F. Mackay, and S. T. Grey. 2008. Marginal-zone B-cells of nonobese diabetic mice expand with diabetes onset, invade the pancreatic lymph nodes, and present autoantigen to diabetogenic T- cells. Diabetes 57:395-404. 97. Rubtsov, A. V., C. L. Swanson, S. Troy, P. Strauch, R. Pelanda, and R. M. Torres. 2008. TLR agonists promote marginal zone B cell activation and facilitate T- dependent IgM responses. J Immunol 180:3882-3888. 98. Kuppers, R. 2005. Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer 5:251-262.   54 99. Shaffer, A. L., A. Rosenwald, and L. M. Staudt. 2002. Lymphoid malignancies: the dark side of B-cell differentiation. Nat Rev Immunol 2:920-932. 100. Herreros, B., A. Sanchez-Aguilera, and M. A. Piris. 2008. Lymphoma microenvironment: culprit or innocent? Leukemia 22:49-58. 101. Nishio, M., T. Endo, N. Tsukada, J. Ohata, S. Kitada, J. C. Reed, N. J. Zvaifler, and T. J. Kipps. 2005. Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1alpha. Blood 106:1012-1020. 102. Burger, J. A., and T. J. Kipps. 2006. CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 107:1761-1767. 103. Redondo-Munoz, J., E. Escobar-Diaz, R. Samaniego, M. J. Terol, J. A. Garcia-Marco, and A. Garcia-Pardo. 2006. MMP-9 in B-cell chronic lymphocytic leukemia is up- regulated by alpha4beta1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration. Blood 108:3143-3151. 104. Husson, H., A. S. Freedman, A. A. Cardoso, J. Schultze, O. Munoz, G. Strola, J. Kutok, E. G. Carideo, R. De Beaumont, F. Caligaris-Cappio, and P. Ghia. 2002. CXCL13 (BCA-1) is produced by follicular lymphoma cells: role in the accumulation of malignant B cells. Br J Haematol 119:492-495. 105. Burkle, A., M. Niedermeier, A. Schmitt-Graff, W. G. Wierda, M. J. Keating, and J. A. Burger. 2007. Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood 110:3316-3325. 106. Dal Porto, J. M., S. B. Gauld, K. T. Merrell, D. Mills, A. E. Pugh-Bernard, and J. Cambier. 2004. B cell antigen receptor signaling 101. Mol Immunol 41:599-613. 107. Kurosaki, T. 2002. Regulation of B-cell signal transduction by adaptor proteins. Nat Rev Immunol 2:354-363. 108. Niiro, H., and E. A. Clark. 2002. Regulation of B-cell fate by antigen-receptor signals. Nat Rev Immunol 2:945-956. 109. Cahalan, M. D., I. Parker, S. H. Wei, and M. J. Miller. 2002. Two-photon tissue imaging: seeing the immune system in a fresh light. Nat Rev Immunol 2:872-880. 110. Phan, T. G., E. E. Gray, and J. G. Cyster. 2009. The microanatomy of B cell activation. Curr Opin Immunol 21:258-265. 111. Batista, F. D., and N. E. Harwood. 2009. The who, how and where of antigen presentation to B cells. Nat Rev Immunol 9:15-27. 112. Carrasco, Y. R., and F. D. Batista. 2007. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27:160-171. 113. Junt, T., E. A. Moseman, M. Iannacone, S. Massberg, P. A. Lang, M. Boes, K. Fink, S. E. Henrickson, D. M. Shayakhmetov, N. C. Di Paolo, N. van Rooijen, T. R. Mempel, S. P. Whelan, and U. H. von Andrian. 2007. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450:110-114. 114. Phan, T. G., I. Grigorova, T. Okada, and J. G. Cyster. 2007. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat Immunol 8:992-1000.   55 115. Martinez-Pomares, L., M. Kosco-Vilbois, E. Darley, P. Tree, S. Herren, J. Y. Bonnefoy, and S. Gordon. 1996. Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers. J Exp Med 184:1927-1937. 116. Batista, F. D., E. Arana, P. Barral, Y. R. Carrasco, D. Depoil, J. Eckl-Dorna, S. Fleire, K. Howe, A. Vehlow, M. Weber, and B. Treanor. 2007. The role of integrins and coreceptors in refining thresholds for B-cell responses. Immunol Rev 218:197-213. 117. Ferguson, A. R., M. E. Youd, and R. B. Corley. 2004. Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells. Int Immunol 16:1411-1422. 118. Suzuki, K., I. Grigorova, T. G. Phan, L. M. Kelly, and J. G. Cyster. 2009. Visualizing B cell capture of cognate antigen from follicular dendritic cells. J Exp Med 206:1485- 1493. 119. Qin, D., J. Wu, M. C. Carroll, G. F. Burton, A. K. Szakal, and J. G. Tew. 1998. Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J Immunol 161:4549-4554. 120. Fischer, M. B., M. Ma, N. C. Hsu, and M. C. Carroll. 1998. Local synthesis of C3 within the splenic lymphoid compartment can reconstitute the impaired immune response in C3-deficient mice. J Immunol 160:2619-2625. 121. Fischer, M. B., S. Goerg, L. Shen, A. P. Prodeus, C. C. Goodnow, G. Kelsoe, and M. C. Carroll. 1998. Dependence of germinal center B cells on expression of CD21/CD35 for survival. Science 280:582-585. 122. Molina, H., V. M. Holers, B. Li, Y. Fung, S. Mariathasan, J. Goellner, J. Strauss- Schoenberger, R. W. Karr, and D. D. Chaplin. 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proc Natl Acad Sci U S A 93:3357-3361. 123. Allen, C. D., and J. G. Cyster. 2008. Follicular dendritic cell networks of primary follicles and germinal centers: phenotype and function. Semin Immunol 20:14-25. 124. Szakal, A. K., M. H. Kosco, and J. G. Tew. 1989. Microanatomy of lymphoid tissue during humoral immune responses: structure function relationships. Annu Rev Immunol 7:91-109. 125. Qi, H., J. G. Egen, A. Y. Huang, and R. N. Germain. 2006. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312:1672-1676. 126. Bergtold, A., D. D. Desai, A. Gavhane, and R. Clynes. 2005. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23:503-514. 127. Tomura, M., N. Yoshida, J. Tanaka, S. Karasawa, Y. Miwa, A. Miyawaki, and O. Kanagawa. 2008. Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice. Proc Natl Acad Sci U S A 105:10871- 10876. 128. Pape, K. A., D. M. Catron, A. A. Itano, and M. K. Jenkins. 2007. The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles. Immunity 26:491-502. 129. Roozendaal, R., R. E. Mebius, and G. Kraal. 2008. The conduit system of the lymph node. Int Immunol 20:1483-1487.   56 130. Anderson, A. O., and S. Shaw. 1993. T cell adhesion to endothelium: the FRC conduit system and other anatomic and molecular features which facilitate the adhesion cascade in lymph node. Semin Immunol 5:271-282. 131. Roozendaal, R., T. R. Mempel, L. A. Pitcher, S. F. Gonzalez, A. Verschoor, R. E. Mebius, U. H. von Andrian, and M. C. Carroll. 2009. Conduits mediate transport of low-molecular-weight antigen to lymph node follicles. Immunity 30:264-276. 132. Kurosaki, T., H. Shinohara, and Y. Baba. 2009. B Cell Signaling and Fate Decision. Annu Rev Immunol. 133. Sohn, H. W., P. Tolar, T. Jin, and S. K. Pierce. 2006. Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling. Proc Natl Acad Sci U S A 103:8143-8148. 134. Harwood, N. E., and F. D. Batista. 2008. New insights into the early molecular events underlying B cell activation. Immunity 28:609-619. 135. Crabtree, G. R., and E. N. Olson. 2002. NFAT signaling: choreographing the social lives of cells. Cell 109 Suppl:S67-79. 136. Gold, M. R. 2002. To make antibodies or not: signaling by the B-cell antigen receptor. Trends Pharmacol Sci 23:316-324. 137. McLeod, S. J., and M. R. Gold. 2001. Activation and function of the Rap1 GTPase in B lymphocytes. Int Rev Immunol 20:763-789. 138. Katagiri, K., M. Shimonaka, and T. Kinashi. 2004. Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-gamma1. J Biol Chem 279:11875-11881. 139. Monroe, J. G. 2006. ITAM-mediated tonic signalling through pre-BCR and BCR complexes. Nat Rev Immunol 6:283-294. 140. Brooks, S. R., X. Li, E. J. Volanakis, and R. H. Carter. 2000. Systematic analysis of the role of CD19 cytoplasmic tyrosines in enhancement of activation in Daudi human B cells: clustering of phospholipase C and Vav and of Grb2 and Sos with different CD19 tyrosines. J Immunol 164:3123-3131. 141. Fujimoto, M., Y. Fujimoto, J. C. Poe, P. J. Jansen, C. A. Lowell, A. L. DeFranco, and T. F. Tedder. 2000. CD19 regulates Src family protein tyrosine kinase activation in B lymphocytes through processive amplification. Immunity 13:47-57. 142. Tuveson, D. A., R. H. Carter, S. P. Soltoff, and D. T. Fearon. 1993. CD19 of B cells as a surrogate kinase insert region to bind phosphatidylinositol 3-kinase. Science 260:986-989. 143. Okada, T., A. Maeda, A. Iwamatsu, K. Gotoh, and T. Kurosaki. 2000. BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation. Immunity 13:817-827. 144. Inabe, K., M. Ishiai, A. M. Scharenberg, N. Freshney, J. Downward, and T. Kurosaki. 2002. Vav3 modulates B cell receptor responses by regulating phosphoinositide 3- kinase activation. J Exp Med 195:189-200. 145. Suzuki, H., Y. Terauchi, M. Fujiwara, S. Aizawa, Y. Yazaki, T. Kadowaki, and S. Koyasu. 1999. Xid-like immunodeficiency in mice with disruption of the p85alpha subunit of phosphoinositide 3-kinase. Science 283:390-392. 146. Aiba, Y., M. Kameyama, T. Yamazaki, T. F. Tedder, and T. Kurosaki. 2008. Regulation of B-cell development by BCAP and CD19 through their binding to phosphoinositide 3-kinase. Blood 111:1497-1503.   57 147. Batista, F. D., D. Iber, and M. S. Neuberger. 2001. B cells acquire antigen from target cells after synapse formation. Nature 411:489-494. 148. Lin, J., M. J. Miller, and A. S. Shaw. 2005. The c-SMAC: sorting it all out (or in). J Cell Biol 170:177-182. 149. Weber, M., B. Treanor, D. Depoil, H. Shinohara, N. E. Harwood, M. Hikida, T. Kurosaki, and F. D. Batista. 2008. Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen. J Exp Med 205:853-868. 150. Hou, P., E. Araujo, T. Zhao, M. Zhang, D. Massenburg, M. Veselits, C. Doyle, A. R. Dinner, and M. R. Clark. 2006. B cell antigen receptor signaling and internalization are mutually exclusive events. PLoS Biol 4:e200. 151. Kinashi, T. 2005. Intracellular signalling controlling integrin activation in lymphocytes. Nat Rev Immunol 5:546-559. 152. Hemler, M. E. 1990. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol 8:365-400. 153. Abram, C. L., and C. A. Lowell. 2009. The ins and outs of leukocyte integrin signaling. Annu Rev Immunol 27:339-362. 154. Dustin, M. L., S. Y. Tseng, R. Varma, and G. Campi. 2006. T cell-dendritic cell immunological synapses. Curr Opin Immunol 18:512-516. 155. Hogg, N., A. Smith, A. McDowall, K. Giles, P. Stanley, M. Laschinger, and R. Henderson. 2004. How T cells use LFA-1 to attach and migrate. Immunol Lett 92:51- 54. 156. Evans, R., I. Patzak, L. Svensson, K. De Filippo, K. Jones, A. McDowall, and N. Hogg. 2009. Integrins in immunity. J Cell Sci 122:215-225. 157. Arana, E., A. Vehlow, N. E. Harwood, E. Vigorito, R. Henderson, M. Turner, V. L. Tybulewicz, and F. D. Batista. 2008. Activation of the small GTPase Rac2 via the B cell receptor regulates B cell adhesion and immunological-synapse formation. Immunity 28:88-99. 158. Carrasco, Y. R., S. J. Fleire, T. Cameron, M. L. Dustin, and F. D. Batista. 2004. LFA- 1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20:589-599. 159. Bergmeier, W., T. Goerge, H. W. Wang, J. R. Crittenden, A. C. Baldwin, S. M. Cifuni, D. E. Housman, A. M. Graybiel, and D. D. Wagner. 2007. Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. J Clin Invest 117:1699-1707. 160. Smith, C. W. 2008. 3. Adhesion molecules and receptors. J Allergy Clin Immunol 121:S375-379; quiz S414. 161. Luo, B. H., C. V. Carman, and T. A. Springer. 2007. Structural basis of integrin regulation and signaling. Annu Rev Immunol 25:619-647. 162. Astrof, N. S., A. Salas, M. Shimaoka, J. Chen, and T. A. Springer. 2006. Importance of force linkage in mechanochemistry of adhesion receptors. Biochemistry 45:15020- 15028. 163. McLeod, S. J., A. J. Shum, R. L. Lee, F. Takei, and M. R. Gold. 2004. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem 279:12009-12019.   58 164. Spaargaren, M., E. A. Beuling, M. L. Rurup, H. P. Meijer, M. D. Klok, S. Middendorp, R. W. Hendriks, and S. T. Pals. 2003. The B cell antigen receptor controls integrin activity through Btk and PLCgamma2. J Exp Med 198:1539-1550. 165. Burbach, B. J., R. B. Medeiros, K. L. Mueller, and Y. Shimizu. 2007. T-cell receptor signaling to integrins. Immunol Rev 218:65-81. 166. Medeiros, R. B., D. M. Dickey, H. Chung, A. C. Quale, L. R. Nagarajan, D. D. Billadeau, and Y. Shimizu. 2005. Protein kinase D1 and the beta 1 integrin cytoplasmic domain control beta 1 integrin function via regulation of Rap1 activation. Immunity 23:213-226. 167. Tohyama, Y., K. Katagiri, R. Pardi, C. Lu, T. A. Springer, and T. Kinashi. 2003. The critical cytoplasmic regions of the alphaL/beta2 integrin in Rap1-induced adhesion and migration. Mol Biol Cell 14:2570-2582. 168. Kinashi, T., and K. Katagiri. 2004. Regulation of lymphocyte adhesion and migration by the small GTPase Rap1 and its effector molecule, RAPL. Immunol Lett 93:1-5. 169. Katagiri, K., M. Imamura, and T. Kinashi. 2006. Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat Immunol 7:919-928. 170. Menasche, G., S. Kliche, E. J. Chen, T. E. Stradal, B. Schraven, and G. Koretzky. 2007. RIAM links the ADAP/SKAP-55 signaling module to Rap1, facilitating T-cell- receptor-mediated integrin activation. Mol Cell Biol 27:4070-4081. 171. Jo, E. K., H. Wang, and C. E. Rudd. 2005. An essential role for SKAP-55 in LFA-1 clustering on T cells that cannot be substituted by SKAP-55R. J Exp Med 201:1733- 1739. 172. Kliche, S., D. Breitling, M. Togni, R. Pusch, K. Heuer, X. Wang, C. Freund, A. Kasirer-Friede, G. Menasche, G. A. Koretzky, and B. Schraven. 2006. The ADAP/SKAP55 signaling module regulates T-cell receptor-mediated integrin activation through plasma membrane targeting of Rap1. Mol Cell Biol 26:7130-7144. 173. Togni, M., K. D. Swanson, S. Reimann, S. Kliche, A. C. Pearce, L. Simeoni, D. Reinhold, J. Wienands, B. G. Neel, B. Schraven, and A. Gerber. 2005. Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM. Mol Cell Biol 25:8052-8063. 174. Moser, M., B. Nieswandt, S. Ussar, M. Pozgajova, and R. Fassler. 2008. Kindlin-3 is essential for integrin activation and platelet aggregation. Nat Med 14:325-330. 175. Montanez, E., S. Ussar, M. Schifferer, M. Bosl, R. Zent, M. Moser, and R. Fassler. 2008. Kindlin-2 controls bidirectional signaling of integrins. Genes Dev 22:1325- 1330. 176. Moser, M., M. Bauer, S. Schmid, R. Ruppert, S. Schmidt, M. Sixt, H. V. Wang, M. Sperandio, and R. Fassler. 2009. Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells. Nat Med 15:300-305. 177. Ginsberg, M. H., A. Partridge, and S. J. Shattil. 2005. Integrin regulation. Curr Opin Cell Biol 17:509-516. 178. Porter, J. C., M. Bracke, A. Smith, D. Davies, and N. Hogg. 2002. Signaling through integrin LFA-1 leads to filamentous actin polymerization and remodeling, resulting in enhanced T cell adhesion. J Immunol 168:6330-6335. 179. Baruzzi, A., E. Caveggion, and G. Berton. 2008. Regulation of phagocyte migration and recruitment by Src-family kinases. Cell Mol Life Sci 65:2175-2190.   59 180. Mocsai, A., M. Zhou, F. Meng, V. L. Tybulewicz, and C. A. Lowell. 2002. Syk is required for integrin signaling in neutrophils. Immunity 16:547-558. 181. Mocsai, A., C. L. Abram, Z. Jakus, Y. Hu, L. L. Lanier, and C. A. Lowell. 2006. Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs. Nat Immunol 7:1326-1333. 182. Caveggion, E., S. Continolo, F. J. Pixley, E. R. Stanley, D. D. Bowtell, C. A. Lowell, and G. Berton. 2003. Expression and tyrosine phosphorylation of Cbl regulates macrophage chemokinetic and chemotactic movement. J Cell Physiol 195:276-289. 183. Meng, F., and C. A. Lowell. 1998. A beta 1 integrin signaling pathway involving Src- family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J 17:4391-4403. 184. Dustin, M. L., and T. A. Springer. 1989. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341:619-624. 185. Randall, K. L., T. Lambe, A. Johnson, B. Treanor, E. Kucharska, H. Domaschenz, B. Whittle, L. E. Tze, A. Enders, T. L. Crockford, T. Bouriez-Jones, D. Alston, J. G. Cyster, M. J. Lenardo, F. Mackay, E. K. Deenick, S. G. Tangye, T. D. Chan, T. Camidge, R. Brink, C. G. Vinuesa, F. D. Batista, R. J. Cornall, and C. C. Goodnow. 2009. Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production. Nat Immunol 10:1283-1291. 186. Lin, K. B., S. A. Freeman, S. Zabetian, H. Brugger, M. Weber, V. Lei, M. Dang- Lawson, K. W. Tse, R. Santamaria, F. D. Batista, and M. R. Gold. 2008. The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands. Immunity 28:75-87. 187. Real, E., S. Faure, E. Donnadieu, and J. Delon. 2007. Cutting edge: Atypical PKCs regulate T lymphocyte polarity and scanning behavior. J Immunol 179:5649-5652. 188. Hannigan, M., L. Zhan, Z. Li, Y. Ai, D. Wu, and C. K. Huang. 2002. Neutrophils lacking phosphoinositide 3-kinase gamma show loss of directionality during N- formyl-Met-Leu-Phe-induced chemotaxis. Proc Natl Acad Sci U S A 99:3603-3608. 189. Nombela-Arrieta, C., R. A. Lacalle, M. C. Montoya, Y. Kunisaki, D. Megias, M. Marques, A. C. Carrera, S. Manes, Y. Fukui, A. C. Martinez, and J. V. Stein. 2004. Differential requirements for DOCK2 and phosphoinositide-3-kinase gamma during T and B lymphocyte homing. Immunity 21:429-441. 190. Thelen, M., and J. V. Stein. 2008. How chemokines invite leukocytes to dance. Nat Immunol 9:953-959. 191. Lammermann, T., and M. Sixt. 2009. Mechanical modes of 'amoeboid' cell migration. Curr Opin Cell Biol 21:636-644. 192. Serrador, J. M., M. Nieto, and F. Sanchez-Madrid. 1999. Cytoskeletal rearrangement during migration and activation of T lymphocytes. Trends Cell Biol 9:228-233. 193. Ratner, S., W. S. Sherrod, and D. Lichlyter. 1997. Microtubule retraction into the uropod and its role in T cell polarization and motility. J Immunol 159:1063-1067. 194. Lammermann, T., B. L. Bader, S. J. Monkley, T. Worbs, R. Wedlich-Soldner, K. Hirsch, M. Keller, R. Forster, D. R. Critchley, R. Fassler, and M. Sixt. 2008. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453:51- 55. 195. Friedl, P., and B. Weigelin. 2008. Interstitial leukocyte migration and immune function. Nat Immunol 9:960-969.   60 196. Shulman, Z., V. Shinder, E. Klein, V. Grabovsky, O. Yeger, E. Geron, A. Montresor, M. Bolomini-Vittori, S. W. Feigelson, T. Kirchhausen, C. Laudanna, G. Shakhar, and R. Alon. 2009. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin. Immunity 30:384-396. 197. Forster, R., A. C. Davalos-Misslitz, and A. Rot. 2008. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev Immunol 8:362-371. 198. Stein, J. V., and C. Nombela-Arrieta. 2005. Chemokine control of lymphocyte trafficking: a general overview. Immunology 116:1-12. 199. Zou, Y. R., A. H. Kottmann, M. Kuroda, I. Taniuchi, and D. R. Littman. 1998. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595-599. 200. Ma, Q., D. Jones, P. R. Borghesani, R. A. Segal, T. Nagasawa, T. Kishimoto, R. T. Bronson, and T. A. Springer. 1998. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 95:9448-9453. 201. Pillai, S., A. Cariappa, and S. T. Moran. 2004. Positive selection and lineage commitment during peripheral B-lymphocyte development. Immunol Rev 197:206- 218. 202. Rot, A., and U. H. von Andrian. 2004. Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. Annu Rev Immunol 22:891- 928. 203. Kehrl, J. H. 2006. Chemoattractant receptor signaling and the control of lymphocyte migration. Immunol Res 34:211-227. 204. Bajenoff, M., J. G. Egen, H. Qi, A. Y. Huang, F. Castellino, and R. N. Germain. 2007. Highways, byways and breadcrumbs: directing lymphocyte traffic in the lymph node. Trends Immunol 28:346-352. 205. Sumen, C., T. R. Mempel, I. B. Mazo, and U. H. von Andrian. 2004. Intravital microscopy: visualizing immunity in context. Immunity 21:315-329. 206. Okada, T., V. N. Ngo, E. H. Ekland, R. Forster, M. Lipp, D. R. Littman, and J. G. Cyster. 2002. Chemokine requirements for B cell entry to lymph nodes and Peyer's patches. J Exp Med 196:65-75. 207. von Andrian, U. H., and C. R. Mackay. 2000. T-cell function and migration. Two sides of the same coin. N Engl J Med 343:1020-1034. 208. Davis, M. D., and J. H. Kehrl. 2009. The influence of sphingosine-1-phosphate receptor signaling on lymphocyte trafficking: how a bioactive lipid mediator grew up from an "immature" vascular maturation factor to a "mature" mediator of lymphocyte behavior and function. Immunol Res 43:187-197. 209. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427:355-360. 210. Girkontaite, I., V. Sakk, M. Wagner, T. Borggrefe, K. Tedford, J. Chun, and K. D. Fischer. 2004. The sphingosine-1-phosphate (S1P) lysophospholipid receptor S1P3 regulates MAdCAM-1+ endothelial cells in splenic marginal sinus organization. J Exp Med 200:1491-1501. 211. Sinha, R. K., C. Park, I. Y. Hwang, M. D. Davis, and J. H. Kehrl. 2009. B lymphocytes exit lymph nodes through cortical lymphatic sinusoids by a mechanism   61 independent of sphingosine-1-phosphate-mediated chemotaxis. Immunity 30:434- 446. 212. Allen, S. J., S. E. Crown, and T. M. Handel. 2007. Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol 25:787-820. 213. Thelen, M. 2001. Dancing to the tune of chemokines. Nat Immunol 2:129-134. 214. Ohshima, Y., T. Kubo, R. Koyama, M. Ueno, M. Nakagawa, and T. Yamashita. 2008. Regulation of axonal elongation and pathfinding from the entorhinal cortex to the dentate gyrus in the hippocampus by the chemokine stromal cell-derived factor 1 alpha. J Neurosci 28:8344-8353. 215. Inngjerdingen, M., K. M. Torgersen, and A. A. Maghazachi. 2002. Lck is required for stromal cell-derived factor 1 alpha (CXCL12)-induced lymphoid cell chemotaxis. Blood 99:4318-4325. 216. Ma, Y. C., J. Huang, S. Ali, W. Lowry, and X. Y. Huang. 2000. Src tyrosine kinase is a novel direct effector of G proteins. Cell 102:635-646. 217. Jiang, Y., W. Ma, Y. Wan, T. Kozasa, S. Hattori, and X. Y. Huang. 1998. The G protein G alpha12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain. Nature 395:808-813. 218. Shi, C. S., S. Sinnarajah, H. Cho, T. Kozasa, and J. H. Kehrl. 2000. G13alpha- mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription. J Biol Chem 275:24470-24476. 219. Kurose, H. 2003. Galpha12 and Galpha13 as key regulatory mediator in signal transduction. Life Sci 74:155-161. 220. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7:678-689. 221. Pasvolsky, R., S. W. Feigelson, S. S. Kilic, A. J. Simon, G. Tal-Lapidot, V. Grabovsky, J. R. Crittenden, N. Amariglio, M. Safran, A. M. Graybiel, G. Rechavi, S. Ben-Dor, A. Etzioni, and R. Alon. 2007. A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets. J Exp Med 204:1571-1582. 222. Kooistra, M. R., N. Dube, and J. L. Bos. 2007. Rap1: a key regulator in cell-cell junction formation. J Cell Sci 120:17-22. 223. Manes, S., C. Gomez-Mouton, R. A. Lacalle, S. Jimenez-Baranda, E. Mira, and A. C. Martinez. 2005. Mastering time and space: immune cell polarization and chemotaxis. Semin Immunol 17:77-86. 224. Sasaki, A. T., and R. A. Firtel. 2006. Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR. Eur J Cell Biol 85:873-895. 225. Ward, S. G. 2004. Do phosphoinositide 3-kinases direct lymphocyte navigation? Trends Immunol 25:67-74. 226. Okkenhaug, K., A. Bilancio, G. Farjot, H. Priddle, S. Sancho, E. Peskett, W. Pearce, S. E. Meek, A. Salpekar, M. D. Waterfield, A. J. Smith, and B. Vanhaesebroeck. 2002. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science 297:1031-1034. 227. Reif, K., K. Okkenhaug, T. Sasaki, J. M. Penninger, B. Vanhaesebroeck, and J. G. Cyster. 2004. Cutting edge: differential roles for phosphoinositide 3-kinases, p110gamma and p110delta, in lymphocyte chemotaxis and homing. J Immunol 173:2236-2240.   62 228. Bilancio, A., K. Okkenhaug, M. Camps, J. L. Emery, T. Ruckle, C. Rommel, and B. Vanhaesebroeck. 2006. Key role of the p110delta isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110delta function in B cells. Blood 107:642-650. 229. Durand, C. A., K. Hartvigsen, L. Fogelstrand, S. Kim, S. Iritani, B. Vanhaesebroeck, J. L. Witztum, K. D. Puri, and M. R. Gold. 2009. Phosphoinositide 3-kinase p110delta regulates natural antibody production, marginal zone and B-1 B cell function, and autoantibody responses. J Immunol 183:5673-5684. 230. Hirsch, E., V. L. Katanaev, C. Garlanda, O. Azzolino, L. Pirola, L. Silengo, S. Sozzani, A. Mantovani, F. Altruda, and M. P. Wymann. 2000. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science 287:1049-1053. 231. Sasaki, T., J. Irie-Sasaki, R. G. Jones, A. J. Oliveira-dos-Santos, W. L. Stanford, B. Bolon, A. Wakeham, A. Itie, D. Bouchard, I. Kozieradzki, N. Joza, T. W. Mak, P. S. Ohashi, A. Suzuki, and J. M. Penninger. 2000. Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science 287:1040-1046. 232. Li, Z., H. Jiang, W. Xie, Z. Zhang, A. V. Smrcka, and D. Wu. 2000. Roles of PLC- beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction. Science 287:1046-1049. 233. Jou, S. T., N. Carpino, Y. Takahashi, R. Piekorz, J. R. Chao, D. Wang, and J. N. Ihle. 2002. Essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex. Mol Cell Biol 22:8580-8591. 234. Durand, C. A., K. Hartvigsen, L. Fogelstrand, S. Kim, S. Iritani, B. Vanhaesebroeck, J. L. Witztum, K. D. Puri, and M. R. Gold. 2009. Phosphoinositide 3-kinase p110 delta regulates natural antibody production, marginal zone and B-1 B cell function, and autoantibody responses. J Immunol 183:5673-5684. 235. Croker, B. A., D. M. Tarlinton, L. A. Cluse, A. J. Tuxen, A. Light, F. C. Yang, D. A. Williams, and A. W. Roberts. 2002. The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes. J Immunol 168:3376-3386. 236. Gerard, A., A. E. Mertens, R. A. van der Kammen, and J. G. Collard. 2007. The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization. J Cell Biol 176:863-875. 237. Ludford-Menting, M. J., J. Oliaro, F. Sacirbegovic, E. T. Cheah, N. Pedersen, S. J. Thomas, A. Pasam, R. Iazzolino, L. E. Dow, N. J. Waterhouse, A. Murphy, S. Ellis, M. J. Smyth, M. H. Kershaw, P. K. Darcy, P. O. Humbert, and S. M. Russell. 2005. A network of PDZ-containing proteins regulates T cell polarity and morphology during migration and immunological synapse formation. Immunity 22:737-748. 238. Lee, J. H., T. Katakai, T. Hara, H. Gonda, M. Sugai, and A. Shimizu. 2004. Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation. J Cell Biol 167:327-337. 239. Bardi, G., V. Niggli, and P. Loetscher. 2003. Rho kinase is required for CCR7- mediated polarization and chemotaxis of T lymphocytes. FEBS Lett 542:79-83. 240. Imhof, B. A., and M. Aurrand-Lions. 2004. Adhesion mechanisms regulating the migration of monocytes. Nat Rev Immunol 4:432-444.   63 241. Burridge, K., and K. Wennerberg. 2004. Rho and Rac take center stage. Cell 116:167- 179. 242. Tybulewicz, V. L., and R. B. Henderson. 2009. Rho family GTPases and their regulators in lymphocytes. Nat Rev Immunol 9:630-644. 243. Stossel, T. P., J. Condeelis, L. Cooley, J. H. Hartwig, A. Noegel, M. Schleicher, and S. S. Shapiro. 2001. Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol 2:138-145. 244. Chhabra, E. S., and H. N. Higgs. 2007. The many faces of actin: matching assembly factors with cellular structures. Nat Cell Biol 9:1110-1121. 245. Goley, E. D., and M. D. Welch. 2006. The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol 7:713-726. 246. Oikawa, T., H. Yamaguchi, T. Itoh, M. Kato, T. Ijuin, D. Yamazaki, S. Suetsugu, and T. Takenawa. 2004. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Nat Cell Biol 6:420-426. 247. Nakanishi, O., S. Suetsugu, D. Yamazaki, and T. Takenawa. 2007. Effect of WAVE2 phosphorylation on activation of the Arp2/3 complex. J Biochem 141:319-325. 248. Eisenmann, K. M., R. A. West, D. Hildebrand, S. M. Kitchen, J. Peng, R. Sigler, J. Zhang, K. A. Siminovitch, and A. S. Alberts. 2007. T cell responses in mammalian diaphanous-related formin mDia1 knock-out mice. J Biol Chem 282:25152-25158. 249. Sakata, D., H. Taniguchi, S. Yasuda, A. Adachi-Morishima, Y. Hamazaki, R. Nakayama, T. Miki, N. Minato, and S. Narumiya. 2007. Impaired T lymphocyte trafficking in mice deficient in an actin-nucleating protein, mDia1. J Exp Med 204:2031-2038. 250. Avraham, H., S. Y. Park, K. Schinkmann, and S. Avraham. 2000. RAFTK/Pyk2- mediated cellular signalling. Cell Signal 12:123-133. 251. Andreev, J., J. P. Simon, D. D. Sabatini, J. Kam, G. Plowman, P. A. Randazzo, and J. Schlessinger. 1999. Identification of a new Pyk2 target protein with Arf-GAP activity. Mol Cell Biol 19:2338-2350. 252. Dikic, I., and J. Schlessinger. 1998. Identification of a new Pyk2 isoform implicated in chemokine and antigen receptor signaling. J Biol Chem 273:14301-14308. 253. Behmoaram, E., K. Bijian, S. Jie, Y. Xu, A. Darnel, T. A. Bismar, and M. A. Alaoui- Jamali. 2008. Focal adhesion kinase-related proline-rich tyrosine kinase 2 and focal adhesion kinase are co-overexpressed in early-stage and invasive ErbB-2-positive breast cancer and cooperate for breast cancer cell tumorigenesis and invasiveness. Am J Pathol 173:1540-1550. 254. Lark, A. L., C. A. Livasy, B. Calvo, L. Caskey, D. T. Moore, X. Yang, and W. G. Cance. 2003. Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses. Clin Cancer Res 9:215-222. 255. Lark, A. L., C. A. Livasy, L. Dressler, D. T. Moore, R. C. Millikan, J. Geradts, M. Iacocca, D. Cowan, D. Little, R. J. Craven, and W. Cance. 