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The function of protein tyrosine phosphatase alpha (PTPα) phosphorylation in integrin-mediated signaling Cheng, Suzanne Yuen Shan 2013

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THE FUNCTION OF PROTEIN TYROSINE PHOSPHATASE ALPHA (PTP?) PHOSPHORYLATION IN INTEGRIN-MEDIATED SIGNALING  by  Suzanne Yuen Shan Cheng  B.Sc., The University of British Columbia, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES   (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     December 2013  ? Suzanne Yuen Shan Cheng, 2013  ii Abstract  The integrin signaling network involves over 180 components and regulates a wide range of biological activities including cell adhesion, survival and migration.  However, the details of the molecular mechanisms that govern these cellular processes remain unclear.  Since defective integrin signaling is often associated with diseases such as cancer, a precise understanding of the molecular mechanisms underlying integrin-mediated processes may provide novel insights for developing therapeutics against cancer.  Protein tyrosine phosphatase alpha (PTP?) is a receptor-like PTP that activates Src family kinases (SFKs) upon integrin stimulation.  In addition, its C-terminal tail Tyr789 phosphorylation, mediated by an active Src-FAK complex, promotes integrin-induced cell spreading, focal adhesion (FA) formation, and cell migration.  I hypothesized that PTP?-phosphoTyr789 serves as an SH2-domain binding site to recruit other focal adhesion proteins to regulate cell migration.  Studies involving re-expression of an unphosphorylatable mutant (Y789F) of PTP? in PTP?-null cells revealed that PTP? Tyr789 promotes FA localization and tyrosine phosphorylation of Cas, a key player in cell migration.  Furthermore, BCAR3 was identified as a novel binding partner of PTP?-phosphoTyr789 that mediates Cas association with PTP?, in this way localizing Cas to FAs to promote downstream signaling to regulate cell migration.  The adaptor protein Grb2 also interacts with PTP?-phosphoTyr789 but its role in association with PTP? is unknown.  Using a Grb2-silencing approach, I discovered that Grb2 regulates integrin-induced PTP? Tyr789 phosphorylation via two distinct mechanisms:  1) Grb2 regulates FAK autophosphorylation and thus FAK/Src complex activation and 2) the SH2 and C-terminal SH3 domains of Grb2 mediate the formation of a PTP?-Grb2-FAK complex.   iii Together, these roles of Grb2 promote Src-FAK-mediated PTP? tyrosine phosphorylation.  In summary, my results reveal both upstream molecular mechanisms that regulate PTP? Tyr789 phosphorylation and downstream PTP? Tyr789-dependent events that regulate cell migration.   iv Preface  All of the work presented in this dissertation was conducted in Dr. Catherine J. Pallen's laboratory at the Child and Family Research Institute.  Under the direct supervision of Dr. Pallen, I have performed majority of the experiments presented in this dissertation except for those shown in Chapter 3 (Fig. 3.8), which is used with permission from Sun et al. (2012) of which I am a co-author.  Chapter 3 also includes my work on microscopy that is published in Sun G, Cheng SY, Chen M, Lim CJ, Pallen CJ. (2012) Protein Tyrosine Phosphatase ? Phosphotyrosyl-789 Binds BCAR3 To Position Cas for Activation at Integrin-Mediated Focal Adhesions. Mol. Cell Biol. 2012 Sep;32(18):3776-89. Epub 2012 Jul 16.  I was the primary investigator for the project located in Chapters 4 and 5 of this dissertation where I was responsible for all major areas of concept formation, data collection and analysis, as well as manuscript composition.  The manuscript is currently in revision.    Publication resulting from this research: 1. Sun G, Cheng SY, Chen M, Lim CJ, Pallen CJ. (2012) Protein Tyrosine Phosphatase ? Phosphotyrosyl-789 Binds BCAR3 To Position Cas for Activation at Integrin-Mediated Focal Adhesions. Mol. Cell Biol. 2012 Sep;32(18):3776-89. Epub 2012 Jul 16.   2. Cheng SY, Sun G, Schaelpfer DD, Pallen CJ.  Grb2 is Required for Integrin-induced Focal Adhesion Kinase (FAK) Autophosphorylation and Directs the Phosphorylation of PTP? by the Src-FAK Kinase Complex.  (In revision)  v Table of Contents  Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of Contents .................................................................................................................... v List of Figures .......................................................................................................................... x List of Abbreviations ........................................................................................................... xiii Acknowledgements ............................................................................................................ xviii   Introduction ......................................................................................................... 1 Chapter 1:1.1 The significance and mechanism of cell migration ............................................................... 1 1.1.1 Cell migration in normal and pathological processes ....................................................... 1 1.1.2 Cell migration cycle .......................................................................................................... 2 1.2 Integrins ................................................................................................................................ 3 1.2.1 Overview of integrin structure and function ..................................................................... 4 1.2.2 Integrin signaling: Inside-out and outside-in .................................................................... 5 1.3 Focal adhesions ..................................................................................................................... 8 1.3.1 Structure of focal adhesions .............................................................................................. 8 1.3.2 Components of focal adhesions ........................................................................................ 9 1.3.3 Focal adhesion assembly and maturation ....................................................................... 10 1.3.4 Focal adhesion disassembly and turnover ...................................................................... 11 1.3.5 Regulation of adhesion dynamics ................................................................................... 13 1.4 Major players in integrin signaling ..................................................................................... 15 1.4.1 Major kinases in integrin signaling: FAK and Src ......................................................... 16 1.4.2 Major downstream effectors / scaffolding proteins: Cas and Paxillin ............................ 20  vi 1.4.3 Key adapter proteins: Grb2, Crk, Nck ............................................................................ 25 1.4.4 A novel NSP family adaptor protein:  BCAR3 ............................................................... 29 1.5 Protein tyrosine phosphatases (PTPs) in integrin signaling ................................................ 30 1.5.1 Overview of PTP superfamily ........................................................................................ 31 1.5.2 Regulation of PTPs ......................................................................................................... 33 1.5.3 PTPs involved in integrin signaling ................................................................................ 33 1.6 PTP? and its roles in integrin signaling .............................................................................. 37 ????? Structure and function of PTP? ...................................................................................... 38 ????? Phosphorylation of PTP? ................................................................................................ 40 1.6.3 Roles of PTP? in integrin signaling ................................................................................ 41 1.6.4 Binding partners of PTP?-phosphoTyr789:  Src and Grb2 ............................................ 43 1.7 Rationale and hypothesis .................................................................................................... 45   Materials and Methods ..................................................................................... 56 Chapter 2:2.1 Cell lines and cell culture .................................................................................................... 56 2.2 Cell stimulation and inhibition ............................................................................................ 56 2.2.1 Fibronectin (FN) stimulation .......................................................................................... 56 2.2.2 Serum stimulation ........................................................................................................... 57 2.2.3 Shp2 inhibitor treatment ................................................................................................. 57 2.3 Antibodies and immunological detection reagents ............................................................. 57 2.4 Grb2 expression constructs ................................................................................................. 58 2.5 PTP?, paxillin and Grb2 expression and Grb2/Shp2 depletion .......................................... 60 2.6 Cell lysis, immunoprecipitation, and immunoblot analysis ................................................ 60 2.7 Immunofluorescence and TIRF microscopy ....................................................................... 61 2.8 Kinase assay ........................................................................................................................ 62  vii 2.9 PTP? peptide affinity chromatography ............................................................................... 62 2.9.1 Sample preparation ......................................................................................................... 62 2.9.2 Affinity chromatography ................................................................................................ 63 2.10 Wound healing assay .......................................................................................................... 64 2.11 Focal adhesion enrichment assay ........................................................................................ 64 2.12 Statistical analyses .............................................................................................................. 65   Identification of BCAR3 as a PTP?-phosphoTyr789 Binding Partner that Chapter 3:Mediates Integrin-Induced Cell Migration ........................................................................ 66 3.1 Rationale ............................................................................................................................. 66 3.2 Detection of PTP?-phosphoTyr789 binding partners using phospho-peptide affinity chromatography ............................................................................................................................... 67 3.2.1 Identification of PTP? phosphopeptide-associated proteins ........................................ 68 3.3 In vivo validation of PTP?-phosphoTyr789 candidate binding partners ............................. 70 3.4 PTP? Tyr789 is required for integrin-induced Cas tyrosine phosphorylation .................... 70 3.5 Cas localization to focal adhesions is defective in PTP?-Y789F expressing cells ............. 72 3.6 BCAR3 SH2 and GEF domains physically link integrin-induced phosphoPTP? to Cas ... 73 3.7 BCAR3 localization to focal adhesions is dependent on the BCAR3 SH2 domain and PTP? Tyr789 .................................................................................................................................... 74 3.8 Discussion ........................................................................................................................... 75   The Role of Grb2 in Integrin-Mediated PTP? Tyr789 Phosphorylation ..... 91 Chapter 4:4.1 Rationale ............................................................................................................................. 91 4.2 Integrin stimulation induces PTP?-phosphoTyr789 association with Grb2 ....................... 92 4.3 Integrin-induced PTP? Tyr789 phosphorylation is defective in Grb2 knockdown cells .... 93  viii 4.4 Cas phosphorylation and cell migration are impaired in the absence of Grb2.................... 94 4.5 PTP? translocates to focal adhesions in the absence of Grb2-mediated PTP? Tyr789 phosphorylation ................................................................................................................................ 96 4.6 Detection of transient PTP? phosphorylation in Grb2-depleted cells ................................. 97 4.6.1 Serum stimulation induces PTP? Tyr789 phosphorylation ............................................ 98 4.6.2 Overexpression of BCAR3 in the absence of Grb2 fails to rescue PTP? phosphorylation 98 4.7 Investigation of FAK-Src complex activity in cells lacking Grb2 .................................... 100 4.7.1 Integrin-induced Src phosphorylation and activity are unaffected by Grb2 ................. 100 4.7.2 Integrin-induced FAK phosphorylation and FAK-Src association are impaired in Grb2-depleted cells ............................................................................................................................. 102 4.7.3 Similarities between Grb2 knockdown cells and FAK-Y397F expressing cells .......... 103 4.8 Discussion ......................................................................................................................... 104   Identification of a Second Role of Grb2 in the Regulation of Integrin-Chapter 5:Stimulated PTP? Tyr789 Phosphorylation ...................................................................... 120 5.1 Rationale ........................................................................................................................... 120 5.2 Knockdown or inhibition of Shp2 cannot rescue defective FAK-phosphoTyr397 in Grb2 knockdown cells ............................................................................................................................. 121 5.3 Effects of paxillin overexpression on integrin-induced FAK and PTP? phosphorylation in Grb2-depleted cells ........................................................................................................................ 121 5.3.1 Paxillin expression and association with FAK are impaired in the absence of Grb2 ... 122 5.3.2 Overexpression of paxillin in the absence of Grb2 rescues FAK Tyr397 but not PTP? Tyr789 phosphorylation ............................................................................................................. 123 5.4 Different domains of Grb2 are required for PTP? and FAK tyrosine phosphorylation .... 124  ix 5.5 Enhanced BCAR3 expression fails to restore PTP? Tyr789 phosphorylation under normal FAK phosphorylation ..................................................................................................................... 126 5.6 Association of FAK with PTP? is dependent on Grb2 C-terminal SH3 and SH2 domains 127 5.7 FAK Tyr925 is required for integrin-induced PTP? Tyr789 phosphorylation ................. 128 5.8 Discussion ......................................................................................................................... 129   Discussion and Future Directions .................................................................. 144 Chapter 6:6.1 The interplay between BCAR3, Grb2 and PTP? in integrin signaling ............................. 144 6.1.1 Regulation of integrin-induced PTP? Tyr789 phosphorylation by BCAR3 and Grb2 . 144 6.1.2 Roles of PTP?, BCAR3 and Grb2 in integrin-mediated cell migration ....................... 147 6.1.3 A potential PTP?-Grb2-FAK complex mediator .......................................................... 151 6.2 PTP?, BCAR3 and Grb2 in cancer signaling .................................................................... 153 6.3 Conclusion ........................................................................................................................ 156  Bibliography ........................................................................................................................ 161 Appendix .............................................................................................................................. 194     x List of Figures  Figure 1.1    Schematic diagram of the migration cycle ......................................................... 47 Figure 1.2    Integrin bi-directional signaling ......................................................................... 48 Figure 1.3    Structures of adhesions in a polarized/migrating cell......................................... 49 Figure 1.4    Domain structures of FAK and Src .................................................................... 50 Figure 1.5    Domain structures of Cas and paxillin ............................................................... 51 Figure 1.6    Domain structures of Crk, Nck and Grb2 ........................................................... 52 Figure 1.7    Domain structure of PTP? .................................................................................. 53 Figure 1.8    Two roles of PTP? in integrin signaling ............................................................ 54 Figure 1.9    PTP?-Grb2 association ....................................................................................... 55  Figure 3.1    Schematic diagram of affinity column chromatography with PTP? non-phosphopeptide and phosphopeptide ...................................................................................... 80 Figure 3.2    SH2-domain containing proteins in PTP?-/- MEFs are able to bind to PTP?-phosphopeptide ....................................................................................................................... 81 Figure 3.3    SH2-domain containing proteins in wild type MEFs are able to bind to PTP?-phosphopeptide ....................................................................................................................... 82 Figure 3.4    Shc, Crk and Nck do not co-immunoprecipitate with PTP? .............................. 83 Figure 3.5    PTP? Tyr789 is required for optimal integrin-induced Cas tyrosine phosphorylation....................................................................................................................... 84 Figure 3.6    Integrin-induced Cas-Src association is impaired in PTP?-Y789F expressing cells ......................................................................................................................................... 85 Figure 3.7    PTP? Tyr789 promotes Cas localization to focal adhesions (1) ........................ 86 Figure 3.8    PTP? Tyr789 promotes Cas localization to focal adhesions (2) ........................ 87 Figure 3.9    PTP? Tyr789 and BCAR3 SH2 domain are required for integrin-induced formation of a PTP?-BCAR3-Cas-Src complex ..................................................................... 88 Figure 3.10    The BCAR3 SH2 domain is required for its localization to focal adhesions.. . 89 Figure 3.11    BCAR3 focal adhesion localization is defective in PTP?-Y789F expressing cells ......................................................................................................................................... 90   xi Figure 4.1   Grb2 associates with phosphoPTP? in an integrin-dependent manner ............. 108 Figure 4.2   Grb2 knockdown reduces PTP? Tyr789 phosphorylation ................................ 109 Figure 4.3   Grb2 depletion reduces Cas SD domain phosphorylation at Tyr410 ................ 110 Figure 4.4   Cell migration is reduced in the absence of Grb2 ............................................. 111 Figure 4.5   PTP? localizes to focal adhesions in the absence of Tyr789 phosphorylation. 112 Figure 4.6   Serum stimulation induces reduced and transient PTP? Tyr789 phosphorylation in Grb2 knockdown cells ...................................................................................................... 113 Figure 4.7   BCAR3 overexpression fails to rescue phosphoPTP?-Tyr789 ......................... 114 Figure 4.8   Grb2 knockdown does not prevent integrin-induced Src activation ................. 115 Figure 4.9   Integrin-induced FAK Tyr397 phosphorylation and FAK-Src complex formation are dependent on Grb2 .......................................................................................................... 116 Figure 4.10   Re-expression of WT-Grb2 rescues integrin-stimulated FAK Tyr397 phosphorylation..................................................................................................................... 117 Figure 4.11   FAK-Y397F expressing cells exhibit defective PTP? Tyr789 phosphorylation................................................................................................................................................ 118 Figure 4.12   Potential actions of Grb2 in the activation of FAK ......................................... 119  Figure 5.1    Shp2 silencing or inhibition does not rescue the effects of Grb2 depletion ..... 133 Figure 5.2   Paxillin expression and association with FAK are reduced in Grb2-depleted cells............................................................................................................................................... 134 Figure 5.3   Exogenous paxillin restores FAK activation but not PTP? Ty789 phosphorylation in Grb2-deficient cells................................................................................ 135 Figure 5.4    Grb2-SH2 and -C-terminal SH3 domains are required for PTP? Tyr789 phosphorylation..................................................................................................................... 136 Figure 5.5    Grb2-SH2 and -C-terminal SH3 domains promote cell spreading ................... 137 Figure 5.6    PTP?-Grb2 association is dependent on Grb2-SH2 and ?C-terminal SH3 domains ................................................................................................................................. 