2005. High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype. Mod Pathol 18:1289-1294. 256. Owens, L. V., L. Xu, R. J. Craven, G. A. Dent, T. M. Weiner, L. Kornberg, E. T. Liu, and W. G. Cance. 1995. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 55:2752-2755.   64 257. Tremblay, L., W. Hauck, A. G. Aprikian, L. R. Begin, A. Chapdelaine, and S. Chevalier. 1996. Focal adhesion kinase (pp125FAK) expression, activation and association with paxillin and p50CSK in human metastatic prostate carcinoma. Int J Cancer 68:164-171. 258. Zhang, S., X. Qiu, Y. Gu, and E. Wang. 2008. Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer. Lung Cancer 62:295-301. 259. Brunton, V. G., and M. C. Frame. 2008. Src and focal adhesion kinase as therapeutic targets in cancer. Curr Opin Pharmacol 8:427-432. 260. Lipinski, C. A., and J. C. Loftus. Targeting Pyk2 for therapeutic intervention. Expert Opin Ther Targets 14:95-108. 261. Chen, R., O. Kim, M. Li, X. Xiong, J. L. Guan, H. J. Kung, H. Chen, Y. Shimizu, and Y. Qiu. 2001. Regulation of the PH-domain-containing tyrosine kinase Etk by focal adhesion kinase through the FERM domain. Nat Cell Biol 3:439-444. 262. Poullet, P., A. Gautreau, G. Kadare, J. A. Girault, D. Louvard, and M. Arpin. 2001. Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion. J Biol Chem 276:37686-37691. 263. Jacamo, R. O., and E. Rozengurt. 2005. A truncated FAK lacking the FERM domain displays high catalytic activity but retains responsiveness to adhesion-mediated signals. Biochem Biophys Res Commun 334:1299-1304. 264. Cooper, L. A., T. L. Shen, and J. L. Guan. 2003. Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction. Mol Cell Biol 23:8030-8041. 265. Lietha, D., X. Cai, D. F. Ceccarelli, Y. Li, M. D. Schaller, and M. J. Eck. 2007. Structural basis for the autoinhibition of focal adhesion kinase. Cell 129:1177-1187. 266. Wu, S. S., R. O. Jacamo, S. K. Vong, and E. Rozengurt. 2006. Differential regulation of Pyk2 phosphorylation at Tyr-402 and Tyr-580 in intestinal epithelial cells: roles of calcium, Src, Rho kinase, and the cytoskeleton. Cell Signal 18:1932-1940. 267. Kohno, T., E. Matsuda, H. Sasaki, and T. Sasaki. 2008. Protein-tyrosine kinase CAKbeta/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 alpha2 helix and thus forming its dimer. Biochem J 410:513-523. 268. Lim, S. T., N. L. Miller, J. O. Nam, X. L. Chen, Y. Lim, and D. D. Schlaepfer. 2009. PYK2 inhibition of p53 as an adaptive and intrinsic mechanism facilitating cell proliferation and survival. J Biol Chem. 269. Lim, S. T., X. L. Chen, Y. Lim, D. A. Hanson, T. T. Vo, K. Howerton, N. Larocque, S. J. Fisher, D. D. Schlaepfer, and D. Ilic. 2008. Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation. Mol Cell 29:9- 22. 270. Mitra, S. K., D. A. Hanson, and D. D. Schlaepfer. 2005. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 6:56-68. 271. Hanks, S. K., L. Ryzhova, N. Y. Shin, and J. Brabek. 2003. Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 8:d982-996. 272. Chodniewicz, D., and R. L. Klemke. 2004. Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold. Biochim Biophys Acta 1692:63- 76.   65 273. Schlaepfer, D. D., S. K. Mitra, and D. Ilic. 2004. Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochim Biophys Acta 1692:77-102. 274. Park, S. Y., H. K. Avraham, and S. Avraham. 2004. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279:33315-33322. 275. Ruest, P. J., S. Roy, E. Shi, R. L. Mernaugh, and S. K. Hanks. 2000. Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site. Cell Growth Differ 11:41-48. 276. Salazar, E. P., and E. Rozengurt. 2001. Src family kinases are required for integrin- mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem 276:17788-17795. 277. Sahu, S. N., S. Nunez, G. Bai, and A. Gupta. 2007. Interaction of Pyk2 and PTP- PEST with leupaxin in prostate cancer cells. Am J Physiol Cell Physiol 292:C2288- 2296. 278. Maksumova, L., Y. Wang, N. K. Wong, H. T. Le, C. J. Pallen, and P. Johnson. 2007. Differential function of PTPalpha and PTPalpha Y789F in T cells and regulation of PTPalpha phosphorylation at Tyr-789 by CD45. J Biol Chem 282:20925-20932. 279. Le, H. T., L. Maksumova, J. Wang, and C. J. Pallen. 2006. Reduced NMDA receptor tyrosine phosphorylation in PTPalpha-deficient mouse synaptosomes is accompanied by inhibition of four src family kinases and Pyk2: an upstream role for PTPalpha in NMDA receptor regulation. J Neurochem 98:1798-1809. 280. Zeng, L., X. Si, W. P. Yu, H. T. Le, K. P. Ng, R. M. Teng, K. Ryan, D. Z. Wang, S. Ponniah, and C. J. Pallen. 2003. PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration. J Cell Biol 160:137-146. 281. Tomar, A., and D. D. Schlaepfer. 2009. Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility. Curr Opin Cell Biol 21:676-683. 282. Schlaepfer, D. D., and S. K. Mitra. 2004. Multiple connections link FAK to cell motility and invasion. Curr Opin Genet Dev 14:92-101. 283. Chang, F., C. A. Lemmon, D. Park, and L. H. Romer. 2007. FAK potentiates Rac1 activation and localization to matrix adhesion sites: a role for betaPIX. Mol Biol Cell 18:253-264. 284. Schober, M., S. Raghavan, M. Nikolova, L. Polak, H. A. Pasolli, H. E. Beggs, L. F. Reichardt, and E. Fuchs. 2007. Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics. J Cell Biol 176:667-680. 285. Defilippi, P., P. Di Stefano, and S. Cabodi. 2006. p130Cas: a versatile scaffold in signaling networks. Trends Cell Biol 16:257-263. 286. Tomar, A., S. T. Lim, Y. Lim, and D. D. Schlaepfer. 2009. A FAK-p120RasGAP- p190RhoGAP complex regulates polarity in migrating cells. J Cell Sci 122:1852- 1862. 287. Serrels, B., A. Serrels, V. G. Brunton, M. Holt, G. W. McLean, C. H. Gray, G. E. Jones, and M. C. Frame. 2007. Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex. Nat Cell Biol 9:1046-1056.   66 288. Zhang, X., G. Jiang, Y. Cai, S. J. Monkley, D. R. Critchley, and M. P. Sheetz. 2008. Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat Cell Biol 10:1062-1068. 289. Gupton, S. L., and C. M. Waterman-Storer. 2006. Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration. Cell 125:1361-1374. 290. Iwanicki, M. P., T. Vomastek, R. W. Tilghman, K. H. Martin, J. Banerjee, P. B. Wedegaertner, and J. T. Parsons. 2008. FAK, PDZ-RhoGEF and ROCKII cooperate to regulate adhesion movement and trailing-edge retraction in fibroblasts. J Cell Sci 121:895-905. 291. Ilic, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, and T. Yamamoto. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377:539- 544. 292. Okigaki, M., C. Davis, M. Falasca, S. Harroch, D. P. Felsenfeld, M. P. Sheetz, and J. Schlessinger. 2003. Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration. Proc Natl Acad Sci U S A 100:10740-10745. 293. Owen, K. A., F. J. Pixley, K. S. Thomas, M. Vicente-Manzanares, B. J. Ray, A. F. Horwitz, J. T. Parsons, H. E. Beggs, E. R. Stanley, and A. H. Bouton. 2007. Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase. J Cell Biol 179:1275-1287. 294. Rose, D. M., S. Liu, D. G. Woodside, J. Han, D. D. Schlaepfer, and M. H. Ginsberg. 2003. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2. J Immunol 170:5912-5918. 295. van Seventer, G. A., H. J. Salmen, S. F. Law, G. M. O'Neill, M. M. Mullen, A. M. Franz, S. B. Kanner, E. A. Golemis, and J. M. van Seventer. 2001. Focal adhesion kinase regulates beta1 integrin-dependent T cell migration through an HEF1 effector pathway. Eur J Immunol 31:1417-1427. 296. Bearz, A., G. Tell, S. Formisano, S. Merluzzi, A. Colombatti, and C. Pucillo. 1999. Adhesion to fibronectin promotes the activation of the p125(FAK)/Zap-70complex in human T cells. Immunology 98:564-568. 297. Berg, N. N., and H. L. Ostergaard. 1997. T cell receptor engagement induces tyrosine phosphorylation of FAK and Pyk2 and their association with Lck. J Immunol 159:1753-1757. 298. Haller, H., U. Kunzendorf, K. Sacherer, C. Lindschau, G. Walz, A. Distler, and F. C. Luft. 1997. T cell adhesion to P-selectin induces tyrosine phosphorylation of pp125 focal adhesion kinase and other substrates. J Immunol 158:1061-1067. 299. Bacon, K. B., M. C. Szabo, H. Yssel, J. B. Bolen, and T. J. Schall. 1996. RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells. J Exp Med 184:873-882. 300. Ostergaard, H. L., and T. L. Lysechko. 2005. Focal adhesion kinase-related protein tyrosine kinase Pyk2 in T-cell activation and function. Immunol Res 31:267-282. 301. Park, D., I. Park, D. Lee, Y. B. Choi, H. Lee, and Y. Yun. 2007. The adaptor protein Lad associates with the G protein beta subunit and mediates chemokine-dependent T- cell migration. Blood 109:5122-5128.   67 302. Okabe, S., S. Fukuda, Y. J. Kim, M. Niki, L. M. Pelus, K. Ohyashiki, P. P. Pandolfi, and H. E. Broxmeyer. 2005. Stromal cell-derived factor-1alpha/CXCL12-induced chemotaxis of T cells involves activation of the RasGAP-associated docking protein p62Dok-1. Blood 105:474-480. 303. Sancho, D., M. C. Montoya, A. Monjas, M. Gordon-Alonso, T. Katagiri, D. Gil, R. Tejedor, B. Alarcon, and F. Sanchez-Madrid. 2002. TCR engagement induces proline-rich tyrosine kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity and in an immunoreceptor tyrosine-based activation motif-mediated fashion. J Immunol 169:292-300. 304. Finkelstein, L. D., Y. Shimizu, and P. L. Schwartzberg. 2005. Tec kinases regulate TCR-mediated recruitment of signaling molecules and integrin-dependent cell adhesion. J Immunol 175:5923-5930. 305. Rodriguez-Fernandez, J. L., L. Sanchez-Martin, C. A. de Frutos, D. Sancho, M. Robinson, F. Sanchez-Madrid, and C. Cabanas. 2002. LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca(2)(+)-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells. J Leukoc Biol 71:520-530. 306. Wang, J. F., I. W. Park, and J. E. Groopman. 2000. Stromal cell-derived factor-1alpha stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C. Blood 95:2505-2513. 307. Zhang, X. F., J. F. Wang, E. Matczak, J. A. Proper, and J. E. Groopman. 2001. Janus kinase 2 is involved in stromal cell-derived factor-1alpha-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells. Blood 97:3342-3348. 308. Ganju, R. K., W. C. Hatch, H. Avraham, M. A. Ona, B. Druker, S. Avraham, and J. E. Groopman. 1997. RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes. J Exp Med 185:1055-1063. 309. Collins, M., M. Tremblay, N. Chapman, M. Curtiss, P. B. Rothman, and J. C. Houtman. 2009. The T cell receptor-mediated phosphorylation of Pyk2 tyrosines 402 and 580 occurs via a distinct mechanism than other receptor systems. J Leukoc Biol. 310. Astier, A., H. Avraham, S. N. Manie, J. Groopman, T. Canty, S. Avraham, and A. S. Freedman. 1997. The related adhesion focal tyrosine kinase is tyrosine- phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas. J Biol Chem 272:228-232. 311. Ganju, R. K., S. A. Brubaker, J. Meyer, P. Dutt, Y. Yang, S. Qin, W. Newman, and J. E. Groopman. 1998. The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J Biol Chem 273:23169-23175. 312. Mlinaric-Rascan, I., and T. Yamamoto. 2001. B cell receptor signaling involves physical and functional association of FAK with Lyn and IgM. FEBS Lett 498:26-31. 313. Glodek, A. M., Y. Le, D. M. Dykxhoorn, S. Y. Park, G. Mostoslavsky, R. Mulligan, J. Lieberman, H. E. Beggs, M. Honczarenko, and L. E. Silberstein. 2007. Focal adhesion kinase is required for CXCL12-induced chemotactic and pro-adhesive responses in hematopoietic precursor cells. Leukemia 21:1723-1732.   68 314. Durand, C. A., J. Westendorf, K. W. Tse, and M. R. Gold. 2006. The Rap GTPases mediate CXCL13- and sphingosine1-phosphate-induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes. Eur J Immunol 36:2235-2249. 315. Ozkal, S., J. C. Paterson, S. Tedoldi, M. L. Hansmann, A. Kargi, S. Manek, D. Y. Mason, and T. Marafioti. 2009. Focal adhesion kinase (FAK) expression in normal and neoplastic lymphoid tissues. Pathol Res Pract 205:781-788. 316. Glodek, A. M., M. Honczarenko, Y. Le, J. J. Campbell, and L. E. Silberstein. 2003. Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis. J Exp Med 197:461-473. 317. Le, Y., B. M. Zhu, B. Harley, S. Y. Park, T. Kobayashi, J. P. Manis, H. R. Luo, A. Yoshimura, L. Hennighausen, and L. E. Silberstein. 2007. SOCS3 protein developmentally regulates the chemokine receptor CXCR4-FAK signaling pathway during B lymphopoiesis. Immunity 27:811-823. 318. Zanin-Zhorov, A., G. Tal, S. Shivtiel, M. Cohen, T. Lapidot, G. Nussbaum, R. Margalit, I. R. Cohen, and O. Lider. 2005. Heat shock protein 60 activates cytokine- associated negative regulator suppressor of cytokine signaling 3 in T cells: effects on signaling, chemotaxis, and inflammation. J Immunol 175:276-285. 319. Stork, P. J., and T. J. Dillon. 2005. Multiple roles of Rap1 in hematopoietic cells: complementary versus antagonistic functions. Blood 106:2952-2961. 320. Bos, J. L., J. de Rooij, and K. A. Reedquist. 2001. Rap1 signalling: adhering to new models. Nat Rev Mol Cell Biol 2:369-377. 321. Bos, J. L. 2005. Linking Rap to cell adhesion. Curr Opin Cell Biol 17:123-128. 322. Raaijmakers, J. H., and J. L. Bos. 2009. Specificity in Ras and Rap signaling. J Biol Chem 284:10995-10999. 323. Boettner, B., and L. Van Aelst. 2009. Control of cell adhesion dynamics by Rap1 signaling. Curr Opin Cell Biol 21:684-693. 324. Bos, J. L., H. Rehmann, and A. Wittinghofer. 2007. GEFs and GAPs: critical elements in the control of small G proteins. Cell 129:865-877. 325. Spilker, C., G. A. Acuna Sanhueza, T. M. Bockers, M. R. Kreutz, and E. D. Gundelfinger. 2008. SPAR2, a novel SPAR-related protein with GAP activity for Rap1 and Rap2. J Neurochem 104:187-201. 326. McLeod, S. J., R. J. Ingham, J. L. Bos, T. Kurosaki, and M. R. Gold. 1998. Activation of the Rap1 GTPase by the B cell antigen receptor. J Biol Chem 273:29218-29223. 327. Jeong, H. W., Z. Li, M. D. Brown, and D. B. Sacks. 2007. IQGAP1 binds Rap1 and modulates its activity. J Biol Chem 282:20752-20762. 328. Bos, J. L., K. de Bruyn, J. Enserink, B. Kuiperij, S. Rangarajan, H. Rehmann, J. Riedl, J. de Rooij, F. van Mansfeld, and F. Zwartkruis. 2003. The role of Rap1 in integrin-mediated cell adhesion. Biochem Soc Trans 31:83-86. 329. Ishida, D., L. Su, A. Tamura, Y. Katayama, Y. Kawai, S. F. Wang, M. Taniwaki, Y. Hamazaki, M. Hattori, and N. Minato. 2006. Rap1 signal controls B cell receptor repertoire and generation of self-reactive B1a cells. Immunity 24:417-427. 330. Li, Y., J. Yan, P. De, H. C. Chang, A. Yamauchi, K. W. Christopherson, 2nd, N. C. Paranavitana, X. Peng, C. Kim, V. Munugalavadla, R. Kapur, H. Chen, W. Shou, J. C. Stone, M. H. Kaplan, M. C. Dinauer, D. L. Durden, and L. A. Quilliam. 2007. Rap1a null mice have altered myeloid cell functions suggesting distinct roles for the closely related Rap1a and 1b proteins. J Immunol 179:8322-8331.   69 331. Chu, H., A. Awasthi, G. C. White, 2nd, M. Chrzanowska-Wodnicka, and S. Malarkannan. 2008. Rap1b regulates B cell development, homing, and T cell- dependent humoral immunity. J Immunol 181:3373-3383. 332. McLeod, S. J., A. H. Li, R. L. Lee, A. E. Burgess, and M. R. Gold. 2002. The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor- 1 (CXCL12): potential role for Rap2 in promoting B cell migration. J Immunol 169:1365-1371. 333. Minato, N., and M. Hattori. 2009. Spa-1 (Sipa1) and Rap signaling in leukemia and cancer metastasis. Cancer Sci 100:17-23. 334. Kinashi, T., and K. Katagiri. 2005. Regulation of immune cell adhesion and migration by regulator of adhesion and cell polarization enriched in lymphoid tissues. Immunology 116:164-171. 335. Katagiri, K., A. Maeda, M. Shimonaka, and T. Kinashi. 2003. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat Immunol 4:741-748.      70 2. B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading1 2.1 Introduction  Antibodies (Abs) made by B-lymphocytes play a critical role in host defense against infection.  Antigen-induced signaling by the B cell receptor (BCR) initiates an activation program that leads to B cell proliferation and subsequent differentiation into Ab-producing cells.  BCR clustering by antigens, or by anti-immunoglobulin (anti-Ig) Abs used as surrogate antigens, initiates multiple signaling pathways that control gene expression, cell survival, and proliferation pathways (1-3). BCR signaling also promotes integrin activation (4, 5), localized actin polymerization, reorganization of the actin cytoskeleton, and changes in B cell morphology (6, 7), all of which may facilitate B cell activation.  Integrin activation and cell spreading is critical for the activation of B cells by membrane-bound antigens.  Macrophages, dendritic cells, and follicular dendritic cells (FDCs) can present arrays of captured antigens to B cells (8, 9) and this may be one of the main ways in which B cells encounter antigens (10).  BCR- induced integrin activation prolongs the interaction between the B cell and the antigen- presenting cell and also allows the B cell to spread on the surface of the antigen-presenting cell such that more BCRs can encounter and bind membrane-bound antigens (11). Subsequent contraction of the B cell membrane allows the B cells to gather the BCR-bound antigen into an immune synapse in which clustered antigen-engaged BCRs are surrounded by a ring of ligand-bound integrins.  Formation of this immune synapse reduces the amount of antigen that is required for B cell activation (12, 13). Recent work has shown that B cells in lymphoid organs may contact soluble antigens by extending membrane processes into a highly organized network of lymph-filled conduits  1 A version of this chapter has been published: Tse KW, Dang-Lawson M, Lee RL, Vong D, Bulic A, Buckbinder L, Gold MR. (2009).  B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading.  J Biol Chem.  284(34):22865-77    71 (14).  These conduits are created by fibroblastic reticular cells (FRCs) that partially ensheath collagen fibrils.  In addition to being rich in collagen, fibronectin, and other extracellular matrix (ECM) components, the FRCs that form these conduits express high levels of intercellular adhesion molecule-1 (ICAM-1), the ligand for the αLβ2 integrin (lymphocyte function-associated antigen-1, LFA-1) on B cells (10).  Thus B cells interacting with these conduits are likely to be in contact with integrin ligands and integrin-dependent spreading may enhance the ability of B cells to extend membrane processes into the FRC conduit. In addition to promoting cell spreading, integrins can act as co-stimulatory receptors that enhance signaling by many receptors including the T cell receptor (TCR) and the BCR (15-17).  Thus signaling proteins that regulate B cell spreading, and which are also targets of BCR/integrin co-stimulation, may play a key role in the activation of B cells by membrane- bound antigens as well as soluble antigens that are delivered to lymphoid organs by FRC conduits. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related non- receptor protein tyrosine kinases that integrate signals from multiple receptors and play an important role in regulating cell adhesion, cell morphology and cell migration in many cell types (18-20).  Integrins, receptor tyrosine kinases, antigen receptors, and G protein-coupled chemokine receptors all stimulate tyrosine phosphorylation of Pyk2 and FAK, modifications that increase the enzymatic activity of these kinases and allow them to bind SH2 domain- containing signaling proteins (21).  FAK, which is expressed in almost all tissues (21), is a focal adhesion component that mediates integrin-dependent cell migration (22), cell spreading, and cell adhesion (18) in adherent cells, as well as co-clustering of LFA-1 with the TCR in lymphocytes (23).  Pyk2 is expressed mainly in hematopoietic cells, osteoclasts, and the central nervous system (24) and is critical for chemokine-induced migration of B cells, macrophages and natural killer cells (20, 25, 26), as well as the spreading of osteoclasts on vitronectin (27).  FAK and Pyk2 are thought to mediate overlapping but distinct functions since Pyk2 expression only partially reverses the cell adhesion and migration defects in FAK-deficient fibroblasts (28). In B cells, clustering of the BCR, β1 integrins, or β7 integrins induces tyrosine phosphorylation of both Pyk2 and FAK (29-33).  FAK is involved in the chemokine-induced   72 adhesion of B cell progenitors (34) and Pyk2 is required for chemokine-induced migration of mature B cells (25).  However, the role of these kinases in BCR- and integrin-induced B cell spreading has not been investigated and the signaling pathways that link the BCR and integrins to tyrosine phosphorylation of Pyk2 and FAK have not been elucidated. We have previously shown that the ability of the BCR to induce integrin activation, B cell spreading, and immune synapse formation requires activation of the Rap GTPases (6, 17).  In addition to binding effector proteins such as RapL and RIAM that promote integrin activation (35-37), the active GTP-bound forms of Rap1 and Rap2 bind multiple proteins that control actin dynamics and cell morphology (38).  Moreover, we showed that BCR/integrin- induced phosphorylation of Pyk2 in B cells is dependent on Rap activation (17).  However this previous study did not address how Rap-GTP links the BCR and integrins to Pyk2 phosphorylation, whether Rap activation is important for FAK phosphorylation in B cells, or whether B cell spreading is regulated by Pyk2 or FAK.  We now show that Pyk2 and FAK are differentially expressed and localized in B cells, that Pyk2 and FAK are important for B cell spreading, and that integrin engagement enhances BCR-induced phosphorylation of Pyk2 and FAK, a process that depends on both Rap activation and actin dynamics.   73 2.2 Results 2.2.1 Expression and localization of Pyk2 and FAK in B cells Because Pyk2 and FAK regulate cell morphology in many cell types, we asked whether both of these kinases were expressed in mature B cells from mouse spleen.  Immunoblotting showed that resting B cells from mouse spleen expressed high levels of Pyk2 but only low levels of FAK (Figure 2.1A).  We also asked whether B cell activation altered the expression of Pyk2 or FAK since activated, but not resting, primary B cells undergo dramatic spreading when plated on integrin ligands or on immobilized Abs to CD44, CD23, or the BCR (39-41). Activating splenic B cells with LPS plus IL-4 for 2 days resulted in a 4- to 5-fold decrease in Pyk2 protein levels and a 6-fold increase in FAK levels (Figure 2.1A).  This likely reflects transcriptional regulation since a similar downregulation of pyk2 mRNA and upregulation of fak mRNA occurred upon B cell activation (Figure 2.1B).  A number of murine (WEHI-231, BAL17, A20) and human B lymphoma cell lines (Ramos, Daudi, Raji) expressed both Pyk2 and FAK (Figure 2.1A and Appendix A), consistent with that these cells representing transformed versions of activated B cells.  We also observed that Pyk2 from LPS/IL-4 activated B cells ran as a doublet on SDS-PAGE gels (Figure 2.1A and C).  The higher molecular weight form of Pyk2 may be the unspliced form that has been reported to be highly expressed in brain but not in the spleen (42).  Indeed, RT-PCR showed that both the spliced and unspliced forms of pyk2 mRNA were present in activated B cells whereas only the spliced form was present in resting B cells (Figure 2.1D).  Both isoforms of the Pyk2 protein were tyrosine phosphorylated upon BCR clustering in activated splenic B cells (Figure 2.1C). Confocal microscopy showed that Pyk2 and FAK had distinct subcellular localizations in B cells (Figure 2.1E).  In both resting and activated murine splenic B cells, Pyk2 was uniformly distributed in the cytoplasm with a diffuse pattern.  In contrast, FAK was present in punctate structures in both resting and activated splenic B cells (Figure 2.1E), as well as the A20 B cell line (data not shown).  Activated splenic B cells had more FAK-containing punctae than resting splenic B cells and overall, higher levels of FAK, consistent with the immunblotting data.  These punctate FAK-containing structures also contained LFA-1, and to some extent   74 VLA-4 (α4β1 integrin) (Figure 2.1G), suggesting that FAK associates constitutively with these integrins in B cells.   75  Figure 2.1 Expression and localization of Pyk2 and FAK in B cells. A, Pyk2 and FAK protein levels in cell lysates (40 µg protein) from A20 B lymphoma cells, resting splenic B cells, and splenic B cells that were activated with LPS plus IL-4 for 2 days were analyzed by sequential blotting with Abs to Pyk2, FAK, and Erk1/2 (loading control).  Molecular mass markers (in kDa) are indicated to the right of each blot.  The relative amount of Pyk2 or FAK protein in activated B cells, compared to resting B cells (=1), was determined by quantifying band intensities with ImageJ and normalizing the values to the amount of Erk in the sample.  The data represent the mean ± SEM for three independent experiments. B, The relative amounts of pyk2 and fak mRNA in resting and activated splenic B cells were determined by quantitative RT-PCR.  Values were normalized to the amount of gapdh mRNA in the same sample and are expressed as the amount of mRNA (average ± range for two independent experiments) relative to that in resting B cells (=1). C, Resting and LPS/IL-4-activated splenic B cells were left unstimulated for 30 min (-) or were incubated with 10 µg/ml anti-IgM Abs for 30 min. Anti-Pyk2 immunoprecipitates were analyzed by blotting with the 4G10 anti-P-Tyr antibody (upper panel).  The blots were then stripped and reprobed with an Ab to Pyk2 (lower panel).  One of two experiments that yielded similar results is shown. D, RT-PCR analysis of alternatively spliced pyk2 mRNA in resting splenic B cells and LPS/IL-4-activated B cells.  PCR primers flanking the alternatively spliced exon distinguish the full-length mRNA from the spliced isoform, which is 126 bp shorter.  Data are representative of three experiments with similar results. E&F, Resting and LPS/IL-4-activated splenic B cells were permeabilized and stained with goat anti-Pyk2 or goat anti-FAK plus Alexa488-conjugated secondary Ab.  Nuclei were visualized with DAPI (blue).  No fluorescence was observed when splenic B cells were stained with secondary Ab alone, or with non- specific goat IgG plus secondary Ab. G, LPS/IL-4 activated splenic B cells were permeabilized and stained with Abs to FAK plus Abs to either LFA-1 or VLA-4.  In E-G, each panel shows representative data from one of three experiments with similar results.   76 Figure 2.1       77 2.2.2 Adhesion to ECM enhances BCR-induced tyrosine phosphorylation of Pyk2 and FAK activation To examine the role of Pyk2 and FAK in BCR and integrin signaling in B cells, we used the A20 B lymphoma cell line, which expresses both Pyk2 and FAK.  Consistent with the idea that integrins can act as co-stimulatory receptors that enhance BCR signaling, we showed previously that BCR-induced tyrosine phosphorylation of Pyk2 is substantially greater when A20 cells are plated on a collagen/fibronectin ECM that contains integrin ligands than when the cells are stimulated in suspension (17) (see also Figure 2.2A).  As was the case for Pyk2, BCR-induced tyrosine phosphorylation of FAK was also substantially increased when A20 cells were plated on ECM (Figure 2.2A).  The binding of integrins to ECM ligands did not cause an overall enhancement of BCR signaling but selectively augmented BCR-induced tyrosine phosphorylation of FAK and Pyk2.  BCR-induced serine/threonine phosphorylation of Erk, Akt, and the cytoskeleton-associated adaptor protein paxillin was not enhanced by integrin engagement (Figure 2.2C and D).  The selective targeting of Pyk2 and FAK by BCR/integrin co-stimulation suggests that these kinases may be important for integrin- dependent B cell responses.   78 Figure 2.2 Adhesion of B cells to ECM selectively enhances BCR-induced tyrosine phosphorylation of Pyk2 and FAK.  A20 cells were kept in suspension or plated on collagen/fibronectin ECM for 30 min before being stimulated with 20 µg/ml soluble anti-IgG for the indicated times. For unstimulated controls (-), A20 cells were kept in suspension or plated on collagen/fibronectin ECM for 30 min and then left unstimulated for an additional 45 min before being lysed. A, Immunoprecipitated Pyk2 and FAK were analyzed by immunoblotting with the 4G10 anti-P-Tyr Ab. The blots were then reprobed with Abs to Pyk2 or FAK. B, A mock stimulation of A20 cells with PBS for 15 or 30 min did not increase phosphorylation of Pyk2 and FAK, compared to cells left unstimulated for the entire duration of the experiment. C, Cell lysates were immunoblotted with Abs against the phosphorylated forms of Erk (P-Erk) or Akt (P- Akt) and then reprobed with Abs against total Erk or Akt. D, Cell lysates were immunoblotted with a paxillin Ab.  Serine/threonine phosphorylation of paxillin is indicated by a bandshift on SDS-PAGE gels and was dependent on the activity of the Erk and GSK-3 kinases (data not shown), as in T cells and macrophages (43, 44).  For each panel, similar results were obtained in three experiments. αIgG, anti-IgG Ab; ECM, extracellular matrix   79 Figure 2.2    80 2.2.3 BCR/integrin-induced B cell spreading involves Pyk2 and FAK A20 cells spread dramatically when they are plated on fibronectin and then stimulated with anti-Ig Abs (6, 17).  In this scenario BCR signaling activates β1 integrins (e.g. VLA-4), which bind to the fibronectin, and the combined BCR/integrin signaling leads to cell spreading.  A20 cells also spread when plated on immobilized Abs that cluster the LFA-1 integrin, adopting a morphology similar to that of anti-Ig-activated A20 cells spreading on ICAM-1, the physiological ligand for LFA-1 (6).  This indicates that integrin signaling is sufficient to induce B cell spreading.  Because integrin signaling selectively enhances the ability of the BCR to induce tyrosine phosphorylation of Pyk2 and FAK (Figure 2.2), and can independently induce the phosphorylation of these kinases (see Figure 2.5), we asked whether Pyk2 or FAK played a role in B cell spreading. To test this, we used RNA interference to reduce the expression of Pyk2 or FAK in A20 cells.  We established stable bulk populations of A20 cells containing the GFP-encoding pGIPZ lentiviral vector or derivatives of this vector that also encode short hairpin RNAs (shRNAs) specific for either Pyk2 or FAK.  The resulting cell populations were >95% GFP+ (Figure 2.3A) and immunoblotting showed that the Pyk2 shRNA reduced the expression of Pyk2 by 83% without affecting FAK levels whereas the FAK shRNA reduced the expression of FAK by 67% without affecting Pyk2 levels (Figure 2.3B).  Knocking down the expression of either Pyk2 or FAK caused a 30-40% reduction in the number of A20 cells that developed a spread, elongated morphology when plated on fibronectin and then stimulated with anti-Ig Abs (Figure 2.3C).  The same was true when A20 cells were plated on immobilized anti- LFA-1 Abs (Figure 2.3C).  Thus Pyk2 and FAK both contribute to BCR/integrin- and integrin-induced B cell spreading in A20 cells.    81 Figure 2.3 A20 cell spreading involves both Pyk2 and FAK. Lentiviral transduction was used to establish stable bulk populations of A20 cells expressing the GFP- encoding pGIPZ vector or derivates encoding a Pyk2-specific shRNA or a FAK-specific shRNA. A, Representative FACS plots showing GFP expression by the transduced A20 cells (solid lines), compared to untransduced parental A20 cells (shaded curves).  The percent of transduced cells that were GFP+ is indicated. B, Immunoblot analysis of Pyk2 and FAK protein levels in cell lysates (40 µg protein per lane).  The Pyk2 and FAK blots were reprobed with Abs to actin (loading control). C, A20 cells transduced with the pGIPZ vector, Pyk2 shRNA, or FAK shRNA were plated on wells coated with 2.63 µg/cm2 fibronectin and then stimulated with 10 µg/ml soluble anti-IgG for 4 h (upper panels) or were plated on wells coated with 2.63 µg/cm2 anti-LFA-1 Abs for 4 h (bottom panels).  Representative images are shown. D, The percent of adherent A20 cells that had spread after 2 or 4 h, as indicated by being phase dark with an elongated or irregular shape, was determined.  The data are presented as the average + SEM for >300 cells counted in each of three experiments.  *, p <0.05, **, p <0.01 by Student’s one-tailed paired t-test, compared to vector control cells.  αIgG, anti-IgG Abs; αLFA-1, anti-LFA-1 Ab; FN, fibronectin.   82 Figure 2.3     83 2.2.4 BCR/integrin-induced tyrosine phosphorylation of Pyk2 and FAK depends on activation of the Rap GTPases Because activation of the Rap GTPases is critical for BCR- and integrin-induced B cell spreading (6, 17), and Pyk2 and  FAK contribute to this process, we hypothesized that Rap activation would be important for BCR-induced tyrosine phosphorylation of Pyk2 and FAK. The phosphorylation of Pyk2 and FAK on conserved tyrosine residues increases their kinase activity (21, 45).  The initial event in receptor-induced activation of these kinases is phosphorylation of Pyk2 on Y402 or FAK on Y397.  This is thought to occur via dimerization and transphosphorylation (46).  Src family kinases (SFKs) can then bind via their SH2 domain to the phosphorylated Pyk2 Y402 or FAK Y397 and phosphorylate Pyk2 at Y579/Y580 or FAK at Y576/Y577.  Phosphorylation of Pyk2 and FAK on these activation loop residues is required for maximal activity of these kinases towards substrates (21).  We showed previously that Rap activation is required for BCR/integrin-induced phosphorylation of Pyk2 on Y579/Y580 (17).  However, it was not known whether this reflected a role for Rap activation in Y402 phosphorylation or the SFK-mediated phosphorylation of Y579/Y580.  Moreover, the role of Rap activation in BCR/integrin-induced FAK phosphorylation had not been assessed. To address these questions, we blocked Rap activation in A20 cells by expressing the Rap- specific GTPase-activating protein, RapGAPII (47).  RapGAPII converts the Rap1 and Rap2 GTPases to their inactive GDP-bound state and RapGAPII expression has been widely used to assess the role of Rap activation (48, 49).  We have shown that RapGAPII expression completely blocks anti-Ig-, chemokine- and phorbol ester-induced Rap activation in A20 cells, without inhibiting other signaling reactions such as phosphorylation of MAP kinases or Akt (17, 50). Preventing Rap activation via RapGAPII expression significantly inhibited tyrosine phosphorylation of Pyk2 on Y402 when A20 cells were plated on ECM and stimulated with soluble anti-Ig antibodies (Figure 2.4A).  This corresponded with inhibition of total Pyk2 tyrosine phosphorylation, as assessed using anti-P-Tyr Abs (Figure 2.4A).  Thus BCR/integrin-induced phosphorylation of Pyk2 on Y402, the first step in Pyk2 activation is dependent on Rap activation.  The same was true for FAK.  The use of phosphorylation site-   84 specific Abs showed that blocking Rap activation significantly inhibited BCR/integrin- induced phosphorylation of FAK on Y397 (Figure. 2.4B) as well as the subsequent phosphorylation of FAK on Y576/Y577 (Figure 2.4C).  Consistent with this, the total tyrosine phosphorylation of FAK, as detected using the 4G10 anti-P-Tyr Ab was also inhibited when Rap activation was blocked (Figure 2.4B).  Thus during BCR/integrin co- stimulation, Rap activation is critical for the initial step in the activation of Pyk2 and FAK, phosphorylation of Pyk2 on Y402 and FAK on Y397.  As a consequence Rap activation is also required for the subsequent SFK-mediated phosphorylation of the activation loop tyrosine residues of Pyk2 and FAK. The requirement for Rap activation in BCR/integrin co-stimulation-induced phosphorylation of Pyk2 and FAK could reflect a role for Rap activation in one or more of the following processes: coupling BCR signaling pathways to the phosphorylation of Pyk2 and FAK, activating integrins such that ligand binding initiates outside-in integrin signaling, or coupling integrin signaling pathways to the phosphorylation of Pyk2 and FAK.  We have previously shown that Rap activation is essential for the BCR to stimulate integrin activation (17).  Therefore we now investigated whether Rap activation was also an essential component of the signaling pathways that link the BCR and integrins to the phosphorylation of Pyk2 and FAK.  Since integrin engagement greatly enhances BCR-induced phosphorylation of Pyk2 and FAK, we first tested the hypothesis that integrin signaling induces Pyk2 and FAK phosphorylation in a Rap-dependent manner.    85 Figure 2.4 BCR/integrin-induced tyrosine phosphorylation of Pyk2 and FAK depends on activation of the Rap GTPases.  Vector control and RapGAPII-expressing A20 cells were cultured for 30 min in wells coated with collagen/fibronectin ECM before being stimulated with 20 µg/ml anti-IgG for the indicated times.  For unstimulated controls (-), A20 cells were plated on collagen/fibronectin ECM for 30 min and then left unstimulated for another 30 min before being lysed. A, Anti-Pyk2 immunoprecipitates were probed with an Ab against Pyk2 that is phosphorylated on Y402 (anti-pY402), or with the 4G10 anti-P-Tyr Ab, before being reprobed with an anti-Pyk2 Ab. B, Anti-FAK immunoprecipitates were probed with an Ab that recognizes FAK that is phosphorylated on Y397 (anti-pY397), or with the 4G10 anti-P-Tyr Ab, before being reprobed with an anti-FAK Ab. C, Anti-FAK immunoprecipitates were probed sequentially with Abs that recognize FAK that is phosphorylated on either Y576 or Y577 before being reprobed with an anti-FAK Ab.  The relative levels of Pyk2 and FAK phosphorylation were determined by quantifying band intensities using ImageJ, normalizing the values to the total amount of Pyk2 or FAK in the same lane, and expressing the values (mean ± SEM for three experiments) relative to the Pyk2 or FAK phosphorylation levels in unstimulated vector control cells (= 1).  *, p <0.05 by Student’s one-tailed paired t-test.   86 Figure 2.4    87 2.2.5 Rap activation is important for integrin-induced phosphorylation of Pyk2 and FAK In order to initiate integrin signaling without stimulating the cells through the BCR, we plated A20 cells on wells coated with Abs against the LFA-1 or VLA-4 integrins.  We have previously shown that the ability of A20 cells to spread on immobilized anti-integrin Abs, or on immobilized ICAM-1, is dependent on Rap activation (6).  Moreover, Ab-induced clustering of LFA-1 activates Rap1 in A20 cells (6).  Figure 2.5A shows that plating A20 cells on wells coated with Abs to LFA-1 or VLA-4 resulted in increased Pyk2 phosphorylation, compared to cells plated on wells coated with an isotype-matched control monoclonal Ab against CD40.  Both LFA-1- and VLA-4-induced Pyk2 phosphorylation were substantially reduced in the RapGAPII-expressing A20 cells in which Rap activation was blocked (Figure 2.5A).  Similarly, FAK phosphorylation, which was increased 3- to 4-fold by clustering VLA-4, and to a lesser extent by clustering LFA-1, was significantly reduced when Rap activation was blocked (Figure 2.5B).  Thus Rap activation is required for integrin signaling to induce tyrosine phosphorylation of Pyk2 and FAK.   88    Figure 2.5 Rap activation is important for integrin-induced phosphorylation of Pyk2 and FAK.  Vector control and RapGAPII-expressing A20 cells were plated in wells coated with 3.5µg/cm2 anti-CD40 (control), anti-LFA-1, or anti-VLA-4 monoclonal Abs for 15 or 30 min.  Anti-Pyk2 (A) and anti-FAK (B) immunoprecipitates were analyzed by blotting with the 4G10 anti-P-Tyr Ab.  The blots were then stripped and reprobed with Abs against Pyk2 or FAK.  For FAK phosphorylation, band intensities were normalized to the amount of total FAK for each sample and then expressed as the relative phosphorylation (mean ± SEM for three experiments) compared to that for vector control cells plated on anti-CD40 (= 1).  *, p <0.05 by Student’s one-tailed paired t-test.    89 2.2.6 Rap activation is important for BCR-induced phosphorylation of Pyk2, but not for BCR-induced phosphorylation of FAK When A20 cells were stimulated with anti-Ig Abs while in suspension, BCR clustering induced tyrosine phosphorylation of both Pyk2 and FAK, although to a much lesser extent than when the cells were plated on ECM (Figure 2.2).  Since integrin engagement is likely to be minimal when the cells are in suspension, this may reflect integrin-independent BCR signaling events.  Therefore we asked whether Rap activation was important for integrin- independent phosphorylation of Pyk2 and FAK by the BCR.  When we kept vector control and RapGAPII-expressing A20 cells in suspension and stimulated them with soluble anti-Ig Abs, blocking Rap activation completely abrogated BCR-induced phosphorylation of Pyk2 at Y402 and Y579/Y580 (Figure 2.6A).  In contrast, blocking Rap activation did not impair the ability of the BCR to increase tyrosine phosphorylation of FAK, as judged using the 4G10 anti-P-Tyr Ab, or more specifically, phosphorylation of FAK at Y397 (Figure 2.6B).  Thus both BCR-induced (Figure 2.6A) and integrin-induced (Figure 2.5A) Pyk2 phosphorylation required Rap activation whereas integrin-induced FAK phosphorylation was dependent on Rap activation (Figure 2.5B) but BCR-induced FAK phosphorylation was Rap-independent (Figure 2.6B).   90   Figure 2.6 Rap activation is important for BCR-induced tyrosine phosphorylation of Pyk2 but not FAK.  Vector control and RapGAPII-expressing A20 cells were stimulated in suspension with 20 µg/ml anti-IgG for the indicated times.  For unstimulated controls (-), A20 cells were left in suspension for 30 min without being stimulated. A, Anti-Pyk2 immunoprecipitates were probed with either the 4G10 anti-P-Tyr Ab, an Ab against Pyk2 that is phosphorylated on Y402, or an Ab against Pyk2 that is phosphorylated on Y579/Y580.  The blots were then stripped and reprobed with a Pyk2 Ab. B, Anti-FAK immunoprecipitates were probed with either the 4G10 anti-P-Tyr Ab or an Ab against FAK that is phosphorylated on Y397.  The blots were then stripped and reprobed with a FAK Ab.  Band intensities were normalized to the amount of total Pyk2 or FAK for each sample and then expressed as the relative phosphorylation (mean ± SEM for three experiments) compared to that for unstimulated vector control cells (= 1).  *, p <0.05 by Student’s one-tailed paired t-test.  The values for FAK phosphorylation in vector and RapGAPII-expressing cells were not significantly different by this test.   91 2.2.7 The role of Rap activation in the phosphorylation of Pyk2 and FAK corresponds to a requirement for actin dynamics Because Rap activation is required for maximal BCR-induced increases in polymerized F- actin in A20 cells (17), we hypothesized that Rap might regulate the phosphorylation of Pyk2 and FAK via its ability to promote actin polymerization or stabilize actin filaments.   To test this, we pre-treated A20 cells with latrunculin A, a drug that prevents the addition of actin monomers to existing actin filaments, thereby leading to a loss of F-actin.  Confocal microscopy showed that a 30 min treatment with latrunculin A led to a nearly complete loss of F-actin in A20 cells (data not shown).  In the presence of latrunculin A, anti-Ig-induced phosphorylation of Pyk2 at Y402 and Y579/Y580 was almost completely blocked, both when the cells were stimulated in suspension and when they were stimulated while on ECM (Figure 2.7A).  Similar results were obtained using cytochalasin D (data not shown), another drug that leads to the loss of F-actin.  An intact actin cytoskeleton was not required for other BCR signaling events such as phosphorylation of Erk or Akt (Figure 2.7B).  Importantly, latrunculin A did not block BCR-induced Rap1 activation (Figure 2.7C), consistent with the idea that F-actin acts downstream of Rap activation to promote Pyk2 phosphorylation.   92 Figure 2.7 Rap-dependent phosphorylation of Pyk2 and FAK requires actin dynamics.  A20 cells in suspension or plated on ECM were pre-treated with 10 µM latrunculin A or an equivalent volume of DMSO for 30 min before being stimulated with 20 µg/ml soluble anti-IgG for the indicated times.  For unstimulated controls (-), A20 cells were kept in suspension or plated on collagen/fibronectin ECM for 30 min and then left unstimulated for another 30 min before being lysed. A, Anti-Pyk2 immunoprecipitates were sequentially probed with an Ab that recognizes Pyk2 that is phosphorylated at Y402, an Ab that recognizes Pyk2 that is phosphorylated at Y579/Y580, the 4G10 anti- P-Tyr Ab, and an anti-Pyk2 Ab.  B, Cell lysates were assayed for phosphorylation of Akt and Erk as in Figure 2.2C &D. C, A GST-RalGDS fusion protein was used to selectively precipitate the active GTP-bound form of Rap1, which was detected by immunoblotting with a Rap1 Ab (upper panel).  The lower panel shows the amount of Rap1 in the cell lysates. D, Tyrosine phosphorylation of FAK was assessed by blotting anti-FAK immunoprecipitates with the 4G10 anti-P-Tyr Ab and then reprobing with an anti-FAK Ab.  FAK phosphorylation (mean ± SEM for three experiments) relative to that in unstimulated DMSO-treated cells kept in suspension (= 1) is graphed.  *, p <0.05 by Student’s one-tailed paired t-test. E, A20 cells in suspension were pre-treated with 1 µM jasplakinolide or an equivalent volume of DMSO for 30 min before being stimulated with 20 µg/ml anti-IgG for the indicated times or being left unstimulated for 30 min (-).  Pyk2 and FAK tyrosine phosphorylation, as well as Rap1 activation, was assessed.  For each panel, similar results were obtained in three experiments.  Lat A, latrunculin A; Jas, jasplakinolide.   93 Figure 2.7    94 For FAK phosphorylation, the requirement for an intact actin cytoskeleton paralleled the requirement for Rap activation.  When A20 cells were stimulated with anti-Ig while in suspension, BCR-induced FAK phosphorylation was unaffected by blocking Rap activation (Figure 2.6B) or by disrupting the actin cytoskeleton with latrunculin A (Figure 2.7D).  In contrast, when the cells were stimulated while on ECM, BCR/integrin-induced FAK phosphorylation was significantly reduced by disrupting the actin cytoskeleton with latrunculin A (Figure 2.7D) and by blocking Rap activation (Figure 2.4B).  Thus a Rap- and F-actin-dependent pathway links integrins, but not the BCR, to FAK phosphorylation. The ability of latrunculin A and cytochalasin D to block BCR-induced Pyk2 phosphorylation could reflect a requirement for actin filaments, which may act as signaling platforms, or a requirement for the dynamic assembly and disassembly of actin filaments.  To distinguish these possibilities, we used jasplakinolide, a drug that prevents actin filament disassembly (51).  When A20 cells were stimulated in suspension, jasplakinolide treatment completely inhibited BCR-induced phosphorylation of Pyk2 while having no effect on BCR-induced Rap1 activation (Figure 2.7E).  Thus both disrupting actin filaments and stabilizing actin filaments inhibited the Rap-dependent phosphorylation of Pyk2 by the BCR.  In contrast, BCR-induced phosphorylation of FAK in cells that were kept in suspension, did not require Rap activation and was unaffected by either latrunculin A (Figure 2.7D) or jasplakinolide (Figure 2.7E).  2.2.8 B cell spreading requires Pyk2/FAK kinase activity We have shown that Rap activation is important for BCR/integrin-induced tyrosine phosphorylation of Pyk2 and FAK (Figures 2.4 and 2.5) and for BCR- and integrin-induced B cell spreading (6, 17).  This suggests that activated Rap may promote B cell spreading, at least in part, by facilitating the phosphorylation-dependent activation of Pyk2 and FAK. Indeed, knocking down the expression of either Pyk2 or FAK reduced B cell spreading (Figure 2.3C).  To specifically address the role of Pyk2 and FAK kinase activity in BCR/integrin-induced B cell spreading, we used PF-431396, a potent and highly selective pyrimidine-based inhibitor of both Pyk2 and FAK (52).  Consistent with the idea that the tyrosine phosphorylation of Pyk2 and FAK involves an initial autophosphorylation or   95 transphosphorylation step, treating A20 cells with PF-431396 blocked anti-Ig-induced tyrosine phosphorylation of Pyk2 and FAK when the cells were stimulated in suspension (Figure 2.8A) and when they were stimulated on ECM (Figure 2.8B).  The phosphorylation of Pyk2 and FAK induced by clustering LFA-1 with plate-bound Abs was also inhibited by PF-431396 (Figure 2.8B).  PF-431396 treatment was not cytotoxic, as judged by 7-AAD staining (data not shown), and did not reduce the ability of the BCR to stimulate Erk phosphorylation or overall protein tyrosine phosphorylation (Figure 2.8A), which is dependent on the activation of both SFKs and the Syk tyrosine kinase.  Thus, PF-431396 appeared to selectively inhibit BCR-induced tyrosine phosphorylation of Pyk2 and FAK. Importantly, this correlated with a significant inhibition of A20 cell spreading.  PF-431496 treatment significantly reduced the number of A20 cells that developed an elongated, spread morphology after being stimulated with anti-Ig Abs while on a fibronectin (Figure 2.8C). The spreading of A20 cells plated on immobilized anti-LFA-1 Abs was also significantly reduced by PF-431396 treatment (Figure 2.8D).  Thus the kinase activity of Pyk2 and/or FAK is required for both BCR/integrin- and integrin-induced B cell spreading.    96 Figure 2.8 An inhibitor of Pyk2/FAK activity blocks B cell spreading. A, A20 cells in suspension were treated with the indicated concentrations of PF-431396 or with DMSO for 45 min before being stimulated with 20 µg/ml anti-IgG for 20 min.  For unstimulated controls (-), A20 cells were kept in suspension for 45 min and then left unstimulated for another 20 min before being lysed.  Pyk2 and FAK immunoprecipitates were analyzed by blotting with the 4G10 anti-P-Tyr Ab (left panel) before being reprobed with Abs against Pyk2 or FAK.  The same cell lysates were analyzed for total tyrosine phosphorylation using the 4G10 anti-P-Tyr Ab and for Erk phosphorylation (right panel). B, A20 cells were treated with PF-431396 or DMSO for 45 min.  The cells were then added to fibronectin/collagen ECM-coated wells and stimulated for 30 min with soluble anti-Ig.  Alternatively, the cells were added to wells coated with 2.63 µg/cm2 anti-LFA-1 Abs for 30 min.  Pyk2 and FAK immunoprecipitates were analyzed by blotting with the 4G10 anti-P-Tyr Ab. C, A20 cells were plated on wells coated with 2.63 µg/cm2 fibronectin and stimulated with 10 µg/ml anti- IgG in the presence of DMSO or 2.5 µM PF-431396 for 1, 2, or 4 h.  Representative images of the 4 h time point are shown. D, A20 cells were plated on wells coated with 2.63 µg/cm2 anti-LFA-1 Ab in the presence of DMSO or 2.5 µM PF-431396 for 1 h or 3 h.  Representative images of the 3 h time point are shown.  Cell surface expression of LFA-1 was not affected by PF-431396 treatment (data not shown).  The percent of adherent A20 cells that had spread (mean ± SEM for >150 cells counted in each of three experiments), as indicated by being phase dark with an elongated or irregular shape, was determined for each time point.  *, p <0.05 by Student’s one-tailed paired t-test, compared to DMSO-treated cells.   97 Figure 2.8     98 2.3 Discussion The binding of antigens by B cells often occurs in the context of integrin engagement. Integrin-dependent cell spreading enhances the ability of B cells to contact antigens and integrin signaling may synergize with BCR signaling to promote both B cell spreading and activation.  The Pyk2 and FAK kinases are key regulators of cell morphology and in this report we showed that the kinase activities of Pyk2 and FAK are important for BCR/integrin- induced B cell spreading.  Moreover, we showed that integrins enhance the ability of the BCR to phosphorylate Pyk2 and FAK on their auto/transphosphorylation sites, the initial step in the activation of these kinases.  Finally, we showed that both Rap activation and actin dynamics were critical for BCR/integrin-induced phosphorylation of Pyk2 and FAK. We had previously shown that integrin engagement enhances BCR-induced Pyk2 phosphorylation (17) and we have now shown that the same is true for FAK phosphorylation. Moreover, by clustering integrins with Abs, we showed that integrin signaling was sufficient to induce tyrosine phosphorylation of Pyk2 and FAK in B cells.  Thus signaling by antigen- clustered BCR complexes and ligand-bound integrins can have additive effects on the phosphorylation of Pyk2 and FAK.  This highlights the ability of integrins to act as co- stimulatory receptors that collaborate with lymphocyte antigen receptors.  Pyk2 and FAK appeared to be selective targets of the BCR/integrin collaboration as integrin engagement did not enhance BCR-induced phosphorylation of other signaling proteins such as Erk, Akt and paxillin. We also showed that activation of the Rap GTPases was critical for BCR/integrin signaling to induce the phosphorylation of Pyk2 and FAK on their auto/transphosphorylation sites as well as tyrosine residues in their activation loops.  Although Rap-GTP likely contributes to Pyk2 and FAK phosphorylation by activating integrins on B cells (17), we found that activated Rap also acts downstream of the BCR to promote Pyk2 phosphorylation and downstream of integrins to promote the phosphorylation of Pyk2 and FAK.  The active GTP-bound form of Rap binds multiple effector proteins that promote actin polymerization and the stabilization of F-actin polymers (38).  Many of the downstream consequences of Rap activation may therefore reflect its role in reorganization of the actin cytoskeleton.   99 Indeed, we found that the Rap-dependent steps in Pyk2 and FAK phosphorylation were also blocked by actin-disrupting drugs.  This suggests that Rap-GTP promotes Pyk2 and FAK phosphorylation via its ability to remodel the actin cytoskeleton.  Rap1 activation was not dependent on actin dynamics, suggesting that the requirement for actin remodeling lies downstream of Rap activation. Although Pyk2 phosphorylation has been shown to require an intact actin cytoskeleton in a number of cell types (21), how this contributes to Pyk2 phosphorylation is not clear.  Phosphorylation of Pyk2 on Y402 may involve Pyk2 dimerization and subsequent transphosphorylation (46).  Rap-dependent actin polymerization could create a cytoskeletal platform that promotes Pyk2 dimerization.  However, we found that treating B cells with the actin stabilizing agent jasplakinolide also prevented tyrosine phosphorylation of Pyk2, indicating that polymerized F-actin is not sufficient to support receptor-induced Pyk2 phosphorylation.  Dynamic remodeling of the actin cytoskeleton may be required for efficient Pyk2 dimerization.  Alternatively, Pyk2-dependent phosphorylation in vivo may require cycles of actin polymerization and depolymerization that regulate either the kinase activity of Pyk2 or the accessibility of its catalytic site. In contrast to Pyk2, Rap activation and actin dynamics were required for integrin- induced FAK phosphorylation but not for BCR-induced FAK phosphorylation in A20 B lymphoma cells.  For integrin-induced FAK phosphorylation, Rap activation was required for the initial step in FAK activation, phosphorylation of Y397, an event that is initiated by transphosphorylation and which can be amplified by SFK (53).  How Rap activation and F- actin contribute to integrin-induced FAK Y397 phosphorylation is not clear.  Our microscopy data suggest that FAK constitutively co-localizes with integrins in B cells.  Rap activation and actin polymerization could therefore contribute to the recruitment and/or stabilization of other proteins that regulate FAK Y397 phosphorylation.  In adherent cells that form focal adhesions, integrin activation results in the recruitment of talin to the integrin α and β chain cytoplasmic domains (54).  This allows FAK to interact with paxillin and undergo autophosphorylation.  At the same time, activation of SFKs by PTPα increases the phosphorylation of FAK at Y397.  Further work is required to determine if Rap and F-actin promote integrin-dependent FAK phosphorylation by regulating these steps in B cells.   100 Interestingly, Rap activation and F-actin were not required for BCR-induced phosphorylation of FAK when the cells were in suspension, a situation in which there is minimal integrin engagement.  FAK has been reported to associate constitutively with the SFK Lyn and with the BCR in WEHI-231 B lymphoma cells (33).  FAK phosphorylation could therefore be a proximal Rap-independent BCR signaling event that is initiated by BCR. A key finding was that Pyk2 and FAK are important for B cell spreading that is initiated by BCR/integrin co-stimulation or by integrin clustering.  This is consistent with Pyk2 and FAK being downstream targets of Rap, since blocking Rap activation also prevents B cell spreading (6, 17).  Knocking down the expression of either Pyk2 or FAK in A20 B lymphoma cells reduced the ability of these cells to undergo cell spreading whereas PF- 431396, a dual specificity inhibitor of the kinase activities of both Pyk2 and FAK, substantially inhibited A20 cell spreading.  This suggests that both Pyk2 and FAK contribute to the ability of A20 B lymphoma cells to undergo cell spreading.  Moreover, the use of PF- 431396 showed that the kinase activities of Pyk2 and FAK were critical for B cell spreading. Although it is not known how Pyk2 and FAK promote B cell spreading, these kinases may coordinate the activation of Rac, Cdc42, and RhoA, GTPases that control cytoskeletal organization.  