138 Figure 5.7    Excess BCAR3 is unable to rescue PTP? phosphorylation under normal FAK activation ............................................................................................................................... 139 Figure 5.8    Grb2 is required for PTP?-FAK association .................................................... 140 Figure 5.9    PTP?-FAK association is mediated by Grb2 associaton with phosphoPTP?. . 141  xii Figure 5.10    Integrin-induced PTP? Tyr789 phosphorylation is defective in FAK-Y925F expressing cells ..................................................................................................................... 142 Figure 5.11    Models of Grb2 interactions in the regulation of integrin-induced PTP? Tyr789 phosphorylation..................................................................................................................... 143  Figure 6.1    Regulation of PTP? Tyr789 phosphorylation by BCAR3 and Grb2................ 158 Figure 6.2    Model of phosphoPTP?-associated-Grb2- and -BCAR3-mediated cell migration............................................................................................................................................... 159 Figure 6.3    Interactions between PTP? and other focal adhesion proteins ......................... 160  Figure A.1    PTP? translocation is defective in PTP?-Y789E expressing cells .................. 194     xiii List of Abbreviations  Arp3   actin-related protein 3 Asp   aspartic acid BCAR3  breast cancer antiestrogen resistance 3 BSA   bovine serum albumin Cas   Crk-associated substrate CCH   Cas family C-terminal homology domain CNS   central nervous system C-SH3   C-terminal src homology 3 Csk   c-Src terminal kinase DMEM  Dulbecco?s Modified Eagle Medium Dock180  dedicator of cytokinesis 1 ECD   extracellular domain ECM   extracellular matrix EDTA   ethylenediaminetetraacetic acid EGF   epidermal growth factor ER   estrogen receptor ERK   extracellular signal-regulated kinase FA   focal adhesion  FAK   focal adhesion kinase FAT   focal adhesion targeting FBS   fetal bovine serum FERM   protein 4.1, ezrin, radixin, moesin domain  xiv FGFR2  fibroblast growth factor receptor 2  FIP200  focal adhesion kinase family interacting protein of 200 kDa FN   fibronectin FRET   fluorescence resonance energy transfer FRNK   FAK-related non-kinase domain Gab1   Grb2-associated binder 1 GEF   guanine nucleotide exchange factor GFP   green fluorescent protein Glu   glutamic acid Grb2   growth factor receptor-bound protein 2 HER2   human epidermal growth factor receptor 2 HRP   horseradish peroxidase IGF-1   insulin growth factor-1 Ig   immunoglobulin IgG   immunoglobulin G ILK   integrin-linked kinase LD   leucine-rich domain LIM   Lin-1, Isl1, Mec3 LMW-PTP  low molecular weight protein tyrosine phosphatase MAPK   mitogen-activated protein kinase MEF   mouse embryonic fibroblast MLCK   myosin light chain kinase MS   mass spectrometry  xv Nck   non-catalytic region of tyrosine kinase adaptor protein NP-40   Nonidet P-40 N-SH3   N-terminal src homology 3 NSP   novel SH2 domain-containing protein N-Wasp  Neural Wiskott-Aldrich syndrome protein PAK   p21-activated protein kinase PBS   phosphate-buffered saline PCR   polymerase chain reaction Phe   phenylalanine PI3K   phosphoinositide 3 kinase PIP2   phosphatidylinositol-4,5-bisphosphate PIP3   phosphatidylinositol-3,4,5-trisphosphate PKL   paxillin kinase linker PMSF   phenylmethylsulfonyl fluoride PPII   polyproline type II PR   proline-rich PTB   phosphotyrosine binding PTEN   phosphatase and tensin homolog PTK   protein tyrosine kinase PTP   protein tyrosine phosphatase PTP?? ? ? protein tyrosine phosphatase alpha PVDF   polyvinylidene difluoride Rac1   Ras-related C3 botulinum toxin substrate 1  xvi Rap1   Ras-related protein 1 REF   rat embryo fibroblast RIPA   radioimmunoprecipitation assay ROCK   Rho-associated protein kinase RPTP   receptor-like protein tyrosine phosphatase SBD   Src binding domain SD   substrate domain SDS   sodium dodecyl sulfate SDS-PAGE  sodium dodecyl sulfate polyacrylamide gel electrophoresis SFK   Src family kinase SH2   Src homology 2 SH3   Src homology 3 siRNA   small interfering ribonucleic acid SKAP-Hom  Src kinase associated phosphoprotein 55 homologue SP   signal peptide Sos   son of sevenless Val   valine VN   vitronectin TIRF   total internal reflection fluorescence TM   transmembrane Tyr   tyrosine WAVE  Wasp-family verprolin-homologous protein WIP   Wasp-interacting protein  xvii WT   wild type    xviii Acknowledgements  First of all, I am deeply grateful to my supervisor Dr. Catherine Pallen for her continual guidance, patience, enthusiasm and encouragement throughout my PhD study.  Without her tremendous support and help, this dissertation would not have materialized.  It is my blessing to have a wonderful supervisor like her who always challenges me to do the best I can.  I would like to express my sincere grattitude to my supervisory committee:  Dr. Bruce Verchere, Dr. Calvin Roskelley, Dr. Aly Karsan and Dr. James Lim, for their insightful comments and constructive advice.  I am particularly grateful for the microscopy training given by Dr. Lim.  I would also like to thank the Canadian Institutes of Health Research (CIHR) and Child and Family Research Institute (CFRI) for providing sufficient funding for my research projects.  I also want to genuinely thank all the Pallen Lab members for their warm embraces, companionship and technical help which have made my experience in the lab fun and enjoyable.  Special thanks to my lab manager, Dr. Jing Wang, for her care and support.  My heartfelt appreciation goes to my loving friends and family, especially my parents, my sister, my in-laws, and my beloved husband, Gideon.   I would not have been able to complete my dissertation without their understanding and faith in me.  Last but not least, I want to thank God for walking and growing with me through every turn of this journey.  Glory be to God!    1  Introduction Chapter 1:  1.1 The significance and mechanism of cell migration   Cell migration is an essential process in both normal and pathophysiological contexts.  It refers to the coordinated movement of cells from one location to another in response to the external stimuli in the microenvironment.  This process involves cell surface receptors that survey the extracellular surroundings and transmit signals to other intracellular components to mediate an appropriate response.  Since the biochemical and mechanical compositions of the extracellular milieu can be diverse and subjected to constant changes, it is fascinating to understand how a cell orchestrates all of its elements to regulate cell motility.  As technology advances, many of the macromolecules within a cell can now be visualized in real-time, providing more insights into the complex signaling network of cell migration.  1.1.1 Cell migration in normal and pathological processes   From the time of conception, cell migration is a vital phenomenon that affects our growth and development, and maintains our health.  It controls a number of biological processes, including morphogenesis of the embryo, wound healing and immune responses (Alberts 2008).  During embryonic development, cells migrate as sheets to form layers of the embryo (endoderm, mesoderm and ectoderm) and then translocate from the epithelial layers to other destinations where they differentiate to form specialized organs and tissues (Gilbert 2003).  When the skin is injured, proliferating cells from the basal layer of the epidermis and fibroblasts will migrate towards the wound to repair the damage.  In cases where  2 inflammation is triggered by the injured cells or pathogens (i.e. bacteria), the pro-inflammatory signals given by these cells will recruit leukocytes from the circulation to the site of injury or infection to elicit an inflammatory response.  Cells such as platelets will migrate and aggregate at the wound to stop bleeding by forming fibrin clots.  Other immune cells like macrophages and neutrophils will migrate to destroy foreign microorganisms.   In view of the importance of cell migration in development and homeostasis, deregulation of cell migration results in an array of human diseases.  Impaired cell migration during development may lead to brain and heart abnormalities whereas in wound healing, it may cause delays in tissue repair.  Defective cell migration is also associated with various autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and asthma, and vascular diseases, including atherosclerosis (Ridley et al. 2003).  Tumor metastasis, which is characterized by the dissemination of cancer cells from the primary location to a secondary location, requires cell motility as well (Yamaguchi et al. 2005).  Therefore, a precise understanding of the molecular mechanisms underlying cell migration is essential for the development of effective treatments against these diseases.  1.1.2 Cell migration cycle   Cell migration is a multi-step cyclical process that begins as a cell polarizes and extends membrane protrusions in the direction of migration (Fig. 1.1) (Lauffenburger and Horwitz 1996; Small et al. 2002; Ridley et al. 2003; Parsons et al. 2010).  Actin polymerizes at the leading edge of a cell to form broad, sheet-like protrusions called lamellipodia and spike-like extensions termed filopodia (Pollard and Borisy 2003).  These protrusions are stabilized by the formation of new adhesions that link the actin cytoskeleton to the cell  3 substratum.  Myosin-II motors then slide along the actin filaments to promote actin stress fiber formation. Subsequently, the contraction forces generated by actomyosin (complex of myosin and actin filaments) propel the cell body to move forward (Beningo et al. 2001).  As the cell moves forward, the trailing edge is detached from the cell substratum accompanied by adhesion disassembly (Ridley et al. 2003).  The distinctive events occurring at the front and the rear of a migrating cell indicate that the dynamic spatio-temporal re-organization of the actin cytoskeleton and adhesions is fundamental to the regulation of directed cell movement.    1.2 Integrins   Most of the adhesions involved in cell migration contain a family of transmembrane receptors known as the integrins.  Discovered in 1986 (Tamkun et al. 1986), integrins are metazoan-specific cell adhesion receptors that play important roles in development, immune response, hemostasis and leukocyte transmigration (Hynes 2002).   They are heterodimers consisting of an ? and a ? subunit that are non-covalently linked.  To date, 18? and 8? subunits are identified, forming a family of 24 integrins with differential ligand binding specificities (Zhu et al. 2008; Hu and Luo 2013).  Knockout mouse models of the ? and ? subunits have revealed a distinctive role for each of the integrins (Hynes 2002).  Since defective integrin signaling is associated with various human diseases, including cancer, fibrosis, inflammation and autoimmune diseases, it has been widely studied to gain insights for the development of effective therapeutics against these diseases.  