In T lymphocytes, Pyk2 binds Vav (45), an exchange factor that activates Rac. Both Pyk2 and FAK can interact with the RhoA activator p190RhoGEF (58) and in fibroblasts Pyk2 associates with Wrch1, a Cdc42-like GTPase that promotes the formation of filopodia (55).  Pyk2 and FAK can also bind the p85 subunit of phosphoinositide 3-kinase following integrin ligation (56, 57).  Phosphatidylinositol 3,4,5-trisphosphate produced by phosphoinositide 3-kinase activates Vav and promotes Rac-dependent actin polymerization and cytoskeletal rearrangement.  Pyk2 and FAK can also bind and phosphorylate the scaffolding proteins p130Cas and paxillin, which can then recruit the Rac activators DOCK180 and PIX, leading to Rac-dependent membrane ruffling (57). An interesting observation was that when B cells were activated with LPS plus IL-4, Pyk2 levels decreased but FAK levels increased significantly.  B cells activated in this manner resemble antigen-activated germinal center (GC) B cells, which proliferate within lymphoid organ follicles and undergo somatic hypermutation of their Ig genes.  These GC B cells then compete for limiting amounts of antigen that are displayed on the surface of FDCs,   101 which provide the B cells with survival signals.  GC B cells interacting with FDCs in vivo exhibit a spread morphology with multiple membrane processes (58, 59).  This presumably increases their ability to detect antigens on the surface of the FDC.  The activation-induced increase in FAK expression may reflect a switch from the motile phenotype of a circulating B cell to the more adhesive phenotype of an activated GC B cell.  FAK expression and activation is associated with sustained adhesion, at least in B cell progenitors (34).  A number of adhesion molecules including α6 integrin are upregulated in activated GC B cells (60, 61), and gene expression profiling has shown that fak mRNA levels are elevated in GC B cells (62).  Thus, the increased expression of FAK after B cell activation may be part of a pro- adhesion gene expression program in which FAK promotes integrin-dependent adhesion and cell spreading, which facilitates BCR-antigen interactions that provide survival signals for GC B cells.  Similarly, the change in Pyk2 mRNA splicing in activated B cells may allow Pyk2 to interact with additional proteins that control cell adhesion or cytoskeletal reorganization. In summary, we have shown that Pyk2 and FAK are downstream targets of the Rap GTPases that play an important role in B cell spreading, a process that contributes to B cell activation.    102 2.4 Materials and methods  2.4.1 Antibodies and inhibitors Goat and donkey anti-mouse IgG, as well as goat anti-mouse IgM, were from Jackson ImmunoResearch Laboratories (West Grove, PA).  Rat monoclonal anti-LFA-1 (anti-αL integrin) and anti-CD40 (1C10) were from eBioscience (San Diego, CA).  A rat monoclonal Ab against very late antigen-4 (anti-VLA-4; anti-α4 integrin) was purified from culture supernatants of the PS/2 hybridoma (63) (from Dr. Bosco Chan, Univ. of Western Ontario, London, Ontario, Canada).  Rabbit anti-Erk, goat anti-Pyk2 (sc-1514), and goat anti-FAK (C- 20) were from Santa Cruz Biotechnology (Santa Cruz, CA).  The 4G10 monoclonal anti- phosphotyrosine (P-Tyr) Ab was from Upstate (Charlottesville, VA).  Abs to Y397-, Y576-, and Y577-phosphorylated FAK and Y579/Y580-phosphorylated Pyk2 were from Biosource International (Camarillo, CA).   The rabbit polyclonal Ab against Y402-phosphorylated Pyk2, the murine monoclonal Ab against phosphorylated Erk, and the rabbit monoclonal Ab against S473-phosphorylated Akt were from Cell Signaling Technology (Danvers, MA).  The monoclonal Ab to paxillin was from BD Biosciences (Mississauga, Ontario, Canada). Horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz), goat anti-rabbit IgG (Bio-Rad, Hercules, CA), and goat anti-mouse IgG (GE Healthcare Bio-Sciences, Baie d’Urfe, Quebec, Canada) were used for immunoblotting.  Latrunculin A and jasplakinolide were from Calbiochem (La Jolla, CA).  PF-431396 has been described previously (52).  The pCMV-δR8.91 and pCMV-VSV-G-M5 plasmids were a gift from Dorothee von Laer (Georg-Speyer Haus Chemotherapeutic Institute, Frankfurt, Germany). 2.4.2 Cells B cells were isolated from the spleens of C57BL/6 mice using the MACS B cell isolation kit (Miltenyi Biotec, Auburn, CA) to deplete non-B cells (64).  The resulting cells were >98% B cells, as determined by staining with anti-CD19-FITC (BD Pharmingen).  Activated B cells were obtained by culturing splenic B cells with 25 µg/ml lipopolysaccharide (LPS, Sigma- Aldrich, St. Louis, MO) plus 5 ng/ml IL-4 (R&D Systems, Minneapolis, MN) for 2-3 days. A20 cells (ATCC, Manassas, VA) were maintained as previously described (17).  Bulk   103 populations of A20 cells stably transduced with the empty pMSCVpuro vector (BD Biosciences Clontech, Mountain View, CA) or with pMSCVpuro/RapGAPII have been described previously (17). 2.4.3 Expression of Pyk2 and FAK For immunoblotting with Abs to Pyk2 or FAK, cells were solubilized in RIPA buffer (50). For quantitative RT-PCR, RNA was prepared using the RNAeasy kit with QIAshredder columns (Qiagen, Valencia, CA) and converted into cDNA using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA).  Equivalent amounts of cDNA were combined with TaqMan Fast Universal PCR Master Mix (Applied Biosystems) plus TaqMan Gene Expression Assay primers and probes (Applied Biosystems) specific for Pyk2 (Mm00552840_m1), FAK (Mm00433209_m1), or GAPDH (Mm99999915_g1).  PCR reactions and quantitation were performed using an Applied Biosystems 7500 Fast Real- Time PCR system.  The amount of pyk2 or fak mRNA was normalized to the amount of gapdh mRNA for each sample. 2.4.4 Immunofluorescence Cells were fixed with 3% paraformaldehyde for 20 min and then permeabilized with phosphate-buffered saline (PBS) plus 0.1% Tween-20 for 45 min.  After blocking with PBS containing 2% bovine serum albumin for 10 min, the cells were stained with goat Abs to Pyk2 or FAK for 45 min, followed by Alexa488-conjugated donkey anti-goat IgG (Molecular Probes-Invitrogen, Eugene, OR) for 30 min.  Where indicated, cells were also stained with rat monoclonal Abs to LFA-1 or VLA-4, followed by Alexa568-conjugated donkey anti-rat IgG (Molecular Probes-Invitrogen).  The cells were washed and adhered to poly-L-lysine-coated coverslips, which were treated with Prolong Gold anti-fade reagent containing DAPI (Molecular Probes-Invitrogen) and mounted onto glass slides.  Images were collected using an Olympus IX81/Fluoview1000 confocal microscope and processed using Olympus Fluoview 1.6 software. 2.4.5 Phosphorylation of Pyk2, FAK, ERK, Akt, and Paxillin A20 cells or splenic B cells (1.5 x 107) in 1 ml modified HEPES-buffered saline (64) were stimulated with anti-Ig Abs either while in suspension or 30 min after being added to wells of   104 6-well tissue culture plates coated with a collagen/fibronectin ECM (17, 65).  This ECM was generated by sequentially coating the wells with a 2% gelatin solution and then fetal calf serum.  To initiate integrin signaling, cells were added to wells that had been coated with Abs to LFA-1 or VLA-4, as described (6).  Reactions were terminated by adding 0.25 ml of cold 5X lysis buffer (17).  After 10 min on ice, insoluble material was removed by centrifugation. Where indicated, aliquots of cell lysate were removed to assess total protein tyrosine phosphorylation or the phosphorylation of Erk, Akt, paxillin.  Pyk2 and FAK were immunoprecipitated from cell lysates as described (34,41). 2.4.6 Short hairpin RNA (shRNA)-mediated knockdown of Pyk2 and FAK expression in A20 cells pGIPZ lentiviral vectors encoding GFP as well as shRNAmir’s specific for murine Pyk2 (catalogue no. V2LMM_21947) or FAK (catalogue no. V2LMM_37327) were purchased from Open Biosystems (Huntsville, AL).   Lentiviruses were generated by transfecting 293T cells with the appropriate lentiviral vector (7.5 µg) together with 12.5 µg of pCMV-δR8.91 and 2 µg of pCMV-VSV-G-M5 (66, 67).  Viral supernatants were collected 24 and 48 h after transfection and filtered through a 0.45 µm filter.  A20 cells (6 x 105) were added to wells of a 6-well dish containing 3 ml of viral supernatant and then centrifuged at 2000 rpm for 1 h at 21oC.  Cells were cultured with 5 µg/ml puromycin to select for transduced cells. 2.4.7 Cell spreading Tissue-culture plates were coated overnight at 4oC with a rat anti-mouse LFA-1 monoclonal Ab (6), or with fibronectin (R&D Systems, Minneapolis, MN), and then blocked with PBS containing 2% bovine serum albumin for 1 h.  A20 cells (105 cells in 0.5 ml RPMI-1640 with 2% FCS and 50 µM 2-mercaptoethanol) were pre-treated with DMSO or PF-431396 for 45 min, added to the coated wells, and incubated at 37oC.  Cells scored as spread were phase dark and had an elongated or irregular shape with obvious membrane processes. 2.4.8 Rap activation Rap activation assays were performed as described (17).  A GST-RalGDS fusion protein was used to selectively precipitate the active GTP-bound form of Rap, which was detected by immunoblotting with a Rap1 Ab (Santa Cruz Biotechnology).   105 2.5 References 1. Dal Porto, J. M., S. B. Gauld, K. T. Merrell, D. Mills, A. E. Pugh-Bernard, and J. Cambier. 2004. B cell antigen receptor signaling 101. Mol Immunol 41:599-613. 2. Kurosaki, T. 2002. Regulation of B-cell signal transduction by adaptor proteins. Nat Rev Immunol 2:354-363. 3. Niiro, H., and E. A. Clark. 2002. Regulation of B-cell fate by antigen-receptor signals. Nat Rev Immunol 2:945-956. 4. Spaargaren, M., E. A. Beuling, M. L. Rurup, H. P. Meijer, M. D. Klok, S. Middendorp, R. W. Hendriks, and S. T. Pals. 2003. The B cell antigen receptor controls integrin activity through Btk and PLCgamma2. J Exp Med 198:1539-1550. 5. Harris, E. S., T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 2000. The leukocyte integrins. J Biol Chem 275:23409-23412. 6. Lin, K. B., S. A. Freeman, S. Zabetian, H. Brugger, M. Weber, V. Lei, M. Dang- Lawson, K. W. Tse, R. Santamaria, F. D. Batista, and M. R. Gold. 2008. The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands. Immunity 28:75-87. 7. Westerberg, L., G. Greicius, S. B. Snapper, P. Aspenstrom, and E. Severinson. 2001. Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes. Blood 98:1086-1094. 8. Bergtold, A., D. D. Desai, A. Gavhane, and R. Clynes. 2005. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23:503-514. 9. Qi, H., J. G. Egen, A. Y. Huang, and R. N. Germain. 2006. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312:1672-1676. 10. Bajenoff, M., J. G. Egen, L. Y. Koo, J. P. Laugier, F. Brau, N. Glaichenhaus, and R. N. Germain. 2006. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25:989-1001. 11. Fleire, S. J., J. P. Goldman, Y. R. Carrasco, M. Weber, D. Bray, and F. D. Batista. 2006. B cell ligand discrimination through a spreading and contraction response. Science 312:738-741. 12. Carrasco, Y. R., S. J. Fleire, T. Cameron, M. L. Dustin, and F. D. Batista. 2004. LFA- 1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20:589-599. 13. Carrasco, Y. R., and F. D. Batista. 2006. B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1. Embo J 25:889- 899. 14. Roozendaal, R., T. R. Mempel, L. A. Pitcher, S. F. Gonzalez, A. Verschoor, R. E. Mebius, U. H. von Andrian, and M. C. Carroll. 2009. Conduits mediate transport of low-molecular-weight antigen to lymph node follicles. Immunity 30:264-276. 15. Bachmann, M. F., K. McKall-Faienza, R. Schmits, D. Bouchard, J. Beach, D. E. Speiser, T. W. Mak, and P. S. Ohashi. 1997. Distinct roles for LFA-1 and CD28 during activation of naive T cells: adhesion versus costimulation. Immunity 7:549- 557. 16. Schwartz, M. A., and M. H. Ginsberg. 2002. Networks and crosstalk: integrin signalling spreads. Nat Cell Biol 4:E65-68.   106 17. McLeod, S. J., A. J. Shum, R. L. Lee, F. Takei, and M. R. Gold. 2004. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem 279:12009-12019. 18. Rovida, E., B. Lugli, V. Barbetti, S. Giuntoli, M. Olivotto, and P. Dello Sbarba. 2005. Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF. Biol Chem 386:919-929. 19. van Buul, J. D., E. C. Anthony, M. Fernandez-Borja, K. Burridge, and P. L. Hordijk. 2005. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin- based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation. J Biol Chem 280:21129-21136. 20. Okigaki, M., C. Davis, M. Falasca, S. Harroch, D. P. Felsenfeld, M. P. Sheetz, and J. Schlessinger. 2003. Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration. Proc Natl Acad Sci U S A 100:10740-10745. 21. Avraham, H., S. Y. Park, K. Schinkmann, and S. Avraham. 2000. RAFTK/Pyk2- mediated cellular signalling. Cell Signal 12:123-133. 22. van Seventer, G. A., H. J. Salmen, S. F. Law, G. M. O'Neill, M. M. Mullen, A. M. Franz, S. B. Kanner, E. A. Golemis, and J. M. van Seventer. 2001. Focal adhesion kinase regulates beta1 integrin-dependent T cell migration through an HEF1 effector pathway. Eur J Immunol 31:1417-1427. 23. Giannoni, E., P. Chiarugi, G. Cozzi, L. Magnelli, M. L. Taddei, T. Fiaschi, F. Buricchi, G. Raugei, and G. Ramponi. 2003. Lymphocyte function-associated antigen-1-mediated T cell adhesion is impaired by low molecular weight phosphotyrosine phosphatase-dependent inhibition of FAK activity. J Biol Chem 278:36763-36776. 24. Andreev, J., J. P. Simon, D. D. Sabatini, J. Kam, G. Plowman, P. A. Randazzo, and J. Schlessinger. 1999. Identification of a new Pyk2 target protein with Arf-GAP activity. Mol Cell Biol 19:2338-2350. 25. Guinamard, R., M. Okigaki, J. Schlessinger, and J. V. Ravetch. 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 1:31-36. 26. Gismondi, A., J. Jacobelli, R. Strippoli, F. Mainiero, A. Soriani, L. Cifaldi, M. Piccoli, L. Frati, and A. Santoni. 2003. Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration. J Immunol 170:3065-3073. 27. Lakkakorpi, P. T., A. J. Bett, L. Lipfert, G. A. Rodan, and T. Duong le. 2003. PYK2 autophosphorylation, but not kinase activity, is necessary for adhesion-induced association with c-Src, osteoclast spreading, and bone resorption. J Biol Chem 278:11502-11512. 28. Sieg, D. J., D. Ilic, K. C. Jones, C. H. Damsky, T. Hunter, and D. D. Schlaepfer. 1998. Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration. Embo J 17:5933-5947. 29. Astier, A., H. Avraham, S. N. Manie, J. Groopman, T. Canty, S. Avraham, and A. S. Freedman. 1997. The related adhesion focal tyrosine kinase is tyrosine- phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas. J Biol Chem 272:228-232.   107 30. Freedman, A. S., K. Rhynhart, Y. Nojima, J. Svahn, L. Eliseo, C. D. Benjamin, C. Morimoto, and E. Vivier. 1993. Stimulation of protein tyrosine phosphorylation in human B cells after ligation of the beta 1 integrin VLA-4. J Immunol 150:1645-1652. 31. Manie, S. N., A. Astier, D. Wang, J. S. Phifer, J. Chen, A. I. Lazarovits, C. Morimoto, and A. S. Freedman. 1996. Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells. Blood 87:1855-1861. 32. Glodek, A. M., M. Honczarenko, Y. Le, J. J. Campbell, and L. E. Silberstein. 2003. Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis. J Exp Med 197:461-473. 33. Mlinaric-Rascan, I., and T. Yamamoto. 2001. B cell receptor signaling involves physical and functional association of FAK with Lyn and IgM. FEBS Lett 498:26-31. 34. Glodek, A. M., Y. Le, D. M. Dykxhoorn, S. Y. Park, G. Mostoslavsky, R. Mulligan, J. Lieberman, H. E. Beggs, M. Honczarenko, and L. E. Silberstein. 2007. Focal adhesion kinase is required for CXCL12-induced chemotactic and pro-adhesive responses in hematopoietic precursor cells. Leukemia 21:1723-1732. 35. Katagiri, K., A. Maeda, M. Shimonaka, and T. Kinashi. 2003. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat Immunol 4:741-748. 36. Lafuente, E. M., A. A. van Puijenbroek, M. Krause, C. V. Carman, G. J. Freeman, A. Berezovskaya, E. Constantine, T. A. Springer, F. B. Gertler, and V. A. Boussiotis. 2004. RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev Cell 7:585-595. 37. Han, J., C. J. Lim, N. Watanabe, A. Soriani, B. Ratnikov, D. A. Calderwood, W. Puzon-McLaughlin, E. M. Lafuente, V. A. Boussiotis, S. J. Shattil, and M. H. Ginsberg. 2006. Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3. Curr Biol 16:1796-1806. 38. Bos, J. L. 2005. Linking Rap to cell adhesion. Curr Opin Cell Biol 17:123-128. 39. Santos-Argumedo, L., P. W. Kincade, S. Partida-Sanchez, and R. M. Parkhouse. 1997. CD44-stimulated dendrite formation ('spreading') in activated B cells. Immunology 90:147-153. 40. Elenstrom, C., and E. Severinson. 1989. Interleukin 4 induces cellular adhesion among B lymphocytes. Growth Factors 2:73-82. 41. Davey, E. J., J. Thyberg, D. H. Conrad, and E. Severinson. 1998. Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes. J Immunol 160:5366-5373. 42. Xiong, W. C., M. Macklem, and J. T. Parsons. 1998. Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein. J Cell Sci 111 ( Pt 14):1981-1991. 43. Robertson, L. K., L. R. Mireau, and H. L. Ostergaard. 2005. A role for phosphatidylinositol 3-kinase in TCR-stimulated ERK activation leading to paxillin phosphorylation and CTL degranulation. J Immunol 175:8138-8145. 44. Cai, X., M. Li, J. Vrana, and M. D. Schaller. 2006. Glycogen synthase kinase 3- and extracellular signal-regulated kinase-dependent phosphorylation of paxillin regulates cytoskeletal rearrangement. Mol Cell Biol 26:2857-2868. 45. Ostergaard, H. L., and T. L. Lysechko. 2005. Focal adhesion kinase-related protein tyrosine kinase Pyk2 in T-cell activation and function. Immunol Res 31:267-282.   108 46. Park, S. Y., H. K. Avraham, and S. Avraham. 2004. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279:33315-33322. 47. Christian, S. L., R. L. Lee, S. J. McLeod, A. E. Burgess, A. H. Li, M. Dang-Lawson, K. B. Lin, and M. R. Gold. 2003. Activation of the Rap GTPases in B lymphocytes modulates B cell antigen receptor-induced activation of Akt but has no effect on MAPK activation. J Biol Chem 278:41756-41767. 48. Mochizuki, N., Y. Ohba, E. Kiyokawa, T. Kurata, T. Murakami, T. Ozaki, A. Kitabatake, K. Nagashima, and M. Matsuda. 1999. Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i). Nature 400:891-894. 49. Ohba, Y., N. Mochizuki, K. Matsuo, S. Yamashita, M. Nakaya, Y. Hashimoto, M. Hamaguchi, T. Kurata, K. Nagashima, and M. Matsuda. 2000. Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade. Mol Cell Biol 20:6074- 6083. 50. McLeod, S. J., A. H. Li, R. L. Lee, A. E. Burgess, and M. R. Gold. 2002. The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor- 1 (CXCL12): potential role for Rap2 in promoting B cell migration. J Immunol 169:1365-1371. 51. Bubb, M. R., A. M. Senderowicz, E. A. Sausville, K. L. Duncan, and E. D. Korn. 1994. Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem 269:14869- 14871. 52. Buckbinder, L., D. T. Crawford, H. Qi, H. Z. Ke, L. M. Olson, K. R. Long, P. C. Bonnette, A. P. Baumann, J. E. Hambor, W. A. Grasser, 3rd, L. C. Pan, T. A. Owen, M. J. Luzzio, C. A. Hulford, D. F. Gebhard, V. M. Paralkar, H. A. Simmons, J. C. Kath, W. G. Roberts, S. L. Smock, A. Guzman-Perez, T. A. Brown, and M. Li. 2007. Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis. Proc Natl Acad Sci U S A 104:10619-10624. 53. Zeng, L., X. Si, W. P. Yu, H. T. Le, K. P. Ng, R. M. Teng, K. Ryan, D. Z. Wang, S. Ponniah, and C. J. Pallen. 2003. PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration. J Cell Biol 160:137-146. 54. Schlaepfer, D. D., S. K. Mitra, and D. Ilic. 2004. Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochim Biophys Acta 1692:77-102. 55. Ruusala, A., and P. Aspenstrom. 2008. The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics. Mol Cell Biol 28:1802-1814. 56. Sarkar, S., M. Svoboda, R. de Beaumont, and A. S. Freedman. 2002. The role of Aktand RAFTK in beta1 integrin mediated survival of precursor B-acute lymphoblastic leukemia cells. Leuk Lymphoma 43:1663-1671. 57. Mitra, S. K., D. A. Hanson, and D. D. Schlaepfer. 2005. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 6:56-68. 58. Allen, C. D., T. Okada, H. L. Tang, and J. G. Cyster. 2007. Imaging of germinal center selection events during affinity maturation. Science 315:528-531.   109 59. Hauser, A. E., T. Junt, T. R. Mempel, M. W. Sneddon, S. H. Kleinstein, S. E. Henrickson, U. H. von Andrian, M. J. Shlomchik, and A. M. Haberman. 2007. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns. Immunity 26:655-667. 60. Ambrose, H. E., and S. D. Wagner. 2004. Alpha6-integrin is expressed on germinal centre B cells and modifies growth of a B-cell line. Immunology 111:400-406. 61. Klein, U., Y. Tu, G. A. Stolovitzky, J. L. Keller, J. Haddad, Jr., V. Miljkovic, G. Cattoretti, A. Califano, and R. Dalla-Favera. 2003. Transcriptional analysis of the B cell germinal center reaction. Proc Natl Acad Sci U S A 100:2639-2644. 62. Alizadeh, A. A., M. B. Eisen, R. E. Davis, C. Ma, I. S. Lossos, A. Rosenwald, J. C. Boldrick, H. Sabet, T. Tran, X. Yu, J. I. Powell, L. Yang, G. E. Marti, T. Moore, J. Hudson, Jr., L. Lu, D. B. Lewis, R. Tibshirani, G. Sherlock, W. C. Chan, T. C. Greiner, D. D. Weisenburger, J. O. Armitage, R. Warnke, R. Levy, W. Wilson, M. R. Grever, J. C. Byrd, D. Botstein, P. O. Brown, and L. M. Staudt. 2000. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403:503-511. 63. Miyake, K., I. L. Weissman, J. S. Greenberger, and P. W. Kincade. 1991. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med 173:599-607. 64. Durand, C. A., J. Westendorf, K. W. Tse, and M. R. Gold. 2006. The Rap GTPases mediate CXCL13- and sphingosine1-phosphate-induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes. Eur J Immunol 36:2235-2249. 65. Freundlich, B., and N. Avdalovic. 1983. Use of gelatin/plasma coated flasks for isolating human peripheral blood monocytes. J Immunol Methods 62:31-37. 66. Yee, J. K., T. Friedmann, and J. C. Burns. 1994. Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol 43 Pt A:99-112. 67. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267.    110 3. Small molecule inhibitors of proline-rich tyrosine kinase 2 and focal adhesion kinase modulate marginal zone B cell responses to the chemoattractants sphingosine-1-phosphate and CXCL132 3.1 Introduction  The majority of mature B cells are recirculating follicular B-2 cells that transiently accumulate in the lymphoid follicles of secondary lymphoid organs such as the spleen, lymph nodes, and tonsils.  B-2 cells are responsible for T-dependent immune responses to pathogens.  Their location adjacent to the T cell areas of lymphoid organs allows them to move to the border of the follicles when they are activated and interact with activated T helper cells.  In contrast to conventional B-2 cells, the innate-like B cells, including the marginal zone (MZ) and B-1 B cells, reside in specialized locations, do not circulate, and can make rapid antibody (Ab) responses without T cell help (1-3).  B-1 cells are found mainly in the peritoneal cavity and the spleen.  In the mouse, MZ B cells are found exclusively in the region surrounding the marginal sinuses of the lymphoid follicles in the spleen (4, 5).  Their position makes them the first population of immune cells that encounter blood-borne antigens (Ags), and they play a major role in the early response to microbial infection.  MZ B cells are characterized as being IgMhighIgDlowCD21highCD23lowCD1dhigh, and their Ig repertoires are skewed toward the recognition of both microbial and self-Ags.  These cells are functionally different from conventional B-2 cells and are responsible for rapid Ab response to polysaccharide Ags on the surface of encapsulated bacteria (T-independent type II Ags), as well as the production of natural Abs (6).  Natural Abs are predominantly IgM and are produced spontaneously without the requirement of immunization (7).  They often recognize epitopes on encapsulated Gram-positive bacteria, pathogenic viruses, apoptotic cells, and oxidized low-density lipoproteins, thereby providing immediate protection against infection in addition to preventing inflammation by facilitating the clearance of oxidized lipids, oxidized proteins, and apoptotic cells (7-9).  Accordingly, T-independent immune responses  2 A version of this chapter will be submitted for publication: Tse KW, Lin K, Buckbinder L, and Gold MR (2010). Small molecule inhibitors of proline-rich tyrosine kinase 2 and focal adhesion kinase modulate marginal zone B cell responses to the chemoattractants sphingosine-1-phosphate and CXCL13   111 and natural Ab titers are greatly reduced in knockout mice with a contracted or missing MZ B cell compartment (10-12). In addition to their important role in the host response against bacterial pathogens, MZ B cells have been linked to autoimmune diseases.  In chronic inflammatory diseases such as rheumatoid arthritis and lupus, expansion of MZ B cell populations and increased production of self-reactive Abs are observed (13-15).  In addition to producing auto-Abs, MZ B cells may contribute to autoimmunity by acting as antigen-presenting cells (APCs).  In the non-obese diabetic (NOD) mouse model of type 1 diabetes, activated MZ B cells accumulate in the pancreatic lymph nodes and present self-Ags such as insulin to diabetogenic T cells (16).  Therefore, modulating the activation and the localization of MZ B cells might be a useful approach to treat autoimmune diseases mediated by MZ B cells or auto-Abs that these cells secrete.  It is not completely understood how the development and localization of MZ B cells is regulated, but recent studies have suggested that B cell receptor (BCR) signaling, Notch2, the receptor for B cell-activating factor (BAFF), and the canonical nuclear factor-κB pathway are required for MZ B cell development.  In addition, signals involved in cell migration and adhesion are critical for the proper localization of MZ B cells (2). Chemotatic and adhesive responses are key factors in determining B cell trafficking and localization, and in particular the accumulation of MZ B cells near the marginal sinus of the spleen.  This is supported by the finding that mice deficient in signaling molecules involved in cell migration and adhesion such as Rap1b, Rac2 (a Rho family GTPase), Dock2 (a RacGEF), proline-rich tyrosine kinase (Pyk2), and Lsc (a GEF for RhoA that is activated by receptors for chemoattractive lipids such as lysophosphatidic acid and S1P that signal through the G12/13 trimeric G protein), all result in the loss of MZ B cells (11, 12, 17-19). However, the mechanism involved in MZ B cell retention within the MZ is only partly understood.  The adhesion of MZ B cells to marginal sinus stromal cells depends on the interaction between the lymphocyte integrins, LFA-1 or VLA-4, and ICAM-1 or VCAM-1, their respective ligands on stromal cells (20).  MZ B cells shuttle between the MZ and the B cell follicles in response to S1P and CXCL13, respectively (21, 22).  This shuttling allows MZ B cells to deliver blood-borne Ags that they capture in the MZ to follicular dendritic cells (FDCs) in the lymphoid follicle, a key APC for B-2 cells (23).  The high level of CD21 (also known as complement receptor type 2) on MZ B cells also facilitates the transport of   112 immune complexes from the blood to the splenic follicles, where they can be captured by FDCs (22).  Thus, cellular signals that regulate B cell migration and adhesion have a direct effect on the proper homing, localization, and activation of B cells. Focal adhesion kinase (FAK) and the related kinase Pyk2 play an important role in regulating cell adhesion, morphology, and migration in many cell types (24-26).  They are critical for integrating signals from chemokine receptors and other cell surface receptors and contribute to the activation of multiple downstream signaling enzymes including the ERK, JNK, and p38 mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) (27-31).  In B cells, Pyk2 and FAK are downstream targets of signaling by the BCR, integrins, and chemokine receptors (32-35).  In Pyk2-deficient mice, the MZ B cell compartment is completely absent, and the remaining B-2 B cells isolated from these mice exhibit impaired migration in response to the chemokines CXCL12 (SDF-1), CXCL13 (BLC), and CCL21 (SLC) (12).  FAK is important for B cell progenitors to migrate towards SDF-1 and adhere to VCAM-1 (36).  In contrast, SDF-1 stimulation targets FAK for degradation in mature B cells (36, 37).  Although Pyk2 appears to be crucial for MZ B cell development, the function of Pyk2 in normal MZ B cells in adult animals is not known.  Moreover, the role of FAK in mature B cells has not been characterized. Recently, Pyk2- and FAK-selective small molecule inhibitors have been described (38-41).  These ATP-competitive inhibitors are highly selective, have IC50 values in the nanomolar range, and are able to inhibit the kinase activity as well as tyrosine phosphorylation of Pyk2 and FAK.  I showed in Chapter 2 that the dual Pyk2/FAK kinase inhibitor, PF-431396 (PF-396), can attenuate the spreading of A20 B-lymphoma cells in response to integrin engagement (32).  Other groups have shown that the FAK-selective inhibitor PF-573228 (PF-228) can inhibit FAK kinase activity in vitro, as well as FAK phosphorylation in various cell lines and in primary human platelets, with an IC50 of 0.3-3 µM (40, 42).  Its IC50 for inhibition of Pyk2 kinase activity is 1000 fold higher (40), making PF-228 a highly selective inhibitor of FAK.  PF-396 and PF-228 have no inhibitory effect on a number of other kinases including Akt, Abl, ERK, PKC, PI3K, PKA, and c-Src, suggesting that they are relatively specific for the FAK/Pyk2 family of non-receptor tyrosine kinases. Furthermore, in vivo administration of a related dual Pyk2/FAK inhibitor PF-562271 inhibits   113 tumorigenesis in a mouse model (38, 39), suggesting a potential therapeutic application for these small molecule inhibitors. In this study, we have used the FAK-selective inhibitor, PF-228, as well as a newly developed Pyk2-selective inhibitor, PF-3430719 (PF-719), to study the functions of Pyk2 and FAK in murine B-2 and MZ B cells.  We found that both Pyk2 and FAK are important for these cells to respond to the chemoattractants S1P and CXCL13.  Treatment with the Pyk2 or FAK inhibitors reduced S1P- and CXCL13-induced B cell migration.  Moreover, the ability of B cells to adhere to ICAM-1 was reduced upon treatment with the Pyk2 inhibitor PF-719 whereas the FAK inhibitor PF-228 had no effect.  Finally, we found that FAK is involved in CXCL13-induced activation of Akt/protein kinase B, a kinase that regulates cell proliferation, cell survival, and directional cell migration (43-45).  Collectively, our data suggest that both Pyk2 and FAK are involved in regulating the proper localization of MZ B cells in the splenic compartment.   Our data also support the idea that Pyk2 may be a new target for modulating the functions of MZ B cells, which have been implicated in autoimmune diseases.   114 3.2 Results 3.2.1 S1P and CXCL13 induce Pyk2 and FAK tyrosine phosphorylation in B cells Given the multiple functions of Pyk2 and FAK in regulating cell morphology, and the observation that MZ B cells fail to develop in Pyk2-deficient mice (12, 26, 36), we investigated the role of Pyk2 and FAK in MZ B cell migration.  The chemokine CXCL13 and the lipid chemoattractant S1P are the two major chemoattractants that regulate MZ B cell trafficking and localization.  Previously, I showed that stimulation of the A20 B-lymphoma cell line with S1P or CXCL13 induces tyrosine phosphorylation of Pyk2 (33), a modification that results in maximal Pyk2 kinase activity.  To examine whether FAK is also a target of signaling by the receptors for S1P and CXCL13, I again used the A20 B cell line, which expresses high levels of both Pyk2 and FAK (see Figure 2.1).  I found that both S1P and CXCL13 induced robust tyrosine phosphorylation of FAK (Figure 3.1).  This indicates that FAK is involved in signal transduction by the receptors for S1P and CXCL13 in B cells.   115   Figure 3.1 S1P and CXCL13 induce FAK tyrosine phosphorylation in B cells. A20 B cells were stimulated with 100 nM S1P (upper panel) or 200 nM CXCL13 (lower panel) for the indicated times.  Anti-FAK immunoprecipitates were probed with the 4G10 anti-phosphotyrosine (anti-P- Tyr) monoclonal Ab before being reprobed with an anti-FAK Ab.  For each panel, one of two experiments that yielded similar results is shown.   116 3.2.2 The small molecule inhibitors PF-719 and PF-228 block tyrosine phosphorylation of Pyk2 and FAK Because MZ B cells fail to develop in Pyk2-deficient mice, our approach was to use Pyk2- and FAK-selective small molecule inhibitors to assess the functions of these kinases in MZ B cells from adult mice.  I have previously shown that the dual Pyk2/FAK inhibitor, PF-396, effectively blocks BCR-induced Pyk2 and FAK tyrosine phosphorylation in B cells (32). Since then, a selective inhibitor of Pyk2, PF-719, has been developed.   PF-719 (Pfizer) inhibits the kinase activity of purified Pyk2 with an IC50 of 17 nM (unpublished data from Pfizer).  In contrast, its IC50 is 469 nM for FAK, 6.7 µM for Lyn, and >10 µM for the Src, Fyn, Hck, and Lck SFKs.  To examine the specificities of PF-719 and PF-228 in B cells, we pretreated A20 cells with PF-719 or the FAK-selective inhibitor PF-228 and looked at BCR- induced tyrosine phosphorylation of Pyk2 and FAK.  Pre-treating A20 cells with the Pyk2- selective inhibitor, PF-719, inhibited Pyk2 tyrosine phosphorylation when used at 0.5 to 5 µM (Figure 3.2A; left panels) but had no effect on FAK tyrosine phosphorylation at concentrations up to 1 µM.  At 5 µM, PF-719 did block FAK phosphorylation, indicating that it is only specific for Pyk2 at lower concentrations.  Treatment with the FAK inhibitor, PF- 228, inhibited BCR-induced FAK tyrosine phosphorylation but had no effect on Pyk2 tyrosine phosphorylation (Figure 3.2A; right panels).  Importantly, these Pyk2/FAK inhibitors (PF-396, PF-719, or PF-228) have no apparent effects on other BCR signaling pathways in either A20 cells (Figure 3.2B) or splenic B cells (data not shown), suggesting that these inhibitors do not block proximal BCR signaling events that are dependent on activation of SFKs and the Syk tyrosine kinase.  Finally, treating splenic B cells with PF-719, PF-228, or PF-396 for 8 hours did not increase cell death, suggesting that the inhibitors are not toxic to the cells (Figure 3.2C).  Taken together, these results qualify PF-719 and PF-228 as suitable agents for use in B cells to examine the normal cellular functions of Pyk2 and FAK kinase activity and tyrosine phosphorylation.   117  Figure 3.2 Inhibition of Pyk2 and FAK tyrosine phosphorylation by the small molecule inhibitors PF-719 and PF-228. A, A20 cells were pre-treated with the indicated concentrations of PF-719 (left panels) or PF-228 (right panels) or vehicle control (DMSO) for 1 h before being stimulated with anti-IgG (20 µg/ml) for 15 min. Immunoprecipitated Pyk2 and FAK were analyzed by immunoblotting with the anti-P-Tyr Ab.  The blots were then reprobed with Abs to Pyk2 or FAK. B, Total cell lysates from A20 cells stimulated as in A were analyzed by immunoblotting with the anti-P- Tyr monoclonal Ab and then reprobed with Abs against actin as a loading control. C, Total splenic B cells were treated with the indicated concentration of inhibitors for 8 h in HEPES-buffed saline with 2% FBS.  Cell death was assessed by 7-AAD uptake, which was quantified by flow cytometry.   118 Figure 3.2   119  3.2.3 PF-719 and PF-228 inhibit chemoattractant-induced migration of splenic B-2 and MZ B cells CXCL13 and S1P regulate B cell localization and trafficking in vivo.  CXCL13 directs circulating B-2 cells to enter lymphoid follicles.  For MZ B cells, S1P is critical for their retention in the splenic MZ whereas CXCL13 allows MZ B cells to shuttle into the lymphoid follicles, where they can deliver blood-borne Ags to FDCs (21, 22).  Although it has been shown that Pyk2 and FAK are involved in B cell migration toward the chemokine SDF-1, their roles in MZ B cell migration are not known.  Moreover, the ability of small molecule inhibitors of Pyk2 and FAK to modulate the migration and trafficking of B cells, and thereby modulate B cell function, has not been investigated. To assess the effect of these inhibitors on B cell function, we isolated splenic cells from C57BL/6 mice and pretreated them with either the Pyk2 specific inhibitor (PF-719) or the FAK specific inhibitor (PF-228).  Using Transwell migration assays, we found that 1µM PF- 719 and 1 µM PF-228 both reduced CXCL13-induced migration of MZ B cells and splenic B-2 cells by 30-40% (Figure 3.3A).  Freshly isolated splenic B-2 cells do not respond to S1P, but MZ B cells are highly responsive to S1P in the 10-100 nM range ((19) and data not shown).  We found that both PF-719 and PF-228 inhibited S1P-induced MZ B cell migration by >50% (Figure 3.3B).  Thus, both Pyk2 and FAK contribute to the ability of MZ B cells to migrate efficiently towards CXCL13 and S1P.   120 Figure 3.3 PF-719 and PF-228 inhibit chemoattractant-induced migration of splenic B-2 and MZ B cells.  A, Total spleen cells were pretreated with DMSO, PF-719, or PF-228 for 1 h before being added to Transwells containing either medium or 100 nM CXCL13 in the lower chamber.  After 3 h, the percent migration for each B cell subset was determined by comparison to the input population.  Flow cytometry was used to distinguish B-2 (CD45R+CD23highCD21int; where “int” is “intermediate”) and MZ B cells (CD45R+CD23lowCD21high).  Each bar is the average +/- SD of duplicate samples.  For each panel, similar results were obtained in at least three experiments. B, Total spleen cells were pretreated with DMSO, PF-719, or PF-228 for 1 h before being added to Transwells containing medium or 100 nM S1P in the lower chamber.  Chemotaxis was measured as in A. C, Transwell migration assays using spleen cells were performed as in A, except that the chemoattractant was present either in the bottom chamber only (chemotaxis) or in both the top and bottom chambers (chemokinesis).  The percent of cells that migrated into the lower chamber was calculated as in A. For each panel, similar results were obtained in at least three experiments.  Statistical analysis of the data is shown in Table 3.1.   121 Figure 3.3    122 Chemotaxis (CXCL13) B cell subsets Treatment groups p-values B-2 cells DMSO PF-719 0.004 B-2 cells DMSO PF-228 0.009  MZ B cells DMSO PF-719 0.003 MZ B cells DMSO PF-228 0.049  Chemokinesis (CXCL13) B cell subsets Treatment groups p-values B-2 cells DMSO PF-719 0.046 B-2 cells DMSO PF-228 0.095 (N.S.)  MZ B cells DMSO PF-719 0.046 MZ B cells DMSO PF-228 0.185 (N.S.)  Chemotaxis (S1P) B cell subsets Treatment groups p-values MZ B cells DMSO PF-719 0.030 MZ B cells DMSO PF-228 0.005  Chemokinesis (S1P) B cell subsets Treatment groups p-values MZ B cells DMSO PF-719 0.028 MZ B cells DMSO PF-228 0.160 (N.S.)  Table 3.1 Statistical analysis of the effects of the Pyk2 inhibitor (PF-719) and FAK inhibitor (PF-228) on CXCL13- and S1P-induced chemotaxis and chemokinesis. Statistical analysis of the chemotaxis and chemokinesis responses shown in Figure 3.3C.  The p-values were obtained by Student’s one-tailed paired t-test, compared to the DMSO controls in three independent experiments.  N.S., not significant.   The results show that both Pyk2 and FAK contribute to chemotaxis but that Pyk2 may be more important than FAK for chemokinesis.     123 Chemotaxis is a complex response involving persistent directional movement of cells along a chemoattractant gradient as well as a chemoattractant-induced increase in the speed of cell motility, which is referred to as chemokinesis.  Thus chemotaxis involves both chemokinesis and a decreased rate of turning, which results in more persistent directional migration. Distinct signaling pathways regulate chemokinesis versus chemotaxis.  Signaling molecules such as PI3K and LIM domain kinase (LIMK) are critical for directional migration along a chemokine gradient but not for random cell migration (46, 47).  As the inhibitors for Pyk2 and FAK only partially reduced the chemotactic response of B-2 and MZ B cells, this raised the possibility that Pyk2 and FAK may play a more specific role in directional movement. To determine the relative roles of Pyk2 and FAK in chemotaxis versus chemokinesis, we used a standard approach for assessing chemokinesis.  This involves placing the chemoattractant in both the upper and lower Transwell chambers such that the cells in the upper chamber were exposed to the chemoattractant, but there was no gradient.  Figure 3.3C shows that CXCL13 induced chemokinesis in both B-2 and MZ B cells.  The number of cells migrating from the upper chamber to the lower chamber under chemokinesis conditions was ~50% of that observed under chemotaxis conditions in which CXCL13 was present only in the bottom chamber.  In both B-2 cells and MZ B cells, CXCL13-induced chemokinesis was reduced significantly by pre-treating the cells with the Pyk2 inhibitor PF-719 (Figure 3.3C and Table 3.1).  A similar degree of inhibition was observed for S1P-induced chemokinesis in MZ B cells (Figure 3.3C; lower panel).  The FAK inhibitor caused a slight reduction in chemokinesis but statistical analysis showed that the difference was not significant (Table 3.1).  Thus, Pyk2 appears to be critical for both B cell locomotion and directional migration since the Pyk2 inhibitor reduced both chemokinesis and chemotaxis.  In contrast, FAK seems to have a greater role in establishing the direction of B cell movement as the FAK inhibitor reduced chemotaxis to a greater extent than chemokinesis.   124 3.2.4 PF-719 reduces B-2 and MZ B cell adhesion to ICAM-1 The retention of MZ B cells in the MZ surrounding the lymphoid follicles of the spleen requires strong integrin-mediated adhesion (20, 21).  Chemokines and their G protein- coupled receptors are important regulators of integrin-mediated adhesion in lymphocytes, and the chemokine CXCL13 has been shown to induce integrin-dependent adhesion of B cells (33, 48, 49).  Unstimulated B-2 cells and MZ B cells adhere to some extent to integrin ligands, but this can be enhanced by signals from the BCR or chemokine receptors that cause integrin activation (50).  I have previously shown that Pyk2 and FAK act downstream of integrin signaling in B cells (32) but it is not known whether these kinases also participate in the inside-out signaling that leads to integrin activation.  Therefore we tested whether blocking Pyk2 or FAK activity would impair the ability of B cells to adhere to ICAM-1, the ligand for the LFA-1 integrin. Treating splenic B cells with the Pyk2 inhibitor PF-719 reduced their basal adhesion to ICAM-1 by ~50% (Figure 3.4A), whereas the FAK-selective inhibitor, PF-228, had no effect on their basal adhesion to ICAM-1.  Next, we assessed the effects of these inhibitors on both basal and CXCL13-induced adhesion to ICAM-1 in either B-2 cells or MZ B cells.  Although B-2 and MZ B cells adhered well to ICAM-1 in the absence of CXCL13, treatment with this chemokine resulted in increased adhesion (Figure 3.4B and C; p = 0.01 for B-2 cells and p = 0.002 for MZ B cells, n = 3 experiments).  Importantly, we found that the Pyk2 inhibitor PF- 719 reduced basal and CXCL13-induced adhesion to ICAM-1 in both B-2 and MZ cells, whereas the FAK inhibitor PF-228 had very little effect (Figure 3.4B and C).  Pre-incubation with an anti-LFA-1 Ab blocked both B-2 and MZ B cell adhesion to ICAM-1 (data not shown), showing that this adhesion to ICAM-1 was mediated entirely by LFA-1.  Thus, Pyk2 is involved in the LFA-1-dependent adhesion of B-2 and MZ B cells to ICAM-1 while FAK is dispensable for this function.    125  Figure 3.4 PF-719 reduces B-2 and MZ B cell adhesion to ICAM-1. A, Total spleen cells were pretreated with DMSO, PF-719, or PF-228 for 1 h, and then plated on immobilized ICAM-1 for 1 h.  The percent of CD45R+ B cells that adhered after was determined by comparison to the input number cell population. B & C, Total spleen cells were pretreated as in A and stimulated with CXCL13 before being plated on immobilized ICAM-1.  The percent of MZ B cells (B) or B-2 cells (C) that remained adhered after washing was determined by comparison to the input cell population.  Each bar is the mean +/- SD for triplicate samples.  Similar results were obtained in four experiments.  Statistical analysis is compiled in Table 3.2.   126  MZ B cell adhesion Stimulation Treatment groups p-values ICAM DMSO PF-719 0.005 ICAM + CXCL13 DMSO PF-719 0.019  ICAM DMSO PF-228 0.331 (N.S.) ICAM + CXCL13 DMSO PF-228 0.177 (N.S.)  B-2 cell adhesion Stimulation Treatment groups p-values ICAM DMSO PF-719 0.008 ICAM + CXCL13 DMSO PF-719 0.031  ICAM DMSO PF-228 0.161 (N.S.) ICAM + CXCL13 DMSO PF-228 0.062 (N.S.)  Table 3.2 Statistical analysis of the effects of the Pyk2 and FAK inhibitors on B cell adhesion to ICAM-1. Statistical analysis of the adhesion data shown in Figure 3.4B and C.  The p-values were obtained by Student’s one-tailed paired t-test, compared to the DMSO controls in at least three independent experiments.  N.S., not significant.  The data clearly show that Pyk2, but not FAK, is required for B cell adhesion.    127 3.2.5 The FAK inhibitor PF-228 inhibits chemoattractant- and anti-Ig-induced Akt phosphorylation One of the signaling events downstream of chemoattractant receptors in B cells is activation of the PI3K/Akt pathway, which promotes cell survival, growth, proliferation and directional movement (43, 45).  Pyk2 and FAK undergo tyrosine phosphorylation in response to CXCL13 in B cells (Figure 3.1), and both of these kinases have been reported to bind the SH2 domain-containing p85 subunit of PI3K (51).  Therefore, we assessed the role of Pyk2 and FAK in chemoattractant receptor-induced activation of the PI3K/Akt pathway in B cells, using the phosphorylation of Akt as a readout.  Akt is a serine/threonine kinase, and it exhibits full kinase activity when phosphorylated on threonine 308 in the catalytic domain and serine 473 in the hydrophobic motif (52).  Thus, phospho-specific Abs can be used to assess the activation state of Akt.  Intracellular staining and FACS analysis showed that CXCL13 increased the phosphorylation of Akt on serine 473 in both splenic B-2 and MZ B cells, and that this response was blocked by pre-treating the cells with the FAK specific inhibitor PF-228 (Figure 3.5A and B).  In contrast, the Pyk2 inhibitor PF-719 treatment did not affect CXCL13-induced Akt.  Thus, FAK may play a key role in linking signals generated by chemoattractant receptors to the PI3K/Akt pathway. BCR signaling also results in strong PI3K/Akt activation in B cells (53).  We found that treating MZ and B-2 B cells with the FAK inhibitor PF-228 reduced anti-Ig-induced Akt phosphorylation whereas, again, the Pyk2 inhibitor PF-719 had no effect (Figure 3.5C and D).  Notably, CXCL13-induced Akt phosphorylation appeared to be more sensitive to PF- 228 than anti-Ig-induced Akt phosphorylation.  Our finding that the Pyk2 inhibitor PF-719 had no effect on BCR-induced Akt activation is consistent with our previous finding that blocking the activation of Rap, which is upstream of Pyk2, does not inhibit BCR-induced Akt phosphorylation (32).  Thus, FAK, and not Pyk2, appears to link chemoattractant receptors and the BCR to Akt phosphorylation in B cells.     128 Figure 3.5 PF-228 inhibits chemoattractant- and anti-Ig-induced Akt phosphorylation.  A&C, Total splenic B cells were pretreated with the Pyk2 or FAK inhibitors, or with DMSO (vehicle control), for 1 h before being stimulated with 200 nM CXCL13 for 3 min (A) or 10 µg/ml anti-IgM for 5 min (C).  FACS analysis was used to quantify intracellular phospho-Akt levels and to distinguish splenic B- 2 cells and MZ B cells.  Representative FACS plots are shown. B&D, Quantification of results showed in A&C.  The difference in mean fluorescence intensity (MFI) values between unstimulated cells, and cells stimulated with CXCL13, was used as the 100% value (control response).   The difference in MFI between unstimulated and stimulated cells in the presence of the corresponding inhibitor is expressed as a percentage of the control response.  Each bar is the mean +/- SEM for three experiments.   129 Figure 3.5    130 3.3 Discussion The proper trafficking and localization of MZ B cells is dependent on integrated migration and retention signals provided by the stromal cells in the spleen.  Pyk2 and FAK are regulators of B cell migration, adhesion, and morphology, and Pyk2 is essential for MZ B cell development. In addition to showing that S1P and CXCL13 stimulate Pyk2 and FAK tyrosine phosphorylation, in this study we have now shown for the first time that the kinase activities of Pyk2 and FAK are required for these chemoattractants to promote the migration of both B-2 and MZ B cells.  We also show that Pyk2 kinase activity is important for the adhesion of B-2 and MZ B cells to ICAM-1.  Our finding that Pyk2 is required for MZ B cell migration and adhesion may explain why Pyk2-/- mice lack MZ B cells in the spleen (12). Taken together, our data suggests that selective small molecule inhibitors of Pyk2 may have therapeutic potential for modulating the function of MZ B cells in vivo. The roles of S1P and CXCL13 in directing MZ B cell localization in the spleen have recently been elucidated.  S1P is the agonist for five different receptors (S1P1-5), with S1P1 and S1P3 being the two major S1P receptors expressed by B cells (54, 55).  S1P1 plays a key role in the exit of T and B cells from peripheral lymphoid organs and in the proper positioning of MZ B cells in the spleen (21, 56).  In contrast, S1P3 is expressed at much higher levels on MZ B cells than on follicular B-2 cells and it is the major mediator of MZ B cell chemotaxis.  B cells also express CXCR5, which is the receptor for the chemokine CXCL13, whose primary function is to direct B cells homing to the lymphoid follicles.  Our previous results showed that Pyk2 is the target of both S1P receptor and CXCR5 signaling (33).  Here, we show for the first time that S1P and CXCL13 signaling promote the tyrosine phosphorylation of FAK in B cells.  The phosphorylation of Pyk2 and FAK on conserved tyrosine residues increases their kinase activity (27, 57).  The initial event in receptor- induced activation of these kinases is their auto-phosphorylation on Y397 of FAK or Y402 of Pyk2.  This is thought to occur via dimerization and trans-phosphorylation (58).  Src family kinases can bind to this site via their SH2 domains and then phosphorylate the two tyrosine residues in the activation loop of the kinase domain, resulting in maximal activity of Pyk2 or FAK toward their substrates.  Once Pyk2 and FAK are phosphorylated, they can interact with various signaling molecules such as p85 (the regulatory subunit of PI3K), the suppressor of   131 cytokine signaling protein (SOCS-1 and SOCS-3), SFKs, the Shc and Grb (Grb2 and Grb7) adaptor proteins, PLCγ, and p120 RasGAP (27, 59, 60).  Although it remains to be shown whether the Pyk2 and FAK inhibitors block the interactions of these proteins with Pyk2 and FAK, these kinase inhibitors appear to be an effective way to inhibit Pyk2- and FAK- dependent responses to S1P receptor and CXCR5 signaling. Chemoattractant-induced migration and adhesion control the trafficking and localization of B cells, processes that are critical for B cells to encounter Ag and become activated.  We show here that the enzymatic activities of Pyk2 and FAK are important for B cell migration, indicating that these kinases may be the key regulators of chemotactic responses.  This is also supported by other studies showing that Pyk2 and FAK are involved in signaling by the CCR7 and CXCR4 chemokine receptors, and in migration and polarization of hematopoietic lineage cells (36, 61).  In B cell progenitors, FAK is important for prolonged VLA-4 integrin-mediated adhesion to VCAM-1 in response to SDF-1 (36). This may be a critical step in B cell development as progenitor B cells require contact with the bone marrow stromal cells, which express both SDF-1 and VCAM-1, in order to receive survival and differentiation signals (62).  Conversely, FAK does not seem to be involved in cell adhesion in mature B cells.  We found that blocking FAK activity in splenic B-2 and MZ B cells did not significantly affect their adhesion to ICAM-1.  Thus, FAK seems to play a crucial role in progenitor B cell adhesion but mature B cells may use other signaling molecules such as Pyk2 to mediate integrin activation and cell adhesion.  However, as FAK is involved in MZ B cell migration, it would be interesting to investigate whether MZ B cells can develop and function normally in transgenic mice with conditional FAK deficiency in B cells.  In addition, both Pyk2 and FAK kinase activities are required for B cell migration, but it is not known whether Pyk2 and FAK can compensate for each other.  This can be tested by simultaneously treating B cells with PF-719 and PF-228 and then examining whether these compounds have an additive inhibitory effect on B cell migration. In response to chemoattractant stimulation, B cells become polarized and migrate towards the stimuli (63).  Persistent directional movement along a chemoattractant gradient requires the establishment of proper protrusive leading edge and polarity (63, 64).  The formation of these protrusive structures is controlled by dynamic reorganization of the actin cytoskeleton, whereas cell polarization is promoted by asymmetric distribution of signaling   132 molecules.  In our study, we found that Pyk2 and FAK may play distinct roles in cell migration.  While Pyk2 is important for both B cell locomotion and directional migration, FAK has a greater role in establishing the direction of B cell movement.  Our findings are consistent with the previous observations in other cell types, which showed that Pyk2 controls the speed of cell motility whereas FAK regulates the formation of a leading edge during cell migration (61, 65).  It is not known how FAK controls directional protrusion formation in a migrating cell.  One of the signaling molecules that is crucial for directional cell migration is PI3K (66, 67).  At the leading edge of a migrating cell, PI3K activation creates a localized accumulation of PIP3, which can regulate the distribution of key regulators of actin polymerization such as DOCK2 and Vav (a RacGEFs), WAVE2, WASP, and PIX- Cdc42GEF complexes (67-70), thereby allowing the formation of actin-rich protrusions in along the chemoattractant gradient.  In B cells, p110δ is the dominant isoform of the PI3K catalytic subunit and has shown to be crucial for B cell chemotaxis (10, 71).  The lack of a functional p110δ subunit, as well as treatment with a pharmacological inhibitor of p110δ catalytic activity impairs directional migration along a chemokine gradient, but not random cell motility in B cells (10, 71-74).   Importantly, phosphorylation of FAK at Y397 provides a binding site for the p85 regulatory subunit of PI3K, and PI3K activity is enhanced by the association with FAK (75).  Although the molecular mechanism of how FAK regulates B cell chemotaxis is currently not known, one possibility is that FAK may promote the leading edge formation and maintain the polarity of migrating cell via regulating the PI3K pathway.  Our finding that CXCL13- and S1P-induced activation of Akt, a downstream target of PI3K, depends on FAK suggests that FAK could play a key role in linking these receptors to PI3K signaling.  CXCR5 or S1P receptor signaling at the leading edge of a cell could therefore result in the localized formation of FAK-PI3K complexes that promote directional cell migration. In contrast to the potential role of FAK in regulating the polarity of migrating cells, our results suggest that Pyk2 is involved in both the chemotaxis and chemokinesis responses in B cells.  The specific mechanism by which Pyk2 controls cell migration was not determined, but Pyk2 may regulate cell motility by modulating the activation of the Rho family GTPases RhoA, Rac and Cdc42, which control actin reorganization and polymerization (76).  In particular, Pyk2 can promote the formation of lamellapodia and   133 filopodia by binding p190RhoGEF, Vav, and the Cdc42-liked GTPase Wrch1 (77, 78).  Thus treating cells with the Pyk2 inhibitor could reduce the activation of these GTPases. MZ B cell retention in the spleen is critically dependent on signals from chemoattractant receptors and the LFA-1 and α4β1 (VLA-4) integrins.  The expression of the S1P1 receptor is important for proper MZ B cell localization in the spleen.  Although CXCR5 is not essential for MZ B cell development, a balanced chemotactic gradient between S1P and CXCL13 is required for MZ B cells to remain in the MZ and to shuttle between the MZ and follicles, where they can transfer blood-borne Ags to FDCs (21).  We found that Pyk2 is important for S1P- and CXCL13-induced MZ B cell migration and for MZ B cells to adhere to ICAM-1.  Moreover, since Pyk2 is involved in morphological changes in B cells (32), the loss of MZ B cells seen in Pyk2-/- mice may be due to of the inability to these cells to spread and undergo strong adhesion to the resident cells in the MZ area.  The current model for B cell development suggests that MZ progenitor cells need to bind delta-like 1, the ligand for Notch2, while in the red pulp in order to differentiate into mature MZ B cells (2). It is likely that S1P and other chemotactic signals direct MZ B cell precursors to the red pulp and thereby promote their differentiation.  