As of 2010, five anti-integrin drugs have been approved for clinical use in the treatment of thrombosis, multiple sclerosis and non-small cell lung carcinoma (Goodman and Picard 2012).  4 1.2.1 Overview of integrin structure and function   The ? and ? subunits of integrins are type I transmembrane (TM) glycoproteins featuring a large extracellular domain and a short cytoplasmic domain (~20-50 amino acids) (Humphries et al. 2006; Fu et al. 2012), with the exception of the ?4 subunit which has a ~1000 amino acid long cytoplasmic tail (Hynes 2002).  The extracellular domain mediates integrin binding to its ligands, including extracellular matrix (ECM) proteins (i.e. collagen, laminin, fibronectin and vitronectin), cell surface receptors in the Ig superfamily (i.e. VCAM-1, ICAM-1), and bacteria or viruses.  Based on the structural interactions among the integrin-ligand combinations, the integrins are categorized into four groups (Humphries et al. 2006):  1) RGD-binding integrins (i.e. ?V, ?IIb?3) which recognize ligands containing an RGD tripepetide motif, 2) LDV-binding integrins (i.e. ?4?1, ?4?7 ?9?1) that binds to the LDV acidic motif, 3) A-domain ?1 integrins (i.e. ?1?1, ?2?1, ?10?1, ?11?1) which has an A-domain in the ? subunit that specifically binds to laminin and collagen, and 4) Non-??A-domain-containing laminin-binding integrins (i.e. ?3?1, ?6?1, ?7?1, ?6?4).  While a lot of the integrins can interact with a variety of ligands, many ECM and cell surface proteins can bind to more than one integrin receptor as well (Humphries 1990; Plow et al. 2000; van der Flier and Sonnenberg 2001; Humphries et al. 2006).  The integrin non-enzymatic cytoplasmic domain is essential for its ability to mediate bi-directional signal across the plasma membrane.  It serves as a docking site for the assembly of multiprotein complexes and cytoskeletal proteins (Liu et al. 2000; Zaidel-Bar and Geiger 2010), linking the ECM to the intracellular cytoskeleton to control a multitude of cellular responses such as cell adhesion, migration, proliferation, apoptosis and differentiation (Legate et al. 2009).  On the other hand, its interactions with certain  5 cytoplasmic proteins regulate integrin ligand-binding affinity to mediate inside-out signaling (Harburger and Calderwood 2009), which will be further discussed in the next section.  Phosphorylation of the ? and ? tails on serine or tyrosine residues serves as binding sites for cytoplasmic molecules to regulate integrin-mediated signaling (Fagerholm et al. 2004).  For instance, ?4 integrin is phosphorylated on Ser988 where paxillin binds to regulate cell polarization and migration in leukocytes (Goldfinger et al. 2003; Han et al. 2003).    1.2.2 Integrin signaling: Inside-out and outside-in  ? ?ntegrins can transmit mechanical and biochemical signals across the plasma membrane in a bi-directional fashion (?outside-in? and ?inside-out? signaling) via its interaction with extracellular and intracellular molecules.  This unique integrin feature is evident in various cell types and requires conformational changes in the extracellular, TM and cytoplasmic domains of the integrin (Calderwood 2004).  Inside-Out Signaling  Inside-out signaling is best characterized in platelets and leukocytes where integrins are normally inactive to keep the cells in non-adherent state (Harburger and Calderwood 2009; Srichai 2010).  Upon activation by intracellular signals, integrins switch from low affinity to high affinity state to promote cell-ECM or cell-cell adhesion.  For example, activation of the platelet integrin ?IIb?3 is important for platelet aggregation and thrombosis during hemostasis (Shattil et al. 1998) while activation of ?1 and ?2 integrins is associated with leukocyte trafficking (Hogg et al. 2002; Laudanna et al. 2002; Calderwood 2004).  6  In resting state, the integrin extracellular domains are in an inactive ?bent? conformation stabilized by the interfaces between the ? and ? TM and the cytoplasmic domains (Fig. 1.2A) (Xiong et al. 2001; Takagi et al. 2002; Zhu et al. 2008).  Disruption of the ? and ? TM/cytoplasmic heterodimerization results in integrin activation (O'Toole et al. 1991; Lu et al. 2001; Luo et al. 2004; Luo et al. 2005), inducing the ?switchblade? shift in the extracellular domain from a bent to an extended conformation (Takagi and Springer 2002; Qin et al. 2004).  The active conformation exposes the ligand-binding site, increasing ligand-binding affinity to facilitate outside-in signaling.  Talin is a key player in integrin activation.  It is a large actin-binding protein composed of a 50 kDa head domain (THD) and a 220 kDa rod domain.  THD binds strongly to the conserved membrane-proximal NPXY motif in the ? integrin cytoplasmic tail to activate integrins (Calderwood et al. 2002; Calderwood 2004; Ginsberg et al. 2005; Wegener et al. 2007), most likely by separating the TM domains of the integrins through pistonning, disrupting the ?-? integrin salt bridge or prying open the cytoplasmic clasps via steric hindrance (Ye et al. 2011).  Kindlin is another integrin-interacting protein that is involved in integrin inside-out signaling (Ma et al. 2008; Montanez et al. 2008).  Different from talin, it binds to the membrane-distal NxxY motif (Moser et al. 2008) and is unable to activate integrins by itself (Harburger and Calderwood 2009; Hu and Luo 2013).  Instead, kindlin acts as a co-activator of talin-mediated integrin activation to enhance ligand affinity (Ma et al. 2008; Montanez et al. 2008).     7 Outside-In Signaling  Outside-in signaling refers to the engagement of extracellular ligands to the integrins, triggering integrin clustering (Kim et al. 2004) and intracellular signaling (Fig. 1.2B).  Under physiological conditions, a small portion of integrins are basally activated and ligand binding stabilizes the extended active conformation (Hu and Luo 2013).  Since integrins lack enzymatic activity, they transduce extracellular signals to the interior of the cell through the recruitment of intracellular proteins to their cytoplasmic tail to form transient unstable adhesion contacts (Choi et al. 2008), which later mature into more stable focal adhesions.  These adhesions contain actin-binding proteins thereby linking the integrins to the actin cytoskeleton (with the exception of ?4 integrins which are linked to intermediate filaments) to alter cell shape, adhesion and migration.  Outside-in signaling is characterized by a series of tyrosine phosphorylation events and an increase in lipid second messengers such as phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Ferrell and Martin 1989; Golden et al. 1990; McNamee et al. 1993; Chen and Guan 1994).  Tyrosine and serine/threonine phosphorylation of the integrin tails serve as binding sites for phosphotyrosine-binding (PTB) domain-containing proteins (i.e. talin) (Calderwood et al. 2003) and adaptor proteins (i.e. filamin, 14-3-3) (Takala et al. 2008), which in turn recruit other focal adhesion proteins to the sites of adhesion to mediate downstream signaling responses.  Following fibronectin (FN) stimulation, the initial integrin-cytoskeleton linkage is mediated by talin.  Embryonic stem cells or fibroblasts lacking talin are unable to link integrins to the cytoskeleton (Priddle et al. 1998; Zhang et al. 2008), suggesting that talin is involved in both inside-out and outside-in signaling.  8 1.3 Focal adhesions   More than 40 years ago, focal adhesions were first observed in areas close to the ventral cell membrane and its substratum using interference reflection microscopy (Curtis 1964; Izzard and Lochner 1976).  Later on, they were seen as electron-dense plaques associated with actin filament bundles using electron microscopy (Abercrombie et al. 1971; Heath and Dunn 1978).  To date, focal adhesions are known as integrin-containing multiprotein complexes that connect the actin cytoskeleton to the ECM.  They are formed upon integrin engagement and their composition changes spatially and dynamically depending on external cues.  There are at least 180 components encompassing over 742 interactions that are associated with integrin-mediated adhesions (Zaidel-Bar and Geiger 2010), indicating the complexity of adhesion dynamics.  1.3.1 Structure of focal adhesions   Various structures of adhesion have been classified and they are termed nascent adhesions, focal complexes/focal contacts (FCs), focal adhesions (FAs) and fibrillar adhesions (Fig. 1.3) (Webb et al. 2002; Parsons et al. 2010; Huttenlocher and Horwitz 2011).   Nascent adhesions refer to the initial small, short-lived adhesions that are found at the lamellipodium, immediately behind the leading edge of a migrating cell.  These newly formed adhesions can either turn over or mature to form larger, dot-like FCs at the lamellipodium-lamellum interface, where the lamellum is a region of dense actin behind the lamellopodium.  The FCs can then mature into larger, elongated FAs.  These mature FAs typically reside at the ends of actin stress fibres that extend from the front to the back of the  9 cells (Zimerman et al. 2004).  The sizes of these three types of adhesions range from 0.1 ? 10 ?m2. Fibrillar adhesions are highly elongated and stable adhesions that are usually observed when cells are exposed to integrin ligands for an extended period of time.  They are more important in ECM remodeling than in cell migration.  Although all of these adhesions are architecturally different, they form as a continuum rather than as distinct structures.  In this thesis, the terminologies, focal adhesions and focal complexes, are interchanged to describe the collective structures within these classifications.   1.3.2 Components of focal adhesions   As mentioned above, there are at least 180 proteins that are known to take part in the integrin-mediated adhesion signaling network known as the integrin ?adhesome? (Zaidel-Bar and Geiger 2010).  Among these proteins are adaptor proteins, cytoskeletal proteins, actin-binding proteins, serine/threonine protein kinases, serine/threonine protein phosphatases, tyrosine phosphatases, tyrosine kinases, GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs), transmembrane receptors, adhesion proteins, and small family GTPases (Zaidel-Bar et al. 2007).  While some of these components may act as scaffolding proteins to mediate protein-protein interactions that strengthen the physical linkage between the integrins and the ECM, others may act as regulatory proteins to modulate FA components through their enzymatic activity.  For example, the actin-binding protein, filamin, is also capable of interacting with the cytoplasmic tail of integrins, mediating a direct linkage between the integrins and the actin filament (Harburger and Calderwood 2009) whereas p190RhoGAP inhibits Rho activity to diminish adhesion  10 maturation (Schober et al. 2007).  The interplay between all of these elements governs the assembly and disassembly of focal adhesions to alter downstream signaling events.  1.3.3 Focal adhesion assembly and maturation   Adhesion assembly, maturation and disassembly (at the leading edge for adhesion turnover or at the retrieving end for detachment) are coupled to actin polymerization and actomyosin contraction to enable cell movement.  