As Pyk2 is involved in S1P-induced migration of MZ B cells, it is also possible that the MZ B cell precursors in Pyk2-/- mice are unable to localize to the splenic red pulp and receive the Notch2 signal required for them to develop. The activation of Pyk2 by S1P or CXCL13 depends on activation of the Rap GTPases (33).  Rap is a master regulator of cell adhesion and migration and is critical for chemoattractants to simulate B cell chemotaxis and adhesion, as well as lymphocyte polarization and cytoskeletal rearrangement (33, 79, 80).  Although FAK has shown to be involved in SDF-1-induced Rap1 activation in a progenitor-B cell line (36), it is not known how FAK promotes Rap activation and whether FAK acts upstream or downstream of Rap activation in the context of CXCL13 and S1P signaling in mature B cells.  Further work is required to dissect the connection between FAK and the Rap GTPases in chemoattractant receptor signaling. In this study, we also found that FAK links chemoattractant receptors, and to a lesser extent the BCR, to the PI3K/Akt signaling pathway.  In neutrophils and Dictyostelium discoideum, activated Akt moves from the cytosol to the leading edge of the cell during migration, suggesting a possible role for Akt in chemoattractant-induced migration (81).  Akt   134 can also promote cytoskeletal reorganization during cell migration by phosphorylating the actin binding protein Girdin/APE (45).  Whether Akt contributes to chemoattractant-induced B cell migration can be tested by treating B cells with specific inhibitors of Akt activity such as KP372-1 (82).  Chemoattractant-induced Akt activation might also promote other cellular functions such as growth, proliferation and survival in B cells (43).  Recently, various Pyk2 and FAK inhibitors have been developed and tested in animal models for studies on tumorgenesis and osteoporosis (38-41).  The high potency and low toxicity of this series of small molecules inhibitors give them the potential to be useful tools to dissect the role of Pyk2 and FAK in vivo.  In addition, as the Pyk2 inhibitor did not affect Akt activation, it may be useful for blocking B cell migration to sites where B cells may initiate inflammation by acting as APCs, without affecting B cell survival. MZ B cells contribute to autoimmune diseases via the production of self-reactive Abs and by functioning as APCs.  The production of self-reactive Abs can cause inflammatory responses against host tissues and result in chronic autoimmune diseases.  For example, New Zealand Black mice spontaneously develop an autoimmune disease resembling systemic lupus, and they have an expanded MZ B cell population that is hyperactivated and produces anti-DNA Abs (83, 84).  Moreover, the trafficking of MZ B cells could also contribute to the initiation and the development of autoimmune diseases.  TLR signaling changes the responsiveness of MZ B cells to various chemoattractants (85), possibly due to changes in chemoattractant receptor expression.  This gives them the potential to leave their normal localization in the spleen and migrate to other organs.  For example, MZ B cells may contribute to the initiation of type 1 diabetes by functioning as APCs.  MZ B cells activated by TLR signaling are found in the pancreatic lymph nodes, where they can present auto-Ag insulin to auto-reactive T cells, which then initiate the destruction of pancreatic beta cells (16).  Thus, limiting the trafficking and activation of MZ B cells could be an important strategy for treating and controlling autoimmune diseases. In summary, we have shown that Pyk2 and FAK are important for transmitting signals downstream from CXCR5 and S1P receptors, which play an important role in determining B cell localization in the spleen, a process that controls their Ag encounter and activation.  We have also characterized the effect of highly-selective small molecule inhibitors of Pyk2 and FAK on primary B cells.  Because Pyk2 is involved in the   135 development, trafficking and localization of MZ B cells, which have been implicated in autoimmunity, future work should investigate the ability of PF-719 to control the production of auto-reactive Abs and limit inflammatory responses in mouse models of autoimmune disease.    136 3.4 Materials and methods 3.4.1 Animals, cells, and reagents C57BL/6 mice were used at 6-12 wk of age.  B cells were isolated from the spleens of C57BL/6 mice using the MACS B cell isolation kit (Miltenyi Biotec, Auburn, CA) to deplete non-B cells (33).  The resulting cells were >98% B cells, as determined by staining with anti- CD19-FITC (BD Pharmingen).  The A20 B-lymphoma cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained as described previously (79).  Goat and donkey anti-mouse IgG, as well as goat anti-mouse IgM, were from Jackson ImmunoResearch Laboratories (West Grove, PA).  CXCL13 was from R&D Systems and sphingosine 1-phosphate (S1P) was from BioMol.  7-aminoactinomycin D (7-AAD) was from Sigma-Aldrich.  Goat anti-Pyk2 (sc-1514) and goat anti-FAK (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA).  The 4G10 monoclonal anti-phosphotyrosine (P-Tyr) Ab was from Upstate (Charlottesville, VA).  Horseradish peroxidase-conjugated donkey anti- goat IgG (Santa Cruz), goat anti-rabbit IgG (Bio-Rad, Hercules, CA), and goat anti-mouse IgG (GE Healthcare Bio-Sciences, Baie d’Urfe, Quebec, Canada) were used for immunoblotting.  PF-431396 and PF-573228 have been described previously (40, 41).  PF- 3430719 was obtained from Pfizer.  3.4.2 Western blotting A20 cells or splenic B cells (1.5 x 107) in 1 ml modified HEPES-buffered saline (33) were stimulated with anti-Ig Abs, S1P, or CXCL13.  Reactions were terminated by adding 0.25 ml of cold 5X lysis buffer (79).  After 10 min on ice, insoluble material was removed by centrifugation.  Anti-Pyk2 or anti-FAK Abs were added to the lysates to immunoprecipitate Pyk2 and FAK.  The phosphorylation of Pyk2 and FAK were detected by immunoblotting with the 4G10 anti-P-Tyr monoclonal Ab.  After stripping, blots were reprobed with the appropriate Abs as a loading control.  Where indicated, aliquots of cell lysate were removed to assess total protein tyrosine phosphorylation using the 4G10 Ab.    137 3.4.3 Cell viability assay Total splenic B cells (1 x 106) were incubated in HEPES-buffed saline with 2% FBS and treated with the indicated concentrations of inhibitors for 8 h at 37oC.  The cells were then incubated with 7-AAD staining solution (0.1 mg/ml of 7-AAD in 100 µL PBS) for 20 min, followed by analysis using a BD Bioscience LSRII flow cytometer.  Cell death was assessed by the uptake of 7-AAD.  3.4.4 Chemotaxis and chemokinesis assays Transwell migration assays were performed as described (33, 86).   Total mouse splenocytes (2 x 106) were added to the upper chamber and allowed to migrate through 5-µm pore size Transwell inserts (Corning).  For chemotaxis assays, the lower wells contained 600 µl of RPMI-1640/10 mM HEPES/0.2% BSA with or without 100 nM CXCL13 or S1P.  After 3 h at 37oC, cells that migrated into the lower chamber were incubated with an Fcγ receptor- specific monoclonal Ab (2.4G2, American Type Culture Collection) and then stained with anti-CD45R-pacific blue, anti-CD23-FITC, and anti-CD21-PE (eBioscience).  The cells were then counted for 30 s using a BD Bioscience LSRII flow cytometer.  Data were analyzed with FlowJo software (Tree Star).  The percent migration for follicular (CD45R/B220+, CD21int, CD23high) and MZ (CD45R/B220+, CD21high, CD23low) B cells was determined by comparison to the 100% control in which 2 x 106 cells were added directly to the bottom chamber.  For chemokinesis assays, chemoattractants were added to both the top and bottom chambers of the Transwell.  The migration of cells into the lower chamber was then analyzed as described for chemotaxis assays.  3.4.5 Adhesion assay Adhesion assay were performed as described (86), with minor modifications.  Maxisorp 96- well plates were coated with 50 µg/ml anti-human IgG Fc-specific Ab (Jackson ImmunoResearch) overnight at 4oC.  The plates were then washed once with PBS before adding 2.5 µg/ml mouse ICAM-1-human Fc (R&D Systems) for 2 h at room temperature. After blocking the plates with 2% BSA for 1 h, cells were stimulated in suspension and   138 allowed to adhere to the ICAM-coated wells for 1 h at 37oC.  The wells were then washed with warm buffer (RPMI-1640 with 10 mM HEPES) and adherent cells were detached by adding ice cold RPMI-1640 with 5 mM EDTA and incubating on ice for 20 min.  Fc receptors were blocked with the 2.4G2 monoclonal Ab and the cells were stained with anti- CD45R-pacific blue, anti-CD23-FITC, and anti-CD21-PE Abs.  The cells were then analyzed by flow cytometry, collecting for 1 min with the LSRII cytometer.  The percent adhesion for each condition was determined by comparison to the 100% control in which 106 splenocytes were analyzed directly by FACS.  3.4.6 Phospho-flow analysis Purified splenic cells (106) were stimulated in 0.25 ml modified HEPES-buffered saline (33). The cells were then pelleted, fixed in 3% paraformaldehyde at 37oC for 10 min, and stained as described (87), with minor modifications.  Briefly, the cells were permeabilized with PBS containing 0.2% saponin for 15 min on ice, blocked with 20% donkey serum, and incubated overnight with rabbit anti-phospho-Akt (S473) (Cell Signaling Technologies).  The cells were then stained with biotin-linked donkey anti-rabbit IgG (F(ab’)2-specific, Jackson ImmunoResearch Laboratories) followed by incubation with streptavidin-PE-Cy7 (eBiosciences).  Fc receptors were then blocked with the 2.4G2 monoclonal Ab and the cells were stained with anti-CD45R-Pacific blue, anti-CD23-FITC, and anti-CD21-PE.  Data were acquired using a BD Biosciences LSRII flow cytometer.   139 3.5 References 1. Martin, F., A. M. Oliver, and J. F. Kearney. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14:617-629. 2. Pillai, S., and A. Cariappa. 2009. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 9:767-777. 3. Balazs, M., F. Martin, T. Zhou, and J. Kearney. 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17:341-352. 4. Pillai, S., A. Cariappa, and S. T. Moran. 2005. Marginal zone B cells. Annu Rev Immunol 23:161-196. 5. Martin, F., and J. F. Kearney. 2002. Marginal-zone B cells. Nat Rev Immunol 2:323- 335. 6. Martin, F., and J. F. Kearney. 2000. B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a "natural immune memory". Immunol Rev 175:70-79. 7. Fleming, S. D. 2006. Natural antibodies, autoantibodies and complement activation in tissue injury. Autoimmunity 39:379-386. 8. Ochsenbein, A. F., T. Fehr, C. Lutz, M. Suter, F. Brombacher, H. Hengartner, and R. M. Zinkernagel. 1999. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286:2156-2159. 9. Binder, C. J., M. Y. Chou, L. Fogelstrand, K. Hartvigsen, P. X. Shaw, A. Boullier, and J. L. Witztum. 2008. Natural antibodies in murine atherosclerosis. Curr Drug Targets 9:190-195. 10. Clayton, E., G. Bardi, S. E. Bell, D. Chantry, C. P. Downes, A. Gray, L. A. Humphries, D. Rawlings, H. Reynolds, E. Vigorito, and M. Turner. 2002. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation. J Exp Med 196:753-763. 11. Chen, Y., M. Yu, A. Podd, R. Wen, M. Chrzanowska-Wodnicka, G. C. White, and D. Wang. 2008. A critical role of Rap1b in B-cell trafficking and marginal zone B-cell development. Blood 111:4627-4636. 12. Guinamard, R., M. Okigaki, J. Schlessinger, and J. V. Ravetch. 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 1:31-36. 13. Bugatti, S., V. Codullo, R. Caporali, and C. Montecucco. 2007. B cells in rheumatoid arthritis. Autoimmun Rev 7:137-142. 14. Wither, J. E., A. D. Paterson, and B. Vukusic. 2000. Genetic dissection of B cell traits in New Zealand black mice. The expanded population of B cells expressing up- regulated costimulatory molecules shows linkage to Nba2. Eur J Immunol 30:356- 365. 15. Jongstra-Bilen, J., B. Vukusic, K. Boras, and J. E. Wither. 1997. Resting B cells from autoimmune lupus-prone New Zealand Black and (New Zealand Black x New Zealand White)F1 mice are hyper-responsive to T cell-derived stimuli. J Immunol 159:5810-5820.    140 16. Marino, E., M. Batten, J. Groom, S. Walters, D. Liuwantara, F. Mackay, and S. T. Grey. 2008. Marginal-zone B-cells of nonobese diabetic mice expand with diabetes onset, invade the pancreatic lymph nodes, and present autoantigen to diabetogenic T- cells. Diabetes 57:395-404. 17. Croker, B. A., D. M. Tarlinton, L. A. Cluse, A. J. Tuxen, A. Light, F. C. Yang, D. A. Williams, and A. W. Roberts. 2002. The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes. J Immunol 168:3376-3386. 18. Fukui, Y., O. Hashimoto, T. Sanui, T. Oono, H. Koga, M. Abe, A. Inayoshi, M. Noda, M. Oike, T. Shirai, and T. Sasazuki. 2001. Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412:826-831. 19. Rubtsov, A., P. Strauch, A. Digiacomo, J. Hu, R. Pelanda, and R. M. Torres. 2005. Lsc regulates marginal-zone B cell migration and adhesion and is required for the IgM T-dependent antibody response. Immunity 23:527-538. 20. Lu, T. T., and J. G. Cyster. 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297:409-412. 21. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, and J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat Immunol 5:713-720. 22. Cinamon, G., M. A. Zachariah, O. M. Lam, F. W. Foss, Jr., and J. G. Cyster. 2008. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat Immunol 9:54-62. 23. Suzuki, K., I. Grigorova, T. G. Phan, L. M. Kelly, and J. G. Cyster. 2009. Visualizing B cell capture of cognate antigen from follicular dendritic cells. J Exp Med 206:1485- 1493. 24. Rovida, E., B. Lugli, V. Barbetti, S. Giuntoli, M. Olivotto, and P. Dello Sbarba. 2005. Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF. Biol Chem 386:919-929. 25. van Buul, J. D., E. C. Anthony, M. Fernandez-Borja, K. Burridge, and P. L. Hordijk. 2005. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin- based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation. J Biol Chem 280:21129-21136. 26. Okigaki, M., C. Davis, M. Falasca, S. Harroch, D. P. Felsenfeld, M. P. Sheetz, and J. Schlessinger. 2003. Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration. Proc Natl Acad Sci U S A 100:10740-10745. 27. Avraham, H., S. Y. Park, K. Schinkmann, and S. Avraham. 2000. RAFTK/Pyk2- mediated cellular signalling. Cell Signal 12:123-133. 28. Benbernou, N., K. Muegge, and S. K. Durum. 2000. Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha. J Biol Chem 275:7060-7065. 29. Pandey, P., S. Avraham, S. Kumar, A. Nakazawa, A. Place, L. Ghanem, A. Rana, V. Kumar, P. K. Majumder, H. Avraham, R. J. Davis, and S. Kharbanda. 1999. Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism. J Biol Chem 274:10140-10144. 30. Takino, T., M. Nakada, H. Miyamori, Y. Watanabe, T. Sato, D. Gantulga, K. Yoshioka, K. M. Yamada, and H. Sato. 2005. JSAP1/JIP3 cooperates with focal   141 adhesion kinase to regulate c-Jun N-terminal kinase and cell migration. J Biol Chem 280:37772-37781. 31. Huang, C., K. Jacobson, and M. D. Schaller. 2004. MAP kinases and cell migration. J Cell Sci 117:4619-4628. 32. Tse, K. W., M. Dang-Lawson, R. L. Lee, D. Vong, A. Bulic, L. Buckbinder, and M. R. Gold. 2009. B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading. J Biol Chem 284:22865-22877. 33. Durand, C. A., J. Westendorf, K. W. Tse, and M. R. Gold. 2006. The Rap GTPases mediate CXCL13- and sphingosine1-phosphate-induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes. Eur J Immunol 36:2235-2249. 34. Glodek, A. M., M. Honczarenko, Y. Le, J. J. Campbell, and L. E. Silberstein. 2003. Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis. J Exp Med 197:461-473. 35. Astier, A., H. Avraham, S. N. Manie, J. Groopman, T. Canty, S. Avraham, and A. S. Freedman. 1997. The related adhesion focal tyrosine kinase is tyrosine- phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas. J Biol Chem 272:228-232. 36. Glodek, A. M., Y. Le, D. M. Dykxhoorn, S. Y. Park, G. Mostoslavsky, R. Mulligan, J. Lieberman, H. E. Beggs, M. Honczarenko, and L. E. Silberstein. 2007. Focal adhesion kinase is required for CXCL12-induced chemotactic and pro-adhesive responses in hematopoietic precursor cells. Leukemia 21:1723-1732. 37. Le, Y., B. M. Zhu, B. Harley, S. Y. Park, T. Kobayashi, J. P. Manis, H. R. Luo, A. Yoshimura, L. Hennighausen, and L. E. Silberstein. 2007. SOCS3 protein developmentally regulates the chemokine receptor CXCR4-FAK signaling pathway during B lymphopoiesis. Immunity 27:811-823. 38. Roberts, W. G., E. Ung, P. Whalen, B. Cooper, C. Hulford, C. Autry, D. Richter, E. Emerson, J. Lin, J. Kath, K. Coleman, L. Yao, L. Martinez-Alsina, M. Lorenzen, M. Berliner, M. Luzzio, N. Patel, E. Schmitt, S. LaGreca, J. Jani, M. Wessel, E. Marr, M. Griffor, and F. Vajdos. 2008. Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res 68:1935-1944. 39. Bagi, C. M., G. W. Roberts, and C. J. Andresen. 2008. Dual focal adhesion kinase/Pyk2 inhibitor has positive effects on bone tumors: implications for bone metastases. Cancer 112:2313-2321. 40. Slack-Davis, J. K., K. H. Martin, R. W. Tilghman, M. Iwanicki, E. J. Ung, C. Autry, M. J. Luzzio, B. Cooper, J. C. Kath, W. G. Roberts, and J. T. Parsons. 2007. Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem 282:14845- 14852. 41. Buckbinder, L., D. T. Crawford, H. Qi, H. Z. Ke, L. M. Olson, K. R. Long, P. C. Bonnette, A. P. Baumann, J. E. Hambor, W. A. Grasser, 3rd, L. C. Pan, T. A. Owen, M. J. Luzzio, C. A. Hulford, D. F. Gebhard, V. M. Paralkar, H. A. Simmons, J. C. Kath, W. G. Roberts, S. L. Smock, A. Guzman-Perez, T. A. Brown, and M. Li. 2007. Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis. Proc Natl Acad Sci U S A 104:10619-10624.   142 42. Jones, M. L., A. J. Shawe-Taylor, C. M. Williams, and A. W. Poole. 2009. Characterization of a novel focal adhesion kinase inhibitor in human platelets. Biochem Biophys Res Commun 389:198-203. 43. Manning, B. D., and L. C. Cantley. 2007. AKT/PKB signaling: navigating downstream. Cell 129:1261-1274. 44. Durand, C. A., K. Hartvigsen, L. Fogelstrand, S. Kim, S. Iritani, B. Vanhaesebroeck, J. L. Witztum, K. D. Puri, and M. R. Gold. 2009. Phosphoinositide 3-kinase p110 delta regulates natural antibody production, marginal zone and B-1 B cell function, and autoantibody responses. J Immunol 183:5673-5684. 45. Enomoto, A., H. Murakami, N. Asai, N. Morone, T. Watanabe, K. Kawai, Y. Murakumo, J. Usukura, K. Kaibuchi, and M. Takahashi. 2005. Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell 9:389-402. 46. Sadhu, C., B. Masinovsky, K. Dick, C. G. Sowell, and D. E. Staunton. 2003. Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement. J Immunol 170:2647-2654. 47. Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration. J Cell Biol 171:349-359. 48. Kanemitsu, N., Y. Ebisuno, T. Tanaka, K. Otani, H. Hayasaka, T. Kaisho, S. Akira, K. Katagiri, T. Kinashi, N. Fujita, T. Tsuruo, and M. Miyasaka. 2005. CXCL13 is an arrest chemokine for B cells in high endothelial venules. Blood 106:2613-2618. 49. Laudanna, C., J. Y. Kim, G. Constantin, and E. Butcher. 2002. Rapid leukocyte integrin activation by chemokines. Immunol Rev 186:37-46. 50. Rieken, S., A. Sassmann, S. Herroeder, B. Wallenwein, A. Moers, S. Offermanns, and N. Wettschureck. 2006. G12/G13 family G proteins regulate marginal zone B cell maturation, migration, and polarization. J Immunol 177:2985-2993. 51. Hatch, W. C., R. K. Ganju, D. Hiregowdara, S. Avraham, and J. E. Groopman. 1998. The related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated and participates in colony-stimulating factor-1/macrophage colony-stimulating factor signaling in monocyte-macrophages. Blood 91:3967-3973. 52. Bozulic, L., and B. A. Hemmings. 2009. PIKKing on PKB: regulation of PKB activity by phosphorylation. Curr Opin Cell Biol 21:256-261. 53. Marshall, A. J., H. Niiro, T. J. Yun, and E. A. Clark. 2000. Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase Cgamma pathway. Immunol Rev 176:30-46. 54. Spiegel, S., and S. Milstien. 2003. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 4:397-407. 55. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427:355-360. 56. Lo, C. G., Y. Xu, R. L. Proia, and J. G. Cyster. 2005. Cyclical modulation of sphingosine-1-phosphate receptor 1 surface expression during lymphocyte recirculation and relationship to lymphoid organ transit. J Exp Med 201:291-301. 57. Ostergaard, H. L., and T. L. Lysechko. 2005. Focal adhesion kinase-related protein tyrosine kinase Pyk2 in T-cell activation and function. Immunol Res 31:267-282.   143 58. Park, S. Y., H. K. Avraham, and S. Avraham. 2004. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279:33315-33322. 59. Mitra, S. K., D. A. Hanson, and D. D. Schlaepfer. 2005. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 6:56-68. 60. Liu, E., J. F. Cote, and K. Vuori. 2003. Negative regulation of FAK signaling by SOCS proteins. EMBO J 22:5036-5046. 61. Riol-Blanco, L., N. Sanchez-Sanchez, A. Torres, A. Tejedor, S. Narumiya, A. L. Corbi, P. Sanchez-Mateos, and J. L. Rodriguez-Fernandez. 2005. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J Immunol 174:4070-4080. 62. Nagasawa, T. 2006. Microenvironmental niches in the bone marrow required for B- cell development. Nat Rev Immunol 6:107-116. 63. Kehrl, J. H. 2006. Chemoattractant receptor signaling and the control of lymphocyte migration. Immunol Res 34:211-227. 64. Thelen, M., and J. V. Stein. 2008. How chemokines invite leukocytes to dance. Nat Immunol 9:953-959. 65. Tilghman, R. W., J. K. Slack-Davis, N. Sergina, K. H. Martin, M. Iwanicki, E. D. Hershey, H. E. Beggs, L. F. Reichardt, and J. T. Parsons. 2005. Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells. J Cell Sci 118:2613-2623. 66. Ward, S. G. 2004. Do phosphoinositide 3-kinases direct lymphocyte navigation? Trends Immunol 25:67-74. 67. Stephens, L., L. Milne, and P. Hawkins. 2008. Moving towards a better understanding of chemotaxis. Curr Biol 18:R485-494. 68. Kunisaki, Y., A. Nishikimi, Y. Tanaka, R. Takii, M. Noda, A. Inayoshi, K. Watanabe, F. Sanematsu, T. Sasazuki, T. Sasaki, and Y. Fukui. 2006. DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis. J Cell Biol 174:647- 652. 69. Li, Z., M. Hannigan, Z. Mo, B. Liu, W. Lu, Y. Wu, A. V. Smrcka, G. Wu, L. Li, M. Liu, C. K. Huang, and D. Wu. 2003. Directional sensing requires G beta gamma- mediated PAK1 and PIX alpha-dependent activation of Cdc42. Cell 114:215-227. 70. Oikawa, T., H. Yamaguchi, T. Itoh, M. Kato, T. Ijuin, D. Yamazaki, S. Suetsugu, and T. Takenawa. 2004. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Nat Cell Biol 6:420-426. 71. Okkenhaug, K., A. Bilancio, G. Farjot, H. Priddle, S. Sancho, E. Peskett, W. Pearce, S. E. Meek, A. Salpekar, M. D. Waterfield, A. J. Smith, and B. Vanhaesebroeck. 2002. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science 297:1031-1034. 72. Reif, K., K. Okkenhaug, T. Sasaki, J. M. Penninger, B. Vanhaesebroeck, and J. G. Cyster. 2004. Cutting edge: differential roles for phosphoinositide 3-kinases, p110gamma and p110delta, in lymphocyte chemotaxis and homing. J Immunol 173:2236-2240. 73. Bilancio, A., K. Okkenhaug, M. Camps, J. L. Emery, T. Ruckle, C. Rommel, and B. Vanhaesebroeck. 2006. Key role of the p110delta isoform of PI3K in B-cell antigen   144 and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110delta function in B cells. Blood 107:642-650. 74. Durand, C. A., K. Hartvigsen, L. Fogelstrand, S. Kim, S. Iritani, B. Vanhaesebroeck, J. L. Witztum, K. D. Puri, and M. R. Gold. 2009. Phosphoinositide 3-kinase p110delta regulates natural antibody production, marginal zone and B-1 B cell function, and autoantibody responses. J Immunol 183:5673-5684. 75. Chen, H. C., P. A. Appeddu, H. Isoda, and J. L. Guan. 1996. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3- kinase. J Biol Chem 271:26329-26334. 76. Tybulewicz, V. L., and R. B. Henderson. 2009. Rho family GTPases and their regulators in lymphocytes. Nat Rev Immunol 9:630-644. 77. Gismondi, A., J. Jacobelli, R. Strippoli, F. Mainiero, A. Soriani, L. Cifaldi, M. Piccoli, L. Frati, and A. Santoni. 2003. Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration. J Immunol 170:3065-3073. 78. Lim, Y., S. T. Lim, A. Tomar, M. Gardel, J. A. Bernard-Trifilo, X. L. Chen, S. A. Uryu, R. Canete-Soler, J. Zhai, H. Lin, W. W. Schlaepfer, P. Nalbant, G. Bokoch, D. Ilic, C. Waterman-Storer, and D. D. Schlaepfer. 2008. PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility. J Cell Biol 180:187-203. 79. McLeod, S. J., A. J. Shum, R. L. Lee, F. Takei, and M. R. Gold. 2004. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem 279:12009-12019. 80. Ishida, D., L. Su, A. Tamura, Y. Katayama, Y. Kawai, S. F. Wang, M. Taniwaki, Y. Hamazaki, M. Hattori, and N. Minato. 2006. Rap1 signal controls B cell receptor repertoire and generation of self-reactive B1a cells. Immunity 24:417-427. 81. Van Haastert, P. J., and P. N. Devreotes. 2004. Chemotaxis: signalling the way forward. Nat Rev Mol Cell Biol 5:626-634. 82. Mandal, M., M. Younes, E. A. Swan, S. A. Jasser, D. Doan, O. Yigitbasi, A. McMurphey, J. Ludwick, A. K. El-Naggar, C. Bucana, G. B. Mills, and J. N. Myers. 2006. The Akt inhibitor KP372-1 inhibits proliferation and induces apoptosis and anoikis in squamous cell carcinoma of the head and neck. Oral Oncol 42:430-439. 83. Zeng, D., M. K. Lee, J. Tung, A. Brendolan, and S. Strober. 2000. Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice. J Immunol 164:5000- 5004. 84. Schuster, H., T. Martin, L. Marcellin, J. C. Garaud, J. L. Pasquali, and A. S. Korganow. 2002. Expansion of marginal zone B cells is not sufficient for the development of renal disease in NZBxNZW F1 mice. Lupus 11:277-286. 85. Rubtsov, A. V., C. L. Swanson, S. Troy, P. Strauch, R. Pelanda, and R. M. Torres. 2008. TLR agonists promote marginal zone B cell activation and facilitate T- dependent IgM responses. J Immunol 180:3882-3888. 86. Rieken, S., S. Herroeder, A. Sassmann, B. Wallenwein, A. Moers, S. Offermanns, and N. Wettschureck. 2006. Lysophospholipids control integrin-dependent adhesion in splenic B cells through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11). J Biol Chem 281:36985-36992.   145 87. Firaguay, G., and J. A. Nunes. 2009. Analysis of signaling events by dynamic phosphoflow cytometry. Sci Signal 2:pl3.    146 4. Concluding chapter 4.1 Summary and overview The overall goal of this thesis was to examine the regulation and function of the Pyk2 and FAK tyrosine kinases in B cells.  In chapter two, I investigated how Pyk2 and FAK are activated in response to BCR and integrin activation and their roles in regulating B cell morphology.  Since Rap is an important regulator of B cell morphology, I also examined how Rap regulates Pyk2 and FAK activation.  Finally, with the use of Pyk2- and FAK- specific inhibitors, I studied the roles of Pyk2 and FAK in chemokine-induced signaling, migration and adhesion in primary B cells, particularly MZ B cells.  Overall, I found that Pyk2 and FAK are downstream targets of the Rap GTPases and play a key role in regulating B cell morphology, migration, and adhesion.  Together, these findings highlight the regulation of Pyk2 and FAK as well as their functions in B cell trafficking and morphological regulation. The capture of Ags by B cells often occurs in the context of integrin engagement. Integrin-dependent cell spreading enhances the ability of B cells to contact Ags, and integrin signaling may synergize with BCR signaling to promote B cell spreading and activation. One of the main findings of this thesis is that Pyk2 and FAK are key regulators of B cell morphology, and that the kinase activities of Pyk2 and FAK are important for BCR/integrin- induced B cell spreading.  I also characterized the expression and localization of Pyk2 and FAK in primary B cells.  Pyk2 and FAK have distinct localizations in B cells in that FAK co-localizes with the integrins LFA-1 and VLA-4 whereas Pyk2 is uniformly distributed in the cytoplasm with a diffuse pattern.  I also found that activating B cells results in an up- regulation of FAK expression and down-regulation of Pyk2 expression.  These activated B cells also express a longer isoform of Pyk2, which is normally found in the nervous system. Moreover, I showed that integrin engagement enhances the ability of BCR to phosphorylate Pyk2 and FAK on their auto/transphosphorylation sites, the initial step in the activation of these kinases.  I also found that Rap activation is critical for BCR/integrin-induced phosphorylation of Pyk2 and FAK.  Furthermore, I showed that clustering-induced outside- in integrin signaling leads to the activation of Pyk2 and FAK in a Rap-dependent manner. Together, these results suggest that Rap might regulate B cell morphology via these two   147 kinases.  Interestingly, Pyk2 and FAK are differentially regulated by BCR signaling when integrins are not engaged.  