At the leading edge of a motile cell, nascent adhesions are formed at a rate that is comparable to the protrusion rate (Choi et al. 2008).  In support of the notion that there is a connection between adhesion formation and actin polymerization, the assembly of nascent adhesions is found to be dependent on Arp2/3 complex-mediated actin polymerization as inhibition of actin polymerization with cytochalasin D impairs the formation of nascent adhesions (Alexandrova et al. 2008; Choi et al. 2008).  Although nucleation of nascent adhesions is unclear, two models have been proposed (Parsons et al. 2010).  The first model suggests that integrin-ECM binding results in integrin-clustering and leads to subsequent binding of the new adhesion complexes to the clustered integrin cytoplasmic domain (Calderwood et al. 2000; Carman and Springer 2003).  In contrast, the second model hypothesizes that actin polymerization initiates nascent adhesion formation based on the evidence showing that the early focal adhesion-associated proteins, FAK and vinculin, interact with Arp2/3 prior to adhesion formation (DeMali et al. 2002; Serrels et al. 2007; Vicente-Manzanares et al. 2008).  These seemingly distinct models may indeed function simultaneously to nucleate nascent adhesions, which are enriched in phosphotyrosine.  Some proteins that are found in these newly forming adhesions are talin, vinculin, ?-actinin, paxillin and FAK (Choi et al. 2008).    11  As the leading edge of a migratory cell continues to move forward, nascent adhesions can either elongate and mature into focal complexes at the border of the lamellipodium and lamellum, or turn over at the rear of disassembling actin filaments.  Elongation occurs along the thin actin filament bundles that are associated with the actin cross-linking protein ?-actinin, which serves to restrain adhesions to the ends of the filament.  ?-actinin is the earliest component detected in maturing adhesions.  Paxillin and vinculin are then sequentially recruited to the growing adhesions as well (Pasapera et al. 2010).  Unlike nascent adhesions, the formation of mature adhesions requires the actin bundling and contractile activities of myosin-II (Vicente-Manzanares et al. 2009).  The tension generated by myosin-II drives conformational changes in tension-sensitive focal adhesion proteins, unmasking new regions for protein-binding or post-translational modification to transduce downstream signaling (Sawada et al. 2006; Vogel 2006).  1.3.4 Focal adhesion disassembly and turnover   Directed cell migration features asymmetrical adhesion dynamics with nascent adhesions forming at the leading edge and focal adhesions disassembling at the rear to facilitate tail retraction.  Indeed, adhesion disassembly is not limited to the rear end of a polarized cell, it can occur at the front as well to promote adhesion turnover.  Near the leading edge, disassembly primarily occurs at the rear of the lamellipodium where more Arp2/3, the actin filament-nucleating protein complex, is bound to the polymerizing actin filaments (Parsons et al. 2010; Huttenlocher and Horwitz 2011).  Correspondingly, this process coincides with actin severing and reorganization.  FAK and Src tyrosine kinases have been implicated in focal adhesion turnover.  FAK-deficient fibroblasts exhibit reduced cell  12 migration and form increased number and size of peripheral adhesions that can be restored by re-expression of wild type FAK, revealing a FAK-dependent defect in adhesion turnover (Ilic et al. 1995; Sieg et al. 1999).  Both the autophosphorylation of FAK at Tyr397 and its kinase activity are important for FAK-mediated adhesion turnover (Webb et al. 2004; Hamadi et al. 2005).  Its ability to inhibit Rho activity also promotes adhesion turnover (Ren et al. 2000).  The catalytic activity and membrane association of Src are required for Src dissociation from FAK as well as adhesion turnover during cell migration (Fincham and Frame 1998).  Since FAK Tyr397 associates with Src, these results suggest that FAK and Src may coordinate with each other to regulate adhesion turnover.  Focal adhesion disassembly associated with the retracting end is not as well understood as that associated with adhesion turnover.  Disassembly and retraction at the rear of the cell is usually characterized by weakening or severing of the integrin-ECM linkage or integrin-cytoskeletal interactions.  Adhesions appear to ?slide? concomitant with the inward movement of the cell edge, and they disperse once the ECM disconnects from the cytoskeleton.  In support of this sliding adhesion model, ?3 integrins are found to be more diffused in the rear end adhesions than the focal adhesions at the leading edge, suggesting that the affinity or avidity of the integrins for its associated proteins is likely to be reduced.  Integrin ?footprints?, but not other focal adhesion components, have also been observed on the substratum, indicating a breakage between integrin and the cytoplasmic components as the cell body translocates (Regen and Horwitz 1992; Smilenov et al. 1999).   Furthermore, myosin-II generated contractile forces are thought to play a part in adhesion disassembly, possibly by applying traction forces to break the ECM-cytoskeleton linkage as it can be blocked by a myosin-II inhibitor (Gallo 2004).  13  Calpain, a calcium-activated protease, has also been reported as an important mediator of adhesion disassembly in migrating fibroblasts.  The loss or inhibition of calpain resulted in long retracting tails, reduced cell migration and large peripheral adhesions (Huttenlocher et al. 1997; Palecek et al. 1998; Glading et al. 2000; Dourdin et al. 2001) Calpains can cleave several focal adhesion proteins, including Talin, integrin ?3, FAK and paxillin (Carragher et al. 1999; Glading et al. 2002; Franco et al. 2004; Franco and Huttenlocher 2005; Chan et al. 2010) and these proteolytic events are associated with the disassembly of focal adhesions.    1.3.5 Regulation of adhesion dynamics   Focal adhesion dynamics can be regulated by biochemical and physical signals.  The Rho family of small GTPases (Rac, Cdc42 and Rho) has emerged as key biochemical regulators of adhesion dynamics, monitoring the balance of actin-mediated protrusion and myosin-II-mediated contraction (Parsons et al. 2010).  In addition, mechanical forces from the external environment or from actomyosin-generated tension can also affect adhesion dynamics (Kuo 2013).  RhoGTPases in adhesion dynamics  In migrating cells, Rac and Cdc42 are activated at the forward protrusions while Rho is primarily activated at the rear (Kurokawa et al. 2005; Parsons et al. 2010).  Although Rac and Cdc42 may have overlapping functions in mediating membrane protrusions (Tapon and Hall 1997), activation of Cdc42 functions prominently in filopodia formation whereas activated Rac is responsible for lamellipodia protrusions.  Rac and Cdc42 induce actin  14 polymerization mostly through the activation of the N-Wasp-WAVE-Arp2/3 cascade (Parsons et al. 2010).  Integrin-mediated activation of RhoA (Ren et al. 1999; Cox et al. 2001) leads to activation of its effector, Rho kinase (ROCK) (Ishizaki et al. 1996; Leung et al. 1996; Matsui et al. 1996).  ROCK in turn activates myosin-II to promote formation of large, stable adhesions, which inhibits protrusion formation (Chrzanowska-Wodnicka and Burridge 1996), consistent with another report demonstrating that Rho is required for the maturation of existing focal complexes (Rottner et al. 1999).  RhoA has also been implicated in the promotion of tail retraction as the inhibition of Rho kinase results in an elongated morphology with impaired detachment (Worthylake et al. 2001).  Contraction mediated by the RhoA-ROCK-myosin-II axis likely contributes to the retraction of the trailing end of migrating cells (Worthylake et al. 2001; Vicente-Manzanares et al. 2007).    The antagonistic effects of Rac and RhoA in the formation of membrane protrusions and focal adhesions indicate that a precise spatiotemporal regulation of the activation of these molecules is critical to enable proper cell migration.    Mechanical forces in adhesion dynamics  In addition to biochemical signals, mechanical forces also play a role in the regulation of adhesion dynamics (Galbraith et al. 2002).  Myosin-II-induced tension is not required for the initial formation of nascent adhesions, however, it promotes subsequent adhesion maturation.  One explanation for its mechanism of action is that the generation of traction forces alters the conformation of force-sensitive molecules, revealing additional sites for binding or posttranslational modification (Kuo 2013).  For example, Cas has been shown to undergo force-dependent conformational change which stretches open its substrate domain to  15 facilitate Src-mediated phosphorylation and recruitment of SH2-domain containing proteins to the adhesion complex (Sawada et al. 2006).  Contractile forces mediated by actomyosin also act as regulators of adhesion disassembly at the retrieving end of a migrating cell to weaken integrin-matrix or integrin-cytoskeletal associations.  These pieces of evidence suggest that mechanical forces can alter the composition of focal adhesions to control adhesion dynamics.  1.4 Major players in integrin signaling   Integrin engagement and clustering trigger the recruitment of intracellular proteins to form adhesion complexes that link the ECM to intracellular signaling to control cytoskeletal reorganization, cell migration, gene regulation and other cellular processes (Hervy et al. 2006).  The extracellular signal determines the components and interactions in downstream signaling network to elicit a specific response to the environment (Li et al. 2005).  The proteins that are involved in integrin-induced focal adhesions can be classified into three main categories: 1) enzymes (i.e. protein tyrosine kinases and protein tyrosine phosphatases), 2) adaptors / scaffolding proteins that lack intrinsic enzymatic activity and 3) integrin-binding proteins (i.e. talin) (Berrier and Yamada 2007).  In this section, the early integrin-associated central kinases, FAK and Src, and their major downstream effectors will be discussed in more detail.       16 1.4.1 Major kinases in integrin signaling: FAK and Src  Focal Adhesion Kinase (FAK)  FAK is a ubiquitously expressed 125 kDa non-receptor tyrosine kinase that is highly conserved (Ilic et al. 1995; Mitra et al. 2005).  The structure of FAK is comprised of an N-terminal FERM (protein 4.1, ezrin, radixin and moesin homology) domain, a central catalytic domain, a proline-rich region (PR1 and PR2) and a C-terminal focal adhesion targeting (FAT) domain (Fig. 1.4).  In the absence of stimuli, the FERM domain inhibits FAK activity through its interaction with the kinase domain.  This autoinhibitory FERM-kinase interaction can be released by an integrin-induced conformational change in FAK as observed in a biosensor study (Papusheva et al. 2009), though the exact molecular mechanism of release is unclear.  