I showed that Rap activation is required for the BCR to induce Pyk2 phosphorylation but is not required for BCR-stimulated FAK phosphorylation. Finally, I showed that the requirement for Rap activation in BCR- and BCR/integrin- induced Pyk2 and FAK tyrosine phosphorylation correlates with a requirement for actin dynamics, suggesting that Rap might regulate Pyk2 and FAK by modulating the actin cytoskeleton.  Thus, in response to integrin engagement, Pyk2 and FAK are activated in a coordinated manner and then act to promote B cell spreading. In chapter three, I extended my findings from B-lymphoma cells to primary B cells in which I examined the roles of Pyk2 and FAK in B cell migration and adhesion.  I showed that the chemoattractants S1P and CXCL13 induce the phosphorylation of both Pyk2 and FAK on their tyrosine residues.  Using the newly developed small molecule inhibitors with high specificity against Pyk2 and FAK, I showed that Pyk2 and FAK are required for conventional B-2 cells to migrate in response to CXCL13.  I also showed that Pyk2 and FAK are required for MZ B cell migration towards S1P and CXCL13.  Pyk2 appears to regulate general B cell migration rather than specifically regulate directional movement (chemotaxis) because both chemotaxis and chemokinesis were reduced in the presence of Pyk2 inhibitors.  Furthermore, I found that the LFA-1-dependent adhesion of both follicular B-2 and MZ B cells required Pyk2 activity but not FAK activity.  Chemokine receptor signaling activates the PI3K/Akt pathway, which is important for B cell activation and survival and I showed that FAK is required for chemoattractant-induced Akt activation in B cells.  Together, the results suggest that Pyk2 and FAK play an important role in propagating signals from the receptors for S1P and CXCL13 and in regulating B-2 and MZ B cell migration (chapter 3). In summary, Pyk2 and FAK have an important function in integrating signals from the BCR, integrins, and chemoattractant receptors and translating these signals into changes in cell morphology, adhesion, and migration.  The Rap GTPases, master regulators of B cell spreading, migration, and adhesion (1, 2), link receptor signaling to phosphorylation of both Pyk2 and FAK, possibly by regulating actin dynamics.  Since MZ B cells are missing in Pyk2-/- mice (3), the function of Pyk2 in MZ B cells had not been addressed previously, but it was assumed that it is important for adhesion interactions that retain MZ B cells in the   148 MZ.  The results from this thesis demonstrate for the first time that Pyk2 is involved in the migration and adhesion of MZ B cells in response to chemoattractants.  Therefore, Pyk2 could regulate MZ B cell trafficking and retention in vivo.  Together with the recent implication of MZ B cells in the development of autoimmune disease, the findings in this thesis suggest that Pyk2 and FAK could be potential targets for drugs that could be used to treat B cell-mediated autoimmune diseases and limit the metastatic spread of malignant B cells.  Pyk2 inhibitors may be particularly useful because Pyk2 is not ubiquitously expressed like FAK.  The normal development of Pyk2-deficient mice (3, 4) indicates that Pyk2 activity is not essential for the majority of normal cellular processes, whereas FAK knockout mice exhibit embryonic lethality (5).  Thus, targeting Pyk2 may be a useful approach for modulating immune cell trafficking and function, without affecting the survival and normal function of other cell types.   149 4.2 Discussion and future directions 4.2.1 The in vivo functions of Pyk2 in MZ B cells The absence of MZ B cells in Pyk2-deficient mice could be due to their failure to localize to the MZ, processes that require cell adhesion and migration.  The ability to mediate proper chemotaxis and adhesion is crucial for MZ B cell localization, as illustrated by studies showing that blocking the binding of integrins to their ligands or inhibiting the Gi heterotrimeric G protein results in the release of MZ B cells from the MZ (3, 6).  In chapter three, I showed that blocking Pyk2 kinase activity with a Pyk2-specific inhibitor reduced MZ B cell migration to CXCL13 and S1P, as well as their adhesion to ICAM-1.  Thus a key experiment is to test whether Pyk2 activity is required for proper MZ B cell positioning in the spleen in vivo.  A recent study on the role of the p110δ isoform of the PI3K catalytic subunit in innate-liked B cells showed that administering a p110δ-selective inhibitor to mice resulted in substantially reduced numbers of MZ B cells surrounding the follicles (7).  Thus, one can test the in vivo functions of Pyk2 by treating wild-type mice with the Pyk2-selective inhibitor and then performing immunostaining and fluorescence microscopy on spleen sections to examine whether it causes the loss of MZ B cells from the MZ area in the spleen. An alternate explanation for the loss of MZ B cells in Pyk2-knockout mice is that Pyk2 activity is required for the differentiation of MZ B cells.  Recent observations in knockout mice with altered MZ B cell compartments revealed a number of requirements for MZ B cell development.  In the spleen, the differentiation from T2 B cells into MZ precursor (MZP) B cells and then MZ B cells requires weak BCR signal strength, signaling via the receptor for BAFF, a cytokine that promotes B cell development and survival, activation of the canonical NFκB pathway (presumably by BAFF-R signaling), and Notch2 activation by the DL1 family of Notch ligands (8).  The interplay of these signals with adhesion and migration signals that promote the proper localization of MZ B cells is not fully understood.  For example, MZ B cells may need to localize to the MZ in order to be exposed to optimal levels of BAFF.  Transgenic mice deficient in Rap1b or Lsc (9, 10), which are important for cell migration and adhesion, have reduced numbers of MZ B cell numbers, suggesting that localization is important for MZ B cell development and maintenance.  However, Pyk2-/- mice exhibit a complete loss of MZ B cells, suggesting that   150 Pyk2 might be involved in the development of MZ B cells, in addition to its functions in cell migration and adhesion (3).  Although Pyk2 is a downstream target of BCR signaling, it is unlikely that Pyk2 functions to limit BCR signaling such that a weak BCR signal promotes MZ B cell lineage commitment occurs.  My preliminary results showed that BCR signaling events appear to be normal in A20 B-lymphoma cells in which Pyk2 expression was knocked down using shRNA constructs (unpublished result).  Thus, it would be interesting to investigate whether Pyk2 is involved in BAFF-R or Notch2 signaling in purified MZ B cells and their precursors. FAK is highly expressed in the GC and MZ B cells but the role of FAK in MZ B cells is not known (11).  In chapter three, I showed that FAK is involved in MZ B cell migration towards the chemoattractants S1P and CXCL13.  Signals from the S1P receptor are required for proper positioning of MZ B cell in the spleen (12).  Therefore, it would be interesting to examine whether FAK is required for proper MZ B cell development.  Since FAK-/- mice exhibit embryonic lethality (5), one can test this by crossing the mb1-Cre mice with the floxed FAK transgenic mice to generate a mouse strain with a B cell-specific disruption of the FAK gene (13-15).  This idea can also be tested by treating mice with the FAK-selective inhibitor and examining whether blocking FAK kinase activity results in the loss of MZ B cells from the spleen in vivo. B cells contribute to the inflammatory response and to autoimmune disease by producing auto-Abs and inflammatory cytokines, and by acting as APCs (16).  Since Pyk2 is involved in B cell migration and is required for the generation of effective humoral immune response (3), Pyk2 could be involved in the development of B cell-mediated autoimmune diseases.  MZ B cells have been implicated in the initiation of type-1 diabetes (17), an autoimmune disease resulting from the destruction of insulin-producing islet cells by the immune system (18).  In NOD mice, the MZ B compartment is expanded and MZ B cells are found in the pancreatic lymph node where they can present self-Ags to auto-reactive diabetogenic T cells.  These activated T cells can subsequently migrate to the pancreas and destroy the insulin-producing pancreatic beta cells.  TLR signaling in MZ B cells alters their chemoattractant receptor expression and allows MZ B cells to exit the spleen and migrate to other sites (19).  Therefore, blocking MZ B cell migration might be able to delay the onset or decrease the severity of type-1 diabetes by preventing MZ B cells from acting as APCs in   151 the pancreatic lymph nodes.  This hypothesis can be tested by determining whether treating NOD mice with the Pyk2-specific inhibitor can delay the onset, or reduce the incidence, of diabetes.  4.2.2 The role of Pyk2 and FAK in B cell immune synapse formation, polarization, and proliferation In this thesis, I examined the role of Pyk2 and FAK in B cell morphological regulation, migration and adhesion, but it is not known whether these two kinases are involved in other cellular processes for B cells.  B cell recognition of membrane-bound Ag leads to immune synapse formation.  When B cells encounter Ag on an APC, BCR signaling induces rapid cell spreading and subsequent contraction that results in Ag-bound BCRs being gathered into the center of the synapse (20).  BCR signaling also triggers activation of the LFA-1 integrin, leading to ICAM-1 binding and organization of LFA-1 into a ring-liked structure surrounding the center of the synapse.  This integrin-dependent interaction with the APC increases the contact area between the B cell and the Ag-bearing membrane, thereby allowing more BCRs to bind Ags (21).  In T cells, Pyk2 is relocalized to the immune synapse upon Ag receptor activation.  Also, since Rap activation is required for B cell immune synapse formation, as well as BCR-induced activation of Pyk2 and FAK (22), Pyk2 and FAK might function in the same Rap-dependent pathway that leads to the immune synapse formation.  This can be examined by using the planar lipid bilayer system described by Batista and colleagues (21), in which fluorescently labeled anti-κ light chain Abs as surrogate Ags, and fluorescently-labeled GPI-linked ICAM-1 as an LFA-1 ligand, are incorporated into the lipid bilayer, allowing the formation of the immune synapse to be visualized by fluorescence microscopy.  With the use of Pyk2- and FAK-selective inhibitors, as well as the Pyk2- and FAK-knockdown variants of the A20 cell line that I generated, the contribution of Pyk2 and FAK to immune synapse formation can be studied. Multivalent particulate Ags such as viral particles can activate B cells by initiating BCR signaling at a focused contact site, thereby establishing a cell polarity that is accompanied by localized cytoskeletal reorganization and signaling (23).  This type of Ag stimulation can be mimicked using anti-Ig-coated beads, which induces the formation of   152 actin-rich cups and membrane protrusions that extend around the beads (22).  This process is also dependent on Rap activation and we have shown that the RapL adaptor protein, which binds activated Rap and contribute to integrin activation, accumulates at the contact site with the bead (22).  Thus one can test whether inhibiting Pyk2 and FAK kinase activities with inhibitors, or knocking down the expression of Pyk2 or FAK, prevents the formation of these F-actin cups, as well as the subsequent relocalization of RapL to the contact site with the anti-Ig-coated bead. TLR ligands are potent activators of B cells.  TLR ligands increase the expression of the B cell activation markers CD69 and CD86 and promote the survival and proliferation of B cells (7, 24, 25).  A recent study in macrophages has shown that Pyk2 can interact with MyD88 (26), a crucial signaling adaptor protein involved in signaling by all TLRs except TLR3.  This interaction contributes to LPS-induced NF-κB activation and signaling in macrophages (26, 27).  However, the functions of Pyk2 and FAK in TLR-induced B cell activation and proliferation has not been investigated.  B cells primarily express TLR9, which recognizes unmethylated bacterial DNA (CpG DNA), and TLR4, which recognizes LPS (24, 25).  Thus it would be interesting to test whether treating splenic B cells, or purified MZ B cells, with Pyk2- or FAK-selective inhibitors would inhibit the ability of CpG DNA or LPS to induce their proliferation and differentiation into Ab-secreting cells (7, 28).  CFSE dilution could be used to assess in vitro proliferation and ELISA could be used to assess Ab secretion.  If Pyk2 and FAK are involved in these processes, then inhibitors of these kinases may be useful for limiting B cell activation and Ab secretion in autoimmune inflammatory diseases such as lupus.  4.2.3 Pathways upstream of Pyk2 and FAK in B cells 4.2.3.1 The regulation of Pyk2 and FAK activation by Rap  In chapter two, I showed that Rap-GTP promotes Pyk2 phosphorylation via its ability to remodel the actin cytoskeleton.  Although Pyk2 phosphorylation requires an intact actin cytoskeleton in a number of cell types (29), how this contributes to Pyk2 phosphorylation is not clear.  Park et al. showed that Pyk2 undergoes trans- autophosphorylation at Y402 to create a docking site for SFKs (30), allowing the subsequent   153 phosphorylation of other tyrosine sites in the catalytic domain.  Since the first step of Pyk2 autophosphorylation depends on their dimerization, Rap-dependent actin remodeling could create a cytoskeletal platform that facilitates the dimerization of Pyk2.  This can be examined by co-expressing the HA-tagged and the FLAG-tagged wildtype Pyk2 in the same cell and the dimer formation can be shown by co-immunoprecipitation of the two differently-tagged forms of Pyk2.  Accordingly, the role of Rap can be determined by examining whether RapGAPII expression inhibits the ability of Pyk2 to form a dimer after receptor activation.  Using a similar co-immunoprecipitation approach, Kohno et al. showed that Pyk2 binding to the Ca2+/calmodulin complex promotes its dimerization (31). However, the role of Rap in promoting intracellular Ca2+ flux has not yet defined in B cells. Thus, testing whether RapGAPII expression inhibits intracellular Ca2+ flux and Pyk2 dimerization will give better insights into how Rap regulates Pyk2 activation. The actin-interacting protein hematopoietic lineage cell-specific protein 1 (HS1), which plays an important role in regulating cytoskeleton dynamics and cell polarity, has been shown to modulate Pyk2 tyrosine phosphorylation (32, 33).  HS1 is the hematopoietic lineage-restricted homolog of the actin-binding protein cortactin (34).  Both cortactin and HS1 are substrates of SFK and by binding Arp2/3 and actin filaments they promote the formation and stabilization of a branched filament network (32, 35, 36).  HS1 is required for the formation of immune synapses in T cells, as well as for chemotaxis, adhesion, and cell polarization in NK cells (33, 37).  Phosphorylation of HS1 creates binding sites for Vav1, such that HS1 can contribute to Cdc42 and Rac1 activation (32).  Interestingly, HS1- deficient NK cells exhibit impaired phosphorylation of SFKs, Lyn, Pyk2 and Vav1in response to β1 integrin engagement (33), suggesting a role of HS1 in regulating Pyk2 activation.  Both HS1 and Pyk2 control the ability of cells to send out protrusions in the direction of migration.  HS1-knockdown cells exhibit randomly oriented protrusions similar to the manner in which Pyk2-/- macrophages extend multiple lamellipodia in different directions when stimulated with chemokine (4, 37).  Moreover, HS1 is important for chemokine-induced migration (33).  In NK cells, CXCL12 induces robust phosphorylation of HS1.  By expressing a non-phosphorylatable mutant form of HS1 in HS-1 deficient NK cells, Butler et al. showed that the phosphorylation of HS1 is required for chemotaxis as well as the activation of Vav1, Rac1 and Cdc42 (33).  Rap-GTP-deficient cells share many   154 similar phenotypes with HS1-deficicent cells in term of cell polarization, migration, adhesion, and actin polymerization (1, 33, 38, 39).  Therefore, Rap and HS1 may work in the same pathway to modulate actin dynamics and other cellular processes.  This pathway could involve Pyk2 or FAK.  Notably, Pyk2 and FAK can bind to the SH3 domain of HS-1 via their proline-rich regions (33).  Since both Rap and HS-1 are important for Pyk2 and FAK tyrosine phosphorylation, it is worthwhile to examine whether Rap is required for HS- 1 phosphorylation and whether HS-1 is important for actin cytoskeleton remodeling and thereby facilitates Pyk2 and FAK activation.  4.2.3.2 Identification of new regulators of Pyk2 and FAK phosphorylation by high- throughput screening  Although the list of Pyk2 and FAK interacting proteins is continuously growing, how Pyk2 and FAK are activated by different surface receptors is still not completely understood.  The signaling molecules that lie upstream of Pyk2 and FAK activation need to be identified in order to have a full understanding of the signaling mechanisms leading to Pyk2 and FAK phosphorylation and activation.  One approach would be to perform high- throughput screening for potential upstream signaling molecules using RNAi libraries.  This can be done by using robotic liquid handling methods to treat cells with the different siRNAs, followed by the use of flow cytometry to measure Pyk2 and FAK phosphorylation with phospho-specific Abs.  This approach could potentially identify new signaling molecules that link the BCR or chemoattractant receptors to Pyk2 and FAK activation. Importantly, because the activation of Pyk2 and FAK by different receptors may involve different signaling molecules, this approach could reveal distinct pathways by which different cell surface receptors such as the BCR, chemoattractant receptors, and integrins activate these kinases.  As Pyk2 and FAK activation has been linked to tumorgenesis and other disease models (40-47), understanding the molecular mechanisms underlying the regulation of these kinases by different cell surface receptors will provide insights into alterations that might lead to disease states.   155 4.2.4 Potential signaling pathways downstream of Pyk2 and FAK in B cells Cell spreading and migration are complex processes that require dynamic spatiotemporal integration of signals from surface receptors to coordinate cell polarity and cytoskeletal remodeling.  In this thesis, I showed that Pyk2 and FAK are important regulators of B cell morphology and migration, and that FAK is involved in chemokine- induced activation of the Akt pro-survival kinase.  Future studies are required to understand how Pyk2 and FAK control these cellular processes in B cells.  Some potential downstream targets of Pyk2 and FAK in B cell are summarized in Figure 4.1 and discussed below.   Figure 4.1 Potential downstream targets of Pyk2 and FAK in B cells. Pyk2 and FAK can interact with multiple signaling molecules to modulate cytoskeletal structure, to promote cell adhesion and spreading, and to establish cell polarity.  Pyk2 and FAK may regulate actin cytoskeleton dynamics by coordinating the activation of the Rac, Cdc42, and RhoA GTPases.  Pyk2 can bind to Wrch1, which is a Cdc42-liked GTPase, and thereby promote filopodia formation.  Pyk2 and FAK can also control the cycle of RhoA inhibition and activation by modulating the activities of p190RhoGAP and p190RhoGEF, respectively.   RhoA controls the formation of adhesive points (also known as focal dots), which are transient clusters of activated integrins that form in lymphocytes.  FAK has been shown to control cell spreading and the formation of protrusive lamellipodia via a direct association with the Arp2/3 complex.  Also, Pyk2 may promote the stability of actin filaments by negatively regulating the actin severing proteins cofilin and gelsolin.  Finally, Pyk2 and FAK may promote polarization by interacting with Dlg1, a component of the Scribble polarity complex.   156 Recent work in other cell types has shown that Pyk2 and FAK regulate actin cytoskeleton dynamics by coordinating the activation of the Rac, Cdc42, and RhoA GTPases via several mechanisms.  In fibroblasts, Pyk2 promotes the formation of filopodia via its association with Wrch1, a Cdc42-like GTPase (48).  In T cells, activation of the chemokine receptor CXCR4 causes Pyk2 to bind Vav, an exchange factor that activates Rac (49).  Recently, Tomar et al. showed that phosphorylation of FAK at Y397 allows p120RasGAP to bind via its SH2 domain and that this facilitates phosphorylation of p190RhoGAP by FAK, thereby leading to cell polarization following integrin engagement (50).  By inhibiting RhoA activity at cell protrusions by activating RhoGAP, FAK stabilizes adhesion sites at the leading edge of the cell and facilitates cell spreading and migration. Conversely, both Pyk2 and FAK can also interact with the RhoA activator p190RhoGEF, and some RhoA activation is detected at lamellipodial protrusions (51).  During cell migration and spreading, the regulation of RhoA is probably cyclical because cells actively form and remodel their adhesive point within membrane protrusions.  Thus by controlling the cycle of RhoA inhibition and activation, Pyk2 and FAK may connect receptor signaling to cytoskeletal changes that underlie cell migration and cell spreading. Integrin engagement during cell attachment regulates local actin assembly and membrane protrusion at the leading edge.  Actin filament production at lamellipodia is controlled by the Arp2/3 complex (52), which promotes the addition of actin monomers to filament branch-points, leading to the formation of a branched dendritic actin network that provides the force for membrane protrusion (53, 54).  The Arp2/3 complex can be activated by upstream signaling from Rac and Cdc42 though the regulation of the Scar/WAVE or WASP adaptor proteins (32).  Recently, Serrels et al. showed that FAK can control cell spreading and the formation of protrusive lamellipodia via a direct association with the Arp2/3 complex (55).  The FERM domain of FAK binds directly to Arp3 and this association can enhance Apr2/3-dependent actin polymerization.  Therefore, FAK could be a direct link between integrin signaling and the actin polymerization machinery in B cells, thereby promoting integrin-induced cell spreading.  To measure the formation of new actin filament, B cells can be permeabilized and with the incorporation of purified monomeric actin conjugated a fluorescent dye can be measured over time by confocal microscopy (56). Using this assay, the role of FAK in promoting actin polymerization can be tested by   157 comparing control cells to cells treated with the FAK-specific inhibitor or cells in which FAK expression has been knocked down using shRNA. Recently, our lab showed that another actin binding protein, cofilin, plays a crucial role in B cell spreading.  When cofilin is in its dephosphorylated active form, it can sever actin filaments (57-59).  Cofilin can be phosphorylated and inactivated by LIMK whereas the Slingshot phosphatase can activate cofilin by dephosphorylating it.  By overexpressing a constitutively inactive form of Slingshot protein in A20 B cells, cofilin remains phosphorylated and inactive.  Importantly, without the active form of cofilin present, B cells were unable to spread and remodel their cytoskeleton (S. Freeman and M. Gold, unpublished results).  Because knocking down Pyk2 expression and treating B cells with the Pyk2 inhibitor inhibit cell spreading, one can propose that Pyk2 regulates B cell spreading by promoting the dephosphorylation and activation of cofilin.  This idea can be tested using both shRNA knockdown of Pyk2 and the Pyk2 inhibitor. It has been suggested that there are two different cofilin populations that exist simultaneously in cells during chemotaxis: one that is locally activated, allowing sensing and localized protrusion, and one that is globally phosphorylated and usually inhibited, possibly for the maintenance of cell body rigidity (60).  This balance of local excitation and global inhibition results in an asymmetric distribution of cofilin activity, promoting cell protrusion formation and movement.  Chemokine stimulation results in global inhibition of cofilin activity via Rho/ROCK/LIMK-induced phosphorylation of cofilin (see Chapter 1.5 for more information).  Although the mechanism underlying how chemoattractant receptor activation leads to cofilin phosphorylation and lymphocyte migration is not well characterized, a study done in DC showed that the chemokine receptor CCR7 modulates migratory speed via a Rho/Pyk2/cofilin pathway (61).  Similar to what I showed for CXCR5 and the S1P receptor (62), CCR7 signaling results in a very rapid (<1 min) phosphorylation of Pyk2 (61).  Also, expression of a dominant negative mutant of Pyk2 (PRNK) results in a decrease in the migratory speed of DC, as well as a block in chemokine-induced cofilin phosphorylation (61).  Because Pyk2 promotes cofilin phosphorylation in DCs, Pyk2 might promote the formation of cell protrusion in B cells by promoting the asymmetric distribution of cofilin activity during B cell migration and spreading.   158 Pyk2 can also regulate actin cytoskeletal organization via another actin binding protein, gelsolin.  Gelsolin modifies actin-filament length by severing pre-existing filaments and by capping the fast-growing barbed ends (63).  In osteoclasts, integrin activation promotes the binding of Pyk2 to gelsolin, resulting in the phosphorylation of gelsolin (64). Gelsolin phosphorylation promotes its association with phosphatidylinositiol, thereby preventing it from capping actin filaments at barbed ends and thereby allowing the growth of actin filaments.  Similarly, Pyk2 activation promotes the phosphorylation of cofilin and thereby prevents actin being severed (discussed above).  Thus Pyk2 may modulate the activities of multiple actin regulators to control lamellipodial dynamics during B cell adhesion and migration.  Notably, neutrophils from gelsolin-deficient mice are defective in migration (65).  It would be interesting to examine whether B cells isolated from these mice also have defects in adhesion and migration. Discs large 1 (Dlg1 or SAP97) is another potential Pyk2-interacting protein.  Dlg1 is an actin-binding protein that is important for establishing cell polarity and actin polymerization.  It is a scaffolding protein that consists of multiple signaling domains (32). In T cells, Dlg1 is recruited to cortical actin upon TCR engagement and transiently forms complexes with TCR signaling components including Lck, Zap70, and WASP (66, 67). Interestingly, Pyk2 has been reported to associate with Zap70 in T cells (49), suggesting that Pyk2 and Dlg1 could be in the same signaling complex.  Pyk2 has also been reported to interact via its proline-rich domain with PSD-95 and SAP102, homologues of Dlg1, (68). Whether Pyk2 interacts with Dlg1 in B cells during migration or particulate Ag encounter would be interesting to investigate.  Dlg1 is part of a conserved polarity complex involving Scribble, Lethal giant larvae and Dlg.  The Scribble complex is necessary for the initial polarization events necessary for directed migration in multiple cell types such as astrocytes, epithelial cells, and T cells (69).  Our lab has recently shown that Rap activation controls the localization of Scribble in B cells (V. Lei and M. Gold, unpublished results).  Because Pyk2 is involved in B cell migration and macrophage polarization (4), interacting with the Dlg1/Scribble complex could be one of the ways that Pyk2 promotes cell polarization.    159 4.2.5 Role of Pyk2 and FAK in tumor dissemination and development Chemokine-induced cell migration and integrin-induced adhesion are important for malignant cells to home to different organs and initiate the formation of new tumors, a process known as metastasis.  Malignant B cells home to different organs in response to chemokines such as CXCL12, CCL19, CCL21, and CXCL13, which are normally immobilized on the surface of vascular endothelial cells (70, 71).  The directional cue from the chemokine and the adhesive signal from the integrins are both required for malignant B cells to migrate across the endothelium and establish growth in the new tissue (70). FAK is highly expressed in most human B cell leukemias and lymphomas but is not expressed in human T cell lymphomas (11).  FAK expression is also detected in numerous murine B cell lines that arose from different stages in B cell development (see Appendix A). As demonstrated in this thesis, Pyk2 and FAK can transmit signals from integrins and chemoattractant receptors, resulting in cell migration and cytoskeletal remodeling.  