It has been suggested that FERM interacting molecules, such as PIP2 and Src, may displace FERM from the kinase domain to mediate FAK activation (Frame et al. 2010).  Integrin ?-tail was also thought to associate with the FERM domain to activate FAK (Schaller et al. 1995) but later reports suggest that integrin-FAK association is likely mediated through paxillin and talin (Klingbeil et al. 2001; Hayashi et al. 2002; Brakebusch and Fassler 2003; Schlaepfer and Mitra 2004; Geiger et al. 2009), as the FAT region is responsible for focal adhesion localization of FAK (Mitra et al. 2005) and interaction with paxillin and talin (Parsons 2003).   The autophosphorylation of FAK at Tyr397, which occurs either in cis or in trans (Sieg et al. 2000; Toutant et al. 2002), is one of the earliest events that occur upon integrin stimulation.  PhosphoFAK-Tyr397 binds to Src SH2 domain and Src phosphorylates Tyr576/577 in the activation loop of FAK to obtain maximal activation of the Src-FAK complex (Schlaepfer et al. 1994; Calalb et al. 1995; Calalb et al. 1996; Thomas et al. 1998;  17 Hanks et al. 2003; Legate et al. 2009).  The fully activated Src-FAK in turn phosphorylates other signaling proteins to propagate downstream signaling cascades.    As a signaling kinase and also a scaffolding protein (Mitra et al. 2005), FAK functions in integrin signaling to regulate cytoskeleton remodeling, formation and disassembly of focal adhesions, and cell migration.  FAK can directly phosphorylate neural Wiskott-Aldrich syndrome protein (N-Wasp) to modulate actin cytoskeletal rearrangement (Fonseca et al. 2004) and has been shown to interact with Arp2/3 to control lamellipodia formation and cell spreading (Serrels et al. 2007).    FAK gene knockout mice is embryonic lethal and FAK-null cells displayed increased number of focal adhesions and reduced cell motility upon FN stimulation, indicating a role for FAK in FA turnover and cell migration (Ilic et al. 1995; Mitra et al. 2005).  Activated FAK is observed in growing focal adhesions at the periphery but not in stable retracting focal adhesions, suggesting that FAK is involved in polarized cell migration (Papusheva et al. 2009).    This is further supported by FAK regulation of the Rho-family GTPases (RhoA, cdc42, Rac-1) (Tomar and Schlaepfer 2009; Schaller 2010), which are key molecular switches involved in directional cell movement (Jaffe and Hall 2005).  The FAT domain of FAK is capable of binding to p190RhoGEF to activate RhoA (Zhai et al. 2003).  On the other hand, FAK Tyr397 phosphorylation and activity are required for integrin-induced formation of a FAK-p120RasGAP-p190RhoGAP complex at the leading-edge focal adhesions and FAK-mediated tyrosine phosphorylation of p190RhoGAP to induce cell polarity (Tomar and Schlaepfer 2009) that is linked to RhoA inhibition (Bernards and Settleman 2004).  One of the downstream targets of Src-FAK is p130Cas (Cas), which interacts with the proline-rich domain of FAK via its SH3 domain to promote Rac-dependent cell migration (Hanks et al.  18 2003; Chodniewicz and Klemke 2004), and the SH3-domain-mediated binding of Cas to FAK is enhanced by Src-mediated FAK Tyr861 phosphorylation (Lim et al. 2004).  FAK has also been implicated as a mechanosensor to transduce internal or external mechanical forces (i.e. shear stress, stretching) to induce an appropriate response to the stimuli (Katsumi et al. 2004).    FAK is phosphorylated by Src at Tyr925 as well and creates an SH2-binding site for Grb2 to activate the Ras-ERK-MAPK pathway (Schlaepfer et al. 1998).  Integrin-mediated ERK2 activation modulates both FA dynamics through myosin light chain kinase (MLCK), and cell proliferation, cell cycle progression, and survival through PI3K (Chen et al. 1996; Ridley et al. 2003; Webb et al. 2004; Walker and Assoian 2005).  The binding of Grb2 to FAK has also been suggested to displace paxillin from the FAT domain to promote dissociation of FAK from focal adhesions, hence FA turnover (Mitra et al. 2005).   FIP200 is a protein that inhibits FAK-mediated cell migration and proliferation via its interaction with the kinase domain of FAK (Abbi et al. 2002).  FIP200-FAK association is reduced upon integrin stimulation concomitant with FAK activation.  Another isoform of FAK known as the FAK-related non-kinase domain (FRNK), which includes FAK PR1, PR2 and FAT domain, is also a negative regulator of FAK (Parsons 2003)  Src  Src is a 60kDa non-receptor tyrosine kinase that belongs to the Src family kinases (SFKs) (Playford and Schaller 2004).  Of the nine members in this family, Src, Fyn and Yes are ubiquitously expressed while the others are hematopoietic (Legate et al. 2009; Hu and Luo 2013).  Src has a myristoylated N-terminal domain, an SH3 and SH2 domain, a tyrosine  19 kinase domain and a C-terminal tyrosine residue (Tyr527) that forms an intramolecular interaction with its SH2 domain to autoinhibit Src activity (Fig. 1.4) (Thomas and Brugge 1997).  Both the myristoylation of Src at the N-terminus (Resh 1994) and the interaction between its polybasic amino acids and the acidic phospholipids on the inner leaflet of the membrane bilayer are required to anchor Src to the plasma membrane (Sigal et al. 1994; McLaughlin and Aderem 1995; Murray et al. 1998; Resh 1999; Resh 2004; Patwardhan and Resh 2010).  This membrane localization of Src is required for the dephosphorylation of Tyr527 (Bagrodia et al. 1993), and hence is important for regulating its kinase activity.  Src is constitutively associated with the cytoplasmic tail of ?3 integrins via its SH3 domain (de Virgilio et al. 2004; Arias-Salgado et al. 2005).  Upon integrin ligation and clustering, Src is autophosphorylated at Tyr416 (Thomas and Brugge 1997) and dephosphorylated at the inhibitory Tyr527 to become activated.  Dephosphorylation of Src Tyr527 can be catalyzed by the protein tyrosine phosphatases, PTP? and PTP1B (Zheng et al. 1992; den Hertog et al. 1993; Pallen 2003; Liang et al. 2005).  In PTP?-null cells, Src and Fyn activities are reduced concomitant with an increase in Src Tyr527 phosphorylation (Ponniah et al. 1999; Su et al. 1999).  In cells expressing catalytically inactive mutant PTP1B, tyrosine phosphorylation of Src is enhanced with a reduction in Src activity (Arregui et al. 1998).  In addition, c-Src terminal kinase (Csk), a tyrosine kinase that phosphorylates Src Tyr527, also plays a role in the regulation of Src activation (Cooper et al. 1986; Okada and Nakagawa 1989).  In platelets, ?II?3 integrin activation causes the dissociation of Csk from the integrin, resulting in Src dephosphorylation at Tyr527 and autophosphorylation at Tyr416 (Obergfell et al. 2002).  20  Src is known to regulate membrane protrusion and FA turnover in cell migration (Craven et al. 2003; Hanks et al. 2003; Parsons 2003; Schlaepfer and Mitra 2004).  FN- or VN-induced Src-null fibroblasts exhibited reduced adhesion and spreading (Kaplan et al. 1995; Felsenfeld et al. 1999).  The inhibition of SFKs using small molecules also impaired focal adhesion turnover (Webb et al. 2004).  Src binds to FAK phosphoTyr397 through its SH2 domain and phosphorylates Tyr576/577 in the activation loop of FAK (Hanks et al. 2003).  The full activation of Src promotes its interaction with and phosphorylation of Cas and paxillin (Hanks et al. 2003).  In SYF cells, which are Src, Yes and Fyn-deficient, the overall tyrosine phosphorylation of many proteins, including FAK, are reduced upon integrin stimulation and Src-null cells have reduced FAK Tyr397 phosphorylation, suggesting that Src may also play a role in the autophosphorylation of FAK Tyr397 (Klinghoffer et al. 1999; Hanks et al. 2003).  1.4.2 Major downstream effectors / scaffolding proteins: Cas and Paxillin  p130Cas  Crk-associated substrate (Cas), also known as breast cancer anti-estrogen resistance-1 (BCAR1) in humans, was first identified as a highly tyrosine phosphorylated protein that can interact with Src and Crk in v-Src and v-Crk-transformed cells respectively (Mayer et al. 1988; Reynolds et al. 1989; Kanner et al. 1990; Matsuda et al. 1990; Sakai et al. 1994).  It is a 130 kDa ubiquitously expressed scaffolding protein with multiple interactive binding domains (Chodniewicz and Klemke 2004).  There are three other members of the Cas family, NEDD9, EFS and CASS4, which are expressed in epithelial tissues, T lymphocytes, and the spleen and lung respectively (Ishino et al. 1995; Defilippi et al. 2006; Singh et al. 2008;  21 Tikhmyanova et al. 2010).  Cas has an N-terminal SH3 domain, a proline-rich (PR) region, a substrate domain (SD) characterized by 15 repeats of the YXXP motif, a serine-rich region, a Src binding domain (SBD) and a C-terminal Cas-family homology (CCH) domain (Fig. 1.5) (O'Neill et al. 2000; Chodniewicz and Klemke 2004; Defilippi et al. 2006).      Cas is a major mechanotransudction protein that undergoes phosphorylation on force-mediated stretching of its SD domain (Sawada et al. 2006).  The tyrosine phosphorylation of Cas upon integrin ligation (Nojima et al. 1995; Petch et al. 1995; Vuori and Ruoslahti 1995) is primarily catalyzed by Src, which directly binds to Cas SBD through its SH3 and SH2 domains (Nakamoto et al. 1996; Vuori et al. 1996; Klinghoffer et al. 1999; Burnham et al. 2000; Ruest et al. 2001).  In the absence of Src, Cas phosphorylation is impaired (Schlaepfer et al. 1994; Polte and Hanks 1995; Vuori et al. 1996; Sakai et al. 1997; Fonseca et al. 2004).  Indeed, Src is responsible for the tyrosine phosphorylation of at least ten sites within the SD domain that are associated with cell migration (Goldberg et al. 2003; Shin et al. 2004).  Cas is known to interact with FAK proline-rich sequences (PR1 and PR2) via its SH3 domain (Polte and Hanks 1997) in an adhesion-dependent manner (Klemke et al. 1998) but rather than acting as a kinase, FAK is implicated to function as a scaffold to enhance Src-induced Cas phosphorylation (Ruest et al. 2001).  This is in accord with findings demonstrating that Cas SH3 domain is required for its FA localization and tyrosine phosphorylation [Nakamoto, 1997; Donato, 2010; Jano?tiak, 2011].  Although the mechanism governing the focal adhesion targeting of Cas is unclear, the LIM family protein Ajuba might play a role in this process as the loss of Ajuba resulted in reduced concentration and tyrosine phosphorylation of Cas at nascent focal complexes that led to impaired cell migration (Pratt et al. 2005).  22  Tyrosine phosphorylation of the Cas SD domain creates multiple SH2- and PTB-domain binding sites that recruit other signaling molecules to mediate downstream signaling.  One of the major SH2-domain containing proteins that interacts with phosphorylated Cas is the adaptor protein, Crk (Vuori et al. 1996).  Integrin-induced Cas-Crk coupling is a key module that regulates lamellipodia formation and cell migration (Cary et al. 1998; Klemke et al. 1998; Cheresh et al. 1999; Gu et al. 2001; Mielenz et al. 2001).  The coupling of Cas-Crk leads to Rac1 activation via the recruitment of the GTPase-activating protein DOCK180 and ELMO, which serves to localize Rac1 to the membrane to induce ruffling (Kiyokawa et al. 1998; Grimsley et al. 2004).  