More importantly, the upstream regulator of Pyk2 and FAK, Rap1, is necessary for the dissemination of B cell lymphomas (72).  Blocking Rap activation by overexpressing RapGAPII in the A20 B cell line reduced the ability of these cells to establish new tumors in vivo after intravenous injection.  Therefore, an interesting area for future study would be to address the role of Pyk2 and FAK in B-lymphoma cell migration across the endothelium as well as in the dissemination of these malignant B cells.   One can test this by doing in vitro transendothelial migration assays in which cells are allowed to migrate through Transwell inserts coated with TNF-α-activated bEnd.3 murine endothelial cells.  One can then examine whether pre-treating the B-lymphoma cells with Pyk2 and FAK inhibitors or knocking down the expression of Pyk2 and FAK prevents them from migrating across the endothelial cell layer.  If so, the roles of Pyk2 and FAK in metastasis can be further examined by injecting stable Pyk2 and FAK knockdown A20 B-lymphoma cells into Balb/c mice and monitoring the ability of these cells to form tumors in different organs.  An important extension of this experiment is to determine whether treating mice with Pyk2- or FAK-selective inhibitors can reduce lymphoma growth or dissemination.  A small molecule inhibitor of FAK activity has been shown to effectively block tumor growth in vivo (73). Therefore, targeting Pyk2 and FAK with inhibitors might be an effective approach for controlling the growth or metastasis of malignant B cells.   160 4.3 Conclusion In this thesis, I made new findings relating to the regulation of Pyk2 and FAK in B cells and the functions of these kinases in controlling B cell morphology, migration, and adhesion.  Future work should focus on identifying new regulators and effectors of Pyk2 and FAK, as well as identifying additional functions for these kinases in B cells.  By using a Pyk2-selective inhibitor, I was able to examine the function of Pyk2 in primary MZ B cells, which do not develop in Pyk2-/- mice.  The ability of Pyk2 and FAK inhibitors to inhibit B cell responses in vitro suggests that these drugs could be used to modulate B cell functions in vivo.  In particular, since unregulated B cell activation can lead to the development of autoimmunity and to tumorgenesis, drugs that target Pyk2 and FAK in B cell may be useful for treating autoimmune diseases and B cell tumors.  With a better understanding of the mechanisms involved in normal and malignant B cell activation, adhesion, morphological regulation, and trafficking, this knowledge can be useful for future clinical applications.    161 4.4 References  1. Bos, J. L. 2005. Linking Rap to cell adhesion. Curr Opin Cell Biol 17:123-128. 2. Kinashi, T., and K. Katagiri. 2004. Regulation of lymphocyte adhesion and migration by the small GTPase Rap1 and its effector molecule, RAPL. Immunol Lett 93:1-5. 3. Guinamard, R., M. Okigaki, J. Schlessinger, and J. V. Ravetch. 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 1:31-36. 4. Okigaki, M., C. Davis, M. Falasca, S. Harroch, D. P. Felsenfeld, M. P. Sheetz, and J. Schlessinger. 2003. Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration. Proc Natl Acad Sci U S A 100:10740-10745. 5. Ilic, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, and T. Yamamoto. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377:539- 544. 6. Lu, T. T., and J. G. Cyster. 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297:409-412. 7. Durand, C. A., K. Hartvigsen, L. Fogelstrand, S. Kim, S. Iritani, B. Vanhaesebroeck, J. L. Witztum, K. D. Puri, and M. R. Gold. 2009. Phosphoinositide 3-kinase p110 delta regulates natural antibody production, marginal zone and B-1 B cell function, and autoantibody responses. J Immunol 183:5673-5684. 8. Pillai, S., and A. Cariappa. 2009. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 9:767-777. 9. Chen, Y., M. Yu, A. Podd, R. Wen, M. Chrzanowska-Wodnicka, G. C. White, and D. Wang. 2008. A critical role of Rap1b in B-cell trafficking and marginal zone B- cell development. Blood 111:4627-4636. 10. Rubtsov, A., P. Strauch, A. Digiacomo, J. Hu, R. Pelanda, and R. M. Torres. 2005. Lsc regulates marginal-zone B cell migration and adhesion and is required for the IgM T-dependent antibody response. Immunity 23:527-538. 11. Ozkal, S., J. C. Paterson, S. Tedoldi, M. L. Hansmann, A. Kargi, S. Manek, D. Y. Mason, and T. Marafioti. 2009. Focal adhesion kinase (FAK) expression in normal and neoplastic lymphoid tissues. Pathol Res Pract 205:781-788. 12. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, and J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat Immunol 5:713-720. 13. Glodek, A. M., Y. Le, D. M. Dykxhoorn, S. Y. Park, G. Mostoslavsky, R. Mulligan, J. Lieberman, H. E. Beggs, M. Honczarenko, and L. E. Silberstein. 2007. Focal adhesion kinase is required for CXCL12-induced chemotactic and pro-adhesive responses in hematopoietic precursor cells. Leukemia 21:1723-1732. 14. Sauer, B. 1998. Inducible gene targeting in mice using the Cre/lox system. Methods 14:381-392. 15. Pelanda, R., E. Hobeika, T. Kurokawa, Y. Zhang, S. Kuppig, and M. Reth. 2002. Cre recombinase-controlled expression of the mb-1 allele. Genesis 32:154-157.   162 16. LeBien, T. W., and T. F. Tedder. 2008. B lymphocytes: how they develop and function. Blood 112:1570-1580. 17. Marino, E., M. Batten, J. Groom, S. Walters, D. Liuwantara, F. Mackay, and S. T. Grey. 2008. Marginal-zone B-cells of nonobese diabetic mice expand with diabetes onset, invade the pancreatic lymph nodes, and present autoantigen to diabetogenic T- cells. Diabetes 57:395-404. 18. Anderson, M. S., and J. A. Bluestone. 2005. The NOD mouse: a model of immune dysregulation. Annu Rev Immunol 23:447-485. 19. Rubtsov, A. V., C. L. Swanson, S. Troy, P. Strauch, R. Pelanda, and R. M. Torres. 2008. TLR agonists promote marginal zone B cell activation and facilitate T- dependent IgM responses. J Immunol 180:3882-3888. 20. Fleire, S. J., J. P. Goldman, Y. R. Carrasco, M. Weber, D. Bray, and F. D. Batista. 2006. B cell ligand discrimination through a spreading and contraction response. Science 312:738-741. 21. Carrasco, Y. R., S. J. Fleire, T. Cameron, M. L. Dustin, and F. D. Batista. 2004. LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20:589-599. 22. Lin, K. B., S. A. Freeman, S. Zabetian, H. Brugger, M. Weber, V. Lei, M. Dang- Lawson, K. W. Tse, R. Santamaria, F. D. Batista, and M. R. Gold. 2008. The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands. Immunity 28:75-87. 23. Batista, F. D., D. Iber, and M. S. Neuberger. 2001. B cells acquire antigen from target cells after synapse formation. Nature 411:489-494. 24. Borsutzky, S., K. Kretschmer, P. D. Becker, P. F. Muhlradt, C. J. Kirschning, S. Weiss, and C. A. Guzman. 2005. The mucosal adjuvant macrophage-activating lipopeptide-2 directly stimulates B lymphocytes via the TLR2 without the need of accessory cells. J Immunol 174:6308-6313. 25. Bekeredjian-Ding, I., and G. Jego. 2009. Toll-like receptors--sentries in the B-cell response. Immunology 128:311-323. 26. Xi, C. X., F. Xiong, Z. Zhou, L. Mei, and W. C. Xiong. 2009. PYK2 interacts with MyD88 and regulates MyD88-mediated NF-{kappa}B activation in macrophages. J Leukoc Biol. 27. Zeisel, M. B., V. A. Druet, J. Sibilia, J. P. Klein, V. Quesniaux, and D. Wachsmann. 2005. Cross talk between MyD88 and focal adhesion kinase pathways. J Immunol 174:7393-7397. 28. Weill, J. C., S. Weller, and C. A. Reynaud. 2009. Human marginal zone B cells. Annu Rev Immunol 27:267-285. 29. Avraham, H., S. Y. Park, K. Schinkmann, and S. Avraham. 2000. RAFTK/Pyk2- mediated cellular signalling. Cell Signal 12:123-133. 30. Park, S. Y., H. K. Avraham, and S. Avraham. 2004. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279:33315-33322. 31. Lev, S., H. Moreno, R. Martinez, P. Canoll, E. Peles, J. M. Musacchio, G. D. Plowman, B. Rudy, and J. Schlessinger. 1995. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature 376:737-745.   163 32. Burkhardt, J. K., E. Carrizosa, and M. H. Shaffer. 2008. The actin cytoskeleton in T cell activation. Annu Rev Immunol 26:233-259. 33. Butler, B., D. H. Kastendieck, and J. A. Cooper. 2008. Differently phosphorylated forms of the cortactin homolog HS1 mediate distinct functions in natural killer cells. Nat Immunol 9:887-897. 34. Kitamura, D., H. Kaneko, Y. Miyagoe, T. Ariyasu, and T. Watanabe. 1989. Isolation and characterization of a novel human gene expressed specifically in the cells of hematopoietic lineage. Nucleic Acids Res 17:9367-9379. 35. Weaver, A. M., A. V. Karginov, A. W. Kinley, S. A. Weed, Y. Li, J. T. Parsons, and J. A. Cooper. 2001. Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation. Curr Biol 11:370-374. 36. Uruno, T., P. Zhang, J. Liu, J. J. Hao, and X. Zhan. 2003. Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex- mediated actin polymerization. Biochem J 371:485-493. 37. Gomez, T. S., S. D. McCarney, E. Carrizosa, C. M. Labno, E. O. Comiskey, J. C. Nolz, P. Zhu, B. D. Freedman, M. R. Clark, D. J. Rawlings, D. D. Billadeau, and J. K. Burkhardt. 2006. HS1 functions as an essential actin-regulatory adaptor protein at the immune synapse. Immunity 24:741-752. 38. McLeod, S. J., A. J. Shum, R. L. Lee, F. Takei, and M. R. Gold. 2004. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem 279:12009- 12019. 39. McLeod, S. J., A. H. Li, R. L. Lee, A. E. Burgess, and M. R. Gold. 2002. The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor- 1 (CXCL12): potential role for Rap2 in promoting B cell migration. J Immunol 169:1365-1371. 40. Lipinski, C. A., and J. C. Loftus. Targeting Pyk2 for therapeutic intervention. Expert Opin Ther Targets 14:95-108. 41. Behmoaram, E., K. Bijian, S. Jie, Y. Xu, A. Darnel, T. A. Bismar, and M. A. Alaoui- Jamali. 2008. Focal adhesion kinase-related proline-rich tyrosine kinase 2 and focal adhesion kinase are co-overexpressed in early-stage and invasive ErbB-2-positive breast cancer and cooperate for breast cancer cell tumorigenesis and invasiveness. Am J Pathol 173:1540-1550. 42. Lark, A. L., C. A. Livasy, B. Calvo, L. Caskey, D. T. Moore, X. Yang, and W. G. Cance. 2003. Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses. Clin Cancer Res 9:215-222. 43. Lark, A. L., C. A. Livasy, L. Dressler, D. T. Moore, R. C. Millikan, J. Geradts, M. Iacocca, D. Cowan, D. Little, R. J. Craven, and W. Cance. 2005. High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype. Mod Pathol 18:1289-1294. 44. Owens, L. V., L. Xu, R. J. Craven, G. A. Dent, T. M. Weiner, L. Kornberg, E. T. Liu, and W. G. Cance. 1995. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 55:2752-2755. 45. Tremblay, L., W. Hauck, A. G. Aprikian, L. R. Begin, A. Chapdelaine, and S. Chevalier. 1996. Focal adhesion kinase (pp125FAK) expression, activation and   164 association with paxillin and p50CSK in human metastatic prostate carcinoma. Int J Cancer 68:164-171. 46. Zhang, S., X. Qiu, Y. Gu, and E. Wang. 2008. Up-regulation of proline-rich tyrosine kinase 2 in non-small cell lung cancer. Lung Cancer 62:295-301. 47. Brunton, V. G., and M. C. Frame. 2008. Src and focal adhesion kinase as therapeutic targets in cancer. Curr Opin Pharmacol 8:427-432. 48. Ruusala, A., and P. Aspenstrom. 2008. The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics. Mol Cell Biol 28:1802-1814. 49. Okabe, S., S. Fukuda, Y. J. Kim, M. Niki, L. M. Pelus, K. Ohyashiki, P. P. Pandolfi, and H. E. Broxmeyer. 2005. Stromal cell-derived factor-1alpha/CXCL12-induced chemotaxis of T cells involves activation of the RasGAP-associated docking protein p62Dok-1. Blood 105:474-480. 50. Tomar, A., S. T. Lim, Y. Lim, and D. D. Schlaepfer. 2009. A FAK-p120RasGAP- p190RhoGAP complex regulates polarity in migrating cells. J Cell Sci 122:1852- 1862. 51. Lim, Y., S. T. Lim, A. Tomar, M. Gardel, J. A. Bernard-Trifilo, X. L. Chen, S. A. Uryu, R. Canete-Soler, J. Zhai, H. Lin, W. W. Schlaepfer, P. Nalbant, G. Bokoch, D. Ilic, C. Waterman-Storer, and D. D. Schlaepfer. 2008. PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility. J Cell Biol 180:187-203. 52. Stradal, T. E., and G. Scita. 2006. Protein complexes regulating Arp2/3-mediated actin assembly. Curr Opin Cell Biol 18:4-10. 53. Svitkina, T. M., and G. G. Borisy. 1999. Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J Cell Biol 145:1009-1026. 54. Mullins, R. D., J. A. Heuser, and T. D. Pollard. 1998. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci U S A 95:6181-6186. 55. Serrels, B., A. Serrels, V. G. Brunton, M. Holt, G. W. McLean, C. H. Gray, G. E. Jones, and M. C. Frame. 2007. Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex. Nat Cell Biol 9:1046-1056. 56. Cai, L., A. M. Makhov, D. A. Schafer, and J. E. Bear. 2008. Coronin 1B antagonizes cortactin and remodels Arp2/3-containing actin branches in lamellipodia. Cell 134:828-842. 57. Huang, Y., and J. K. Burkhardt. 2007. T-cell-receptor-dependent actin regulatory mechanisms. J Cell Sci 120:723-730. 58. Samstag, Y., S. M. Eibert, M. Klemke, and G. H. Wabnitz. 2003. Actin cytoskeletal dynamics in T lymphocyte activation and migration. J Leukoc Biol 73:30-48. 59. Nishita, M., H. Aizawa, and K. Mizuno. 2002. Stromal cell-derived factor 1alpha activates LIM kinase 1 and induces cofilin phosphorylation for T-cell chemotaxis. Mol Cell Biol 22:774-783. 60. Mouneimne, G., V. DesMarais, M. Sidani, E. Scemes, W. Wang, X. Song, R. Eddy, and J. Condeelis. 2006. Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis. Curr Biol 16:2193-2205.   165 61. Riol-Blanco, L., N. Sanchez-Sanchez, A. Torres, A. Tejedor, S. Narumiya, A. L. Corbi, P. Sanchez-Mateos, and J. L. Rodriguez-Fernandez. 2005. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J Immunol 174:4070-4080. 62. Durand, C. A., J. Westendorf, K. W. Tse, and M. R. Gold. 2006. The Rap GTPases mediate CXCL13- and sphingosine1-phosphate-induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes. Eur J Immunol 36:2235-2249. 63. Sun, H. Q., M. Yamamoto, M. Mejillano, and H. L. Yin. 1999. Gelsolin, a multifunctional actin regulatory protein. J Biol Chem 274:33179-33182. 64. Wang, Q., Y. Xie, Q. S. Du, X. J. Wu, X. Feng, L. Mei, J. M. McDonald, and W. C. Xiong. 2003. Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin. J Cell Biol 160:565-575. 65. Witke, W., A. H. Sharpe, J. H. Hartwig, T. Azuma, T. P. Stossel, and D. J. Kwiatkowski. 1995. Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell 81:41-51. 66. Round, J. L., T. Tomassian, M. Zhang, V. Patel, S. P. Schoenberger, and M. C. Miceli. 2005. Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells. J Exp Med 201:419-430. 67. Xavier, R., S. Rabizadeh, K. Ishiguro, N. Andre, J. B. Ortiz, H. Wachtel, D. G. Morris, M. Lopez-Ilasaca, A. C. Shaw, W. Swat, and B. Seed. 2004. Discs large (Dlg1) complexes in lymphocyte activation. J Cell Biol 166:173-178. 68. Seabold, G. K., A. Burette, I. A. Lim, R. J. Weinberg, and J. W. Hell. 2003. Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102. J Biol Chem 278:15040-15048. 69. Humbert, P. O., L. E. Dow, and S. M. Russell. 2006. The Scribble and Par complexes in polarity and migration: friends or foes? Trends Cell Biol 16:622-630. 70. Pals, S. T., D. J. de Gorter, and M. Spaargaren. 2007. Lymphoma dissemination: the other face of lymphocyte homing. Blood 110:3102-3111. 71. Thelen, M., and J. V. Stein. 2008. How chemokines invite leukocytes to dance. Nat Immunol 9:953-959. 72. Lin, K. B., P. Tan, S. A. Freeman, M. Lam, K. M. McNagny, and M. R. Gold. 2009. The Rap GTPases regulate the migration, invasiveness and in vivo dissemination of B-cell lymphomas. Oncogene. 73. Roberts, W. G., E. Ung, P. Whalen, B. Cooper, C. Hulford, C. Autry, D. Richter, E. Emerson, J. Lin, J. Kath, K. Coleman, L. Yao, L. Martinez-Alsina, M. Lorenzen, M. Berliner, M. Luzzio, N. Patel, E. Schmitt, S. LaGreca, J. Jani, M. Wessel, E. Marr, M. Griffor, and F. Vajdos. 2008. Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res 68:1935-1944.    166 A. Appendix:  The expression of Pyk2 and FAK in different mouse and human cell lines A.1 Rationale Pyk2 and FAK are the targets of the BCR, integrins, and chemokine receptors in B cells and play a key role in B cell migration, adhesion, and spreading.   Because the expression of these two kinases could be associated with the ability of malignant B cells to survive and metastasize, I used immunoblotting to assess the expression of Pyk2 and FAK in a panel of mouse and human B cell lines.  The cell lines that were analyzed represent different stages of B cell development including B cell progenitors (mouse: 300-19, K40B.1, 70Z/3; human: REH), IgM+ immature/transitional B cells that are sensitive to anti-Ig-induced apoptosis (mouse: WEHI-231, CH31), surface IgM+ mature B cells (mouse: Bal17, CH12; human: Ramos, Daudi, Raji, BJAB), surface IgG+ B cells that represent class-switched memory B cells (mouse: A20, 2PK3), and a B cell myeloma line that secretes antibodies (mouse: MPC11).   167 A.2 Experimental procedure B cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 50 µM 2-ME, 2 mM glutamine, 1 mM pyruvate, 15 U/ml penicillin, and 50 µg/ml streptomycin.  Cells were then solubilized in RIPA buffer (1).  After 10 min on ice, insoluble material was removed by centrifugation.  Protein concentration of each samples were determined by BCA assay.  Equal amount of protein were resolved in a 8% SDS-PAGE gel. The expression of Pyk2 and FAK was detected by immunoblotting with anti-Pyk2 Ab (Santa Cruz Biotechnology) and anti-FAK Ab (Abcam).  For total loading control, the blots were then stripped and reprobed with Ab against ERK (Santa Cruz Biotechnology).    168 A.3 Results   Figure A.1 Expression of Pyk2 and FAK in mouse and human B cell lines. Pyk2 and FAK protein levels in cell lysates (40 µg protein) from B cell lines were analyzed by sequential blotting with Abs to Pyk2, FAK, and ERK1/2 (loading control).  The cell lines were grouped according to the stages of B cell development that they represent.  All of these cell lines are available from ATCC, with the exception of the 300.19, K40B-1, CH31, and CH12 cell lines, which were a gift from Dr. Anthony DeFranco (Univ. of California, San Francisco).    169 A.4 Conclusions Almost all of these mouse and human B cell lines express Pyk2, with the exception of the BJAB human B lymphoma cell line.  The MPC11 murine myeloma cell line expressed very low levels of Pyk2, as did the CH31 immature B lymphoma cell line.  Thus, Pyk2 is present in most B cell tumor lines.  FAK is expressed to some degree in all of the human B cell lines, including the REH pro-B cell line in which Silberstein and colleagues have studied the regulation of FAK by the chemokine CXCL12 (2, 3).  While FAK is present at significant levels in the WEHI-231, Bal17, and A20 B lymphoma lines, many of the murine B cell lines, in particular the B progenitor cell lines, the MPC11 myeloma cell line, do not express detectable levels of FAK. In B cells, FAK is highly expressed in transformed cells and is upregulated in activated B cells (Figure 2.1).  As reported previously, FAK protein expression is elevated in many highly malignant human cancers (4), and studies have shown that FAK signaling can promote changes in cell shape and the formation of podosomes or invadopodia (5), which leads to an invasive cell phenotype.  On the other hand, normal B cells have a much higher expression of Pyk2 compared to other B lymphoma cells (Figure 2.1).  This suggests that Pyk2 expression may be inversely correlated with the degree of malignancy as observed in prostate cancer cells (6) .  Notably, activating primary B cells with LPS and IL-4 causes an increase in FAK expression and a decrease in Pyk2 expression (Figure 2.1).  The change in expression of Pyk2 and FAK in proliferating tumor cell lines could potentially be a consequence of B cell activation.  As many of these cell lines have been used to study homing and metastasis of B cell tumors, this data might provide some insights into the degree of tumor progression and metastasis when comparing different tumor models.    170 A.5 References 1. McLeod, S. J., A. H. Li, R. L. Lee, A. E. Burgess, and M. R. Gold. 2002. The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor- 1 (CXCL12): potential role for Rap2 in promoting B cell migration. J Immunol 169:1365-1371. 2. Glodek, A. M., M. Honczarenko, Y. Le, J. J. Campbell, and L. E. Silberstein. 2003. Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis. J Exp Med 197:461-473. 3. Le, Y., M. Honczarenko, A. M. Glodek, D. K. Ho, and L. E. Silberstein. 2005. CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro- B Cells. J Immunol 174:2582-2590. 4. Cance, W. G., J. E. Harris, M. V. Iacocca, E. Roche, X. Yang, J. Chang, S. Simkins, and L. Xu. 2000. Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clin Cancer Res 6:2417-2423. 5. Hauck, C. R., D. A. Hsia, D. Ilic, and D. D. Schlaepfer. 2002. v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion. J Biol Chem 277:12487-12490. 6. Picascia, A., R. Stanzione, P. Chieffi, A. Kisslinger, I. Dikic, and D. Tramontano. 2002. Proline-rich tyrosine kinase 2 regulates proliferation and differentiation of prostate cells. Mol Cell Endocrinol 186:81-87.     171 B. Appendix:  The role of Src family kinases in Pyk2 tyrosine phosphorylation B.1 Rationale Maximal activation of FAK’s kinase activity involves auto/transphosphorylation of Y397, the SH2 domain-dependent binding of Src family kinases (SFKs) to these sites, and the subsequent phosphorylation of the activation loop tyrosine residues Y576/Y577 in FAK (1, 2).  FAK-associated SFKs may also amplify FAK activation by phosphorylating Y397 on other FAK molecules (3, 4).  Both BCR activation and integrin clustering can induce Pyk2 tyrosine phosphorylation (Chapter 2).  Pyk2 activation is thought to proceed in the same manner via sequential phosphorylation of Y402, the SFK binding site, and subsequent SFK- dependent phosphorylation of Y579/Y580 in the activation loop (5-7).  To address whether SFKs amplify Pyk2 activation in B cells by contributing to phosphorylation of Y402 in Pyk2, I made use of the SFK inhibitor PP2 and its inactive structural analogue PP3.  The initial step in BCR signaling is activation of SFKs such as Lyn, Fyn, and Blk, which initiate events that result in the activation of PLCγ and Rap GTPases (see Section 1.2.2).  Since PP2 can inhibit the kinase activity of SFK and prevent downstream activation of PLCγ, I bypassed the early BCR signaling events by stimulating the cells with phorbol myristate acetate (PMA), a phorbol ester that mimics the actions of the diacylglycerol (DAG) produced by PLCγ after BCR engagement.  Therefore, the direct requirement of SFKs in Pyk2 tyrosine phosphorylation can be examined.   172 B.2 Experimental procedure  A20 cells (1.5 x 107 cells/ml) in 1 ml HEPES-buffered saline (8) were pretreated with PP2 or PP3 (control) for 30 min.  Cells were then either left in suspension or added to the wells of 6-well tissue culture plates coated a collagen/fibronectin ECM generated by coating the wells first with 2% gelatin solution (type B from bovine skin, Sigma-Aldrich) and then FCS, as described previously (8).  After 30 min at 37oC, the cells were stimulated with PMA (Sigma-Aldrich).  For LFA-1 or VLA-4 clustering, cells pretreated with PP2 or PP3 were added to the wells coated with 30 µg/ml of the TIB213 anti-LFA-1 monoclonal antibody, the PS/2 anti-VLA-4 monoclonal antibody, or BSA (control).  Reactions were terminated by adding 0.25 ml of cold 5X lysis buffer (9), placing the cells on ice for 10 min and then removing insoluble material by centrifugation, as described previously (9).  To immunoprecipitate Pyk2, the cell extracts were mixed with 1 µg of goat anti-Pyk2 Ab (Santa Cruz Biotechnology) for 1 h at 4 °C and then transferred to tubes containing 10 µl of protein G Sepharose beads (Sigma-Aldrich) for 1 h.  Precipitated proteins were separated by SDS- PAGE and followed by immunoblotting with the 4G10 anti-phosphotyrosine monoclonal antibody or antibodies that recognize specific phosphorylation sites on Pyk2.  For ERK phosphorylation, equal amount of protein were analyzed on SDS-PAGE followed by immunoblotting with Abs against P-ERK and ERK (Santa Cruz Biotechnology).   173 B.3 Results  Figure B.1 The effect of SFK inhibitor on Pyk2 tyrosine phosphorylation in response to PMA stimulation and integrin activation.  A, A20 cells were pretreated with 10 µM of PP2 to inhibit Src family kinases or 10 µM of PP3 (control) for 30 minutes at 37oC.  Cells were rested in suspension or were directly plated in wells coated with a collagen/fibronectin ECM and subsequently cells were stimulated with 20 nM of PMA.  Antibody recognizing Pyk2 was used for immunoprecipitation.  Anti-Pyk2 immunoprecipitates were probed with antibody specifically recognize Pyk2 phosphorylation at Y402 (Anti-pY402; upper panel) or with anti- phosphotyrosine monoclonal antibody 4G10 (Anti-P-Tyr; middle panel) and then reprobed with anti-Pyk2 antibody (lower panel). B, A20 cells were prepared and stimulated as described for A.  Anti-Pyk2 immunoprecipitates were probed with antibody specifically recognize Pyk2 phosphorylation at Y579/580 (Anti-pY579/580; upper panel) and then reprobed with anti-Pyk2 antibody (lower panel). C, A20 cells were prepared and stimulated as described for A.  Total cell lysates were probed with antibody against phosphorylated ERK and then reprobed with anti-ERK antibody. D, A20 cells were prepared as described for A.  Cells were then stimulated by plating on Ab-coated wells for the indicated time.  The lysates were then prepared as described in A. Similar results were obtained from three independent experiments.    174 Figure B.1    175 B.4 Conclusions PMA stimulation of A20 cells in the presence of the inactive compound PP3 induced Pyk2 tyrosine phosphorylation.  This response was observed both when the cells were in suspension and when the cells were plated on ECM, with stronger responses occurring when the cells were plated on ECM.  This presumably reflects the ability of PMA to activate integrins in A20 cells and cause Rap activation (9).  In the presence of PP2, PMA-induced tyrosine phosphorylation of Pyk2, as judged using the 4G10 anti-phosphotyrosine monoclonal antibody, was substantially decreased.  This was a specific effect as PMA- induced ERK activation was not affected by PP2 treatment.  Importantly, PMA-induced phosphorylation of Y402 was unaffected, regardless of whether the cells were stimulated in suspension or on ECM.  This indicates that phosphorylation of Pyk2 on Y579/Y580 is dependent on Src family kinases, as expected, whereas Src kinases do not contribute significantly to the phosphorylation of Pyk2 on Y402, the step that initiates Pyk2 activation. In contrast, tyrosine phosphorylation of Pyk2 at Y402 by integrin clustering appears to require SFK activity as PP2 treatment partially reduced integrin-induced Pyk2 phosphorylation.  Thus, there is a differential requirement of SFKs for Pyk2 autophosphorylation in response to BCR signaling versus integrin clustering.    176 B.5 References 1. Hanks, S. K., L. Ryzhova, N. Y. Shin, and J. Brabek. 2003. Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 8:d982-996. 2. Toutant, M., A. Costa, J. M. Studler, G. Kadare, M. Carnaud, and J. A. Girault. 2002. Alternative splicing controls the mechanisms of FAK autophosphorylation. Mol Cell Biol 22:7731-7743. 3. Salazar, E. P., and E. Rozengurt. 2001. Src family kinases are required for integrin- mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem 276:17788-17795. 4. Zeng, L., X. Si, W. P. Yu, H. T. Le, K. P. Ng, R. M. Teng, K. Ryan, D. Z. Wang, S. Ponniah, and C. J. Pallen. 2003. PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration. J Cell Biol 160:137-146. 5. Park, S. Y., H. K. Avraham, and S. Avraham. 2004. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279:33315-33322. 6. Lakkakorpi, P. T., A. J. Bett, L. Lipfert, G. A. Rodan, and T. Duong le. 2003. PYK2 autophosphorylation, but not kinase activity, is necessary for adhesion-induced association with c-Src, osteoclast spreading, and bone resorption. J Biol Chem 278:11502-11512. 7. Qian, D., S. Lev, N. S. van Oers, I. Dikic, J. Schlessinger, and A. Weiss. 1997. Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling. J Exp Med 185:1253-1259. 8. Freundlich, B., and N. Avdalovic. 1983. Use of gelatin/plasma coated flasks for isolating human peripheral blood monocytes. J Immunol Methods 62:31-37. 9. McLeod, S. J., A. J. Shum, R. L. Lee, F. Takei, and M. R. Gold. 2004. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem 279:12009-12019.    177 C. Appendix:  UBC research ethics board’s certificates of approval Listed on following pages (2 total).   178      179 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0069997/manifest

Comment

Related Items