Cas-Crk mediated Rac1 activation has also been shown to induce cell invasion in a collagen-rich matrix and prevent apoptosis (Cho and Klemke 2000).  Since cell migration is crucial for embryonic development and Cas plays an important role in cell migration, it is not surprising that the deletion of Cas in mice results in embryonic lethality (Honda et al. 1998).  Many studies have reported the role of Cas tyrosine phosphorylation in actin cytoskeleton organization, cell spreading and FA formation (Bouton et al. 2001; Cabodi et al. 2010).  Cas-null fibroblasts show delayed spreading, reduced migration, increased numbers of focal adhesions, and defects in stress fiber formation (Honda et al. 1998; Honda et al. 1999).   Using immunofluorescence and live cell microscopy, Cas has been shown to localize to focal adhesions mediated by its SH3 and CCH domains (Sakai et al. 1994; Polte and Hanks 1995; Harte et al. 1996; Donato et al. 2010).  Without Cas, cells exhibit slow disassembly of focal adhesions at the leading edge which is restored by re-expressed Cas (Webb et al. 2004), indicating the involvement of Cas in focal adhesion turnover.     23 Paxillin  Paxillin is a highly conserved 68 kDa focal adhesion protein that was initially identified in v-Src-transformed cells (Glenney and Zokas 1989; Turner et al. 1990; Turner 1998).  Its name is derived from the Latin word paxillus, meaning a stake or peg, analogous to its role as a scaffolding protein that anchors the actin stress fibers to adhesion sites (Brown and Turner 2004; Deakin and Turner 2008).   Paxillin-null mice are embryonic lethal (Schaller 2001; Hagel et al. 2002).    Other members of the paxillin family include Hic-5 (Shibanuma et al. 1997; Brown et al. 1998; Thomas et al. 1999) and leupaxin, a leukocyte specific protein (Lipsky et al. 1998), both of which are structurally similar to paxillin (Turner 1998).  The N-terminal half of paxillin consists of a short proline-rich region and five leucine-rich (LD) motifs featuring the consensus sequence LDXLLXXL, whereas the C-terminal half is comprised of four tandem lin-11, isl-1, mec-3 (LIM) domains, which are double zinc-finger motifs (Fig. 1.5) (Yang et al. 1993; Charest et al. 1995; Brown et al. 1996; Brown et al. 1998; Garton and Tonks 1999; Turner 2000; Brown and Turner 2004).  This multi-domain structure of paxillin enables its interaction with a variety of proteins including kinases, phosphatases, actin-binding proteins, Rho GTPases and integrins to coordinate differential signaling pathways in response to different stimuli (Harburger and Calderwood 2009).  The LD domains of paxillin are able to mediate its association with a number of focal adhesion-associated proteins that are important for cytoskeletal rearrangements and migration, including FAK, vinculin, actopaxin, PKL and GIT1 (Turner and Miller 1994; Brown et al. 1996; Nikolopoulos and Turner 2000; West et al. 2001; Zhang et al. 2008).  The  24 LIM domains mediate the binding of paxillin with tubulin and PTP-PEST, which are associated with paxillin subcellular localization, cell adhesion and motility (Shen et al. 1998; Cote et al. 1999; Brown and Turner 2002; Jamieson et al. 2005).  In particular, serine/threonine phosphorylation within the LIM2/3 domains is linked to paxillin FA localization and adhesion to FN (Brown et al. 1996; Brown et al.).  Paxillin interacts with the ?4 integrin tail to promote cell migration but reduce integrin-mediated cell spreading, FA and stress fiber formation in leukocytes (Liu et al. 1999; Liu et al. 2002).  It also interacts with ?9 to inhibit cell spreading (Liu et al. 2001).  Although in vitro and in vivo experiments have shown that paxillin can associate with ?1 and ?3 integrins, whether the interaction is direct or not is unclear (Schaller et al. 1995; Chen et al. 2000).    Upon integrin-ECM engagement, paxillin is phosphorylated by FAK and Src on Tyr31 and Tyr118 (Burridge et al. 1992; Bellis et al. 1995; Schaller et al. 1995), which serve as binding sites for the SH2 domain of Crk (Birge et al. 1993; Schaller et al. 1995).  The paxillin-Crk complex is coupled to downstream molecules, such as DOCK180, to mediate Rac1 activation and cell migration (Valles et al. 2004).  In collagen-stimulated rat carcinoma cells expressing the non-phosphorylatable Y31F/Y118F paxillin mutant, paxillin-Crk association, as well as cell migration, was defective (Petit et al. 2000).  The SH2 domains of p120RasGAP can also interact with phosphorylated Tyr31/118 (Tsubouchi et al. 2002).  This interaction displaces p190RhoGAP from p120RasGAP, leading to the localized suppression of RhoA in FAs to facilitate membrane ruffling in spreading or migrating cells (Tsubouchi et al. 2002).  In addition to tyrosine phosphorylation, paxillin is actually predominantly phosphorylated by paxillin-interacting serine/threonine kinase(s) on two serine residues,  25 Ser188 and Ser190, upon FN- or VN-stimulation (De Nichilo and Yamada 1996; Bellis et al. 1997).  However, the functional role of these phosphorylations is yet to be determined.  1.4.3 Key adapter proteins: Grb2, Crk, Nck  Grb2  Grb2 is a 25 kDa adapter protein that was initially isolated from a screen for epidermal growth factor receptor (EGFR) interacting proteins (Lowenstein et al. 1992; Giubellino et al. 2008).  It is highly conserved among different species and is ubiquitously expressed (Asada et al. 1999; Law et al. 1999).  Grb2 plays a critical role in early development since Grb2 knockout mice die early during embryogenesis (Cheng et al. 1998).  It is a cytosolic protein consisting of one central SH2 domain flanked by two SH3 domains (Fig. 1.6) (Lowenstein et al. 1992).  The SH2 domain of Grb2 recognizes a pYXNX motif (Songyang et al. 1994) while the SH3 domains bind to proline-rich (PXXP) motifs (Sparks et al. 1996).  Interestingly, the C-terminal SH3 domain (C-SH3) of Grb2 specifically binds to a PXXXRXXKP sequence that is characterized by an RXXK core motif (Lock et al. 2000; Lewitzky et al. 2001).   Grb2 is often associated with growth factor signaling, interacting with the cytoplasmic tail of receptor tyrosine kinases (RTKs) to mediate downstream ERK activation.  Nevertheless, it also plays an important part in integrin-mediated ERK signaling. Once the integrin-dependent Src-FAK complex is activated, Src phosphorylates FAK at several tyrosine residues, including the Tyr925, where the SH2 domain of Grb2 binds.  Through its N-terminal SH3 domain (N-SH3), Grb2 interacts with the consensus PXXPXR motif of the guanine-nucleotide exchange factor (GEF) Sos to promote Ras activation and downstream  26 signaling (Chardin et al. 1993; Schlaepfer et al. 1994; Simon and Schreiber 1995).  However, it has also been shown that independent of FAK-Grb2 association, ERK activation can be mediated through FAK-dependent Shc Tyr317 phosphorylation to promote Grb2-Shc binding (Wary et al. 1996; Schlaepfer et al. 1997; Schlaepfer et al. 1998).  This suggests that Grb2 regulates integrin-activated ERK signaling via multiple interactions with different molecules.  While the N-SH3 of Grb2 interacts with Sos, the atypical C-SH3 of Grb2 has been shown to interact with Grb2-associated binder (Gab) proteins, which are large multidomain docking proteins that function to localize, transduce and amplify extracellular signals mediated by cell surface receptors (Harkiolaki et al. 2009; Wohrle et al. 2009).  The SH3 domains of Grb2 also interact with cytoskeletal associated proteins, Wasp (She et al. 1997) and its homolog N-Wasp, to bring them into proximity with membrane bound proteins such as Cdc42 and signal via a Wasp-Arp2/3 complex to modulate actin polymerization and filopodia formation (Aspenstrom et al. 1996; Carlier et al. 2000; Higgs and Pollard 2001).  Thus, Grb2 acts as a bridge that connects signaling molecules to the actin cytoskeleton.     Crk  There are three members in the Crk family:  CrkI, CrkII and Crk-like protein (CRKL) (Matsuda et al. 1992; ten Hoeve et al. 1993; Cabodi et al. 2010).  Human CrkI and CrkII are alternate transcripts of Crk that were first cloned by Matsuda et al. in 1992.  CrkI encodes for a 28 kDa protein with one SH2 and one SH3 domains whereas CrkII encodes for a 40 kDa protein similar to CrkI but with an extra SH3 domain (Matsuda et al. 1992).  When it is phosphorylated, CrkII appears as a 42 kDa protein (Fig. 1.6) (Matsuda et al. 1992).  Mice  27 that lack both CrkI and CrkII die perinatally (Park et al. 2006).  In this thesis dissertation, Crk refers to the widely expressed CrkII.  The N-terminal SH2 domain of Crk binds to a pYXXP motif (Birge et al. 1993) while the first SH3 domain recognizes a Polyproline Type II (PPII) motif (PXXPXK), where the lysine is critical for binding affinity and specificity (Knudsen et al. 1995; Wu et al. 1995).  The atypical C-SH3 of Crk is unable to bind to PPII motifs (Muralidharan et al. 2006; Jankowski et al. 2012) yet it can be phosphorylated at Tyr251 by Abl kinase, providing binding sites for SH2/PTB-domain containing proteins to mediate non-canonical signaling pathways (Sriram et al. 2011; Sriram and Birge 2012).  It has also been shown to bind in trans to N-SH3 to block N-SH3 binding with PPII ligands (Sarkar et al. 2007; Sarkar et al. 2011; Sriram and Birge 2012).  Crk can be phosphorylated at Tyr221, which resides in the inter-SH3 linker region, and forms an inhibitory interaction with its SH2 domain to prevent binding of the SH2 domain with other tyrosine phosphorylated proteins (Feller et al. 1994; Rosen et al. 1995; Kobashigawa et al. 2007).  These reports reveal the inhibitory nature of the C-terminal region of Crk.  Crk plays a role to link ECM-integrin to GTPase effector signaling pathways to regulate actin dynamics, cell adhesion and motility.  It mediates integrin-induced Rac activation via the assembly of the Cas-Crk-DOCK180-ELMO complex where Crk binds to phosphorylated Cas SD and recruits DOCK180 through its SH2 and first SH3 domains respectively (Feller et al. 1995; Chodniewicz and Klemke 2004; Barrett et al. 2013).  Besides DOCK180, C3G is another GEF that is found in the Cas-Crk complex upon integrin ligation and binds to the SH3 domain of Crk to regulate Rap1 activation (Tanaka et al. 1994; Vuori et al. 1996; Birge et al. 2009).  Conversely, the Abl kinase, which also interacts with Crk SH3  28 domain, phosphorylates Crk at Tyr221 and mediates uncoupling of the Cas-Crk complex to inhibit cell migration (Kain and Klemke 2001).    Nck  The Nck gene was originally isolated from a human melanoma cDNA library, and encodes a ubiquitously expressed 47 kDa cytosolic protein (Lehmann et al. 1990).  Two members of the family, Nck1 and Nck2 (also known as Grb4), were identified in mammalian cells (Chen et al. 1998; Braverman and Quilliam 1999).  Mice that lack either Nck1 or Nck2 are viable but animals with a deficiency in both proteins are not (Bladt et al. 2003).  Fibroblasts derived from Nck1-/-Nck2-/- embryos show defective actin organization in lamellipodia formation and cell migration, suggesting that Nck plays a role in cytoskeletal arrangements and motility (Bladt et al. 2003).    Nck is an SH2/SH3 adaptor that is characterized by three SH3 domains followed by an SH2 domain that recognizes a pYDEP binding motif (Songyang and Cantley 1995).  It functions to couple RTKs or tyrosine phosphorylated docking proteins to regulate the actin cytoskeleton.  The SH2 domain of Nck can bind to ECM-induced phosphorylated Dok (Noguchi et al. 1999) and Cas (Schlaepfer et al. 1997) to mediate downstream signaling through its SH3 domain interactions.  The Cas-Nck complex has been shown to regulate Cdc42 activation and cell polarity, likely through the Nck-PAK-PIX signaling complex (Funasaka et al. 2010), where the LIM4 domain of p21-activated protein kinase (PAK), a serine/threonine kinase, interacts with the second SH3 domain of Nck in an adhesion-dependent manner (Lu et al. 1997; Howe 2001; Velyvis et al. 2003).  Paxillin has also been shown to mediate the focal adhesion localization of an Nck-PAK-PIX-PKL complex, in  29 which Nck binds to phosphorylated paxillin kinase linker (PKL) through its SH2 domain, upon integrin stimulation and in response to active Rac (Brown et al. 2005).     In addition, Nck has been implicated in integrin signaling through its interaction with PINCH (Tu et al. 1998) which binds to integrin-linked kinase (ILK), connecting ILK to downstream actin-based motility (Li et al. 2001; Buday et al. 2002).  Its ability to interact with cytoskeletal-associated proteins, such as the Wasp and the Wasp-interacting protein (WIP), indicates that Nck plays important roles in modulating actin dynamics (Rivero-Lezcano et al. 1995).  A recent microscopic study reveals the role of Nck in the spatiotemporal coordination of Rho GTPases in directional migration and modulation of integrin-ECM adhesion force (Chaki et al. 2013).     1.4.4 A novel NSP family adaptor protein:  BCAR3   Human breast cancer anti-estrogen resistance-3 (BCAR3) was first identified in a screen for genes that promoted Tamoxifen-resistance in hormone-sensitive breast cancer cells (van Agthoven et al. 1998).  It belongs to the novel SH2-containing proteins (NSPs), which includes NSP1 and Chat/SHEP1 (Lu et al. 1999).  BCAR3 is also known as NSP2 and AND-34 (murine homologue). It is a 95 kDa adaptor protein that is expressed in various tissues. The N-terminus of BCAR3 featured an SH2 domain while the C-terminus featured a Cdc25 GEF-like domain (van Agthoven et al. 1998), which interacts with the C-terminus of Cas (Cai et al. 1999; Gotoh et al. 2000).  BCAR3 is phosphorylated upon fibronectin stimulation but the function of this phosphorylation is unclear (Cai et al. 1999).  Fibroblasts that overexpress BCAR3 exhibit increased membrane ruffles, decreased actin stress fibers and a clear polarized phenotype (Cai et al. 2003; Riggins et al. 2003).  30  The presence of the BCAR3-Cas complex positively correlates with the invasiveness of breast cancer cells (Schrecengost et al. 2007).  Enhanced cell migration, BCAR3-Cas complex formation and co-localization at the cell periphery were observed in less invasive breast cancer cells overexpressing BCAR3 (Schrecengost et al. 2007).  The co-overexpression of BCAR3 and Cas in fibroblasts also promoted cell migration concomitant with Cas localization to the leading edge of a migrating cell (Riggins et al. 2003).  These finding suggests that the spatial-temporal regulation of BCAR3-Cas association is important for cell migration.  BCAR3 has been shown to regulate Cas-Src interaction, Src activation, Src-dependent Cas tyrosine phosphorylation, fibronectin-induced cell spreading and migration (Riggins et al. 2003; Schuh et al. 2010).  It is proposed that BCAR3 functions to translocate Cas from the cytosol to the plasma membrane where Cas interacts with and activates Src, which in turn phosphorylates Cas to lead to downstream activation of Rac and Rap1 (Schuh et al. 2010).  The GEF-like domain is associated with the activation of small GTPases RalA, Rap1, R-Ras, Rac and Cdc42, as well as the effector protein Pak1 (Gotoh et al. 2000; Cai et al. 2003).  Both the SH2 and GEF-like domain has been shown to be important for BCAR3-mediated Rac activation (Felekkis et al. 2005).  However, no BCAR3 SH2-domain binding partners have been identified.  1.5 Protein tyrosine phosphatases (PTPs) in integrin signaling   Phosphorylation is a common post-translational modification that is widely observed in cells to transduce signals under normal and pathophysiological conditions.  The protein residues that can be phosphorylated are serine, threonine and tyrosine.  While tyrosine  31 phosphorylation, first discovered in 1979 (Eckhart et al. 1979), contributes to only 3.8% of total cellular phosphorylation (Olsen et al. 2006), it plays important roles in many eukaryotic processes, including cell proliferation, cell cycle progression, metabolic homeostasis, differentiation and development.  Aberrant tyrosine phosphorylation is often associated with cancer.  Reversible protein tyrosine phosphorylation is regulated by two groups of enzymes, protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).  To date, there are ~90 PTKs and over 100 PTPs identified in the human genome (Manning et al. 2002; Hunter et al. 2009; Kim and Ryu 2012).  Intuitively, PTKs are considered as activators of signaling cascades whereas PTPs are viewed as negative regulators that counteract the actions of PTKs, thus PTKs are oncogenic and PTPs are tumor suppressive.  However, many studies have shown that some PTPs are potential oncogenes (Julien et al. 2011; Labbe et al. 2012).  The physiological role of many PTPs was discovered through knockout mouse models.  The loss of some PTPs are embryonic lethal while others may display mild defects (Hendriks et al. 2008), suggesting that some PTPs have non-redundant functions.  In addition, cell lines, such as mouse embryonic fibroblasts, derived from the knockout animals, are also used to study the functions of various PTPs.  In the following section, I will focus on describing the PTP superfamily and a few of the major PTPs that function in the integrin signaling cascade.  1.5.1 Overview of PTP superfamily   The PTP superfamily comprises at least 107 members that are subdivided into 4 classes based on the amino acid sequence of their catalytic motif.  Class I, II and III are cysteine-based while Class IV is aspartic acid-based.  Class I represents the largest group of PTPs and is subdivided into two categories, the tyrosine-specific classical PTPs and the dual  32 specificity PTPs (DUSPs).  The classical PTP domains, composed of ~280 amino acids, are highly conserved and are characterized by the signature catalytic motif (HCX5R) and mobile acid/base loop (WPD) (Andersen et al. 2001).  Of the 38 members in this subgroup, 17 of them are cytosolic while the remaining 21 are receptor-like PTPs (RPTPs) (Tonks 2006; Kim and Ryu 2012).  RPTPs may possess a single or tandem catalytic domain(s).  The extracellular domain of some of these RPTPs contains features that are similar to those in other adhesion receptors, implicating them in mediating cell-cell and cell-ECM interactions.  Unlike classical PTPs, DUSPs are less conserved and have low sequence homology though they share the same cysteine-containing motif and catalytic mechanism as the classical PTPs.  As their name suggests, the DUSPs can dephosphorylate phosphoserine (pSer) and phosphothreonine (pThr), in addition to phosphotyrosine (pTyr).  The MAPK phosphatases (MKPs) and phosphatase of regenerating liver (PRLs) are examples of DUSPs.  Low molecular weight PTP (LW-PTP) is the only Class II PTP member.  It is an 18 kDa cytosolic phosphatase that does not share any sequence homology with other PTPs besides the cysteine-based signature motif (Wang et al. 2003).  The major difference that distinguishes this class of PTP from the others is the location of its active site which is near the N-terminus of the catalytic domain while the others are closer to the C-terminus.     Class III PTPs consists of the cell division cycle 25 (Cdc25) phosphatases that specifically remove the inhibitory phosphates from cyclin-dependent kinases in order to promote cell cycle progression.  Like the LW-PTP, the Cdc25 phosphatases have little sequence homology with other PTPs.  The cysteine-based signature motif in Cdc25 is flanked by two Cdc25 homology (CH2) domains (Fauman et al. 1998).  There are three members in  33 this class of PTPs, Cdc25A, B and C, where the first two are overexpressed in various human cancers (Gasparotto et al. 1997; Kristjansdottir and Rudolph 2004).  Class IV PTPs comprise four members of the cytosolic eyes absent (EYA) phosphatases.  They play dual roles as a transcription factor and an enzyme (Jemc and Rebay 2007).  Rather than using cysteine, the EYA phosphatases use an aspartate as a nucleophile for catalysis. This class of PTP can dephosphorylate both pTyr and pThr through distinctive regions within its haloacid dehalogenase (HAD) domain (Okabe et al. 2009).   1.5.2 Regulation of PTPs   The functional roles of PTPs not only rely on their substrate selectivity but also depend on the spatial organization mediated by protein-protein interactions that modulate subcellular compartmentalization and substrate docking (Sacco et al. 2012).  Post-translational modifications, such as phosphorylation, proteolytic cleavage and reversible oxidation, may also contribute to the regulation of PTP function (Labbe et al. 2012).  1.5.3 PTPs involved in integrin signaling   Integrin-induced adhesion is followed by an increase in protein tyrosine phosphorylation, implicating a general inhibition of PTP activity (Maher 1993).  On the other hand, focal adhesion disassembly is required for cell migration to occur, suggesting an active role for PTPs in focal adhesion turnover.  Thus, PTPs may act proximal to integrin engagement or target downstream signaling molecules to modulate adhesion dynamics and  34 regulate directional cell migration (Burridge et al. 2006).  Here, I will briefly describe several PTPs that are known to play a major role in integrin signaling.   PTP-PEST  PTP-PEST, encoded by the PTPN12 gene, is a ubiquitously expressed cytosolic PTP that belongs to the PEP family (Yang et al. 1993; Garton and Tonks 1994; Charest et al. 1995).  PTP-PEST knockout mice are embryonic lethal (Sirois et al. 2006).  In non-hematopoietic cells, PTP-PEST is a key regulator of cell adhesion and migration (Veillette et al. 2009).   It is activated in an adhesion-dependent manner and localized to the tips of membrane protrusions in spreading fibroblasts (Sastry et al. 2002).  Although both the overexpression of PTP-PEST and its targeted deletion are associated with impaired cell migration, the former is associated with reduced phosphorylation of a major focal adhesion protein, Cas, while the latter displayed enhanced Cas phosphorylation [Garton and Tonks, 1999; Angers-Loustau, 1999].  This intriguing phenomenon suggests that PTP-PEST regulates cell migration through multiple pathways. Since the interaction between paxillin, Cas and PTP-PEST has been shown to facilitate PTP-PEST mediated dephosphorylation of paxillin and Cas (Garton et al. 1997; Shen et al. 2000), and the the expression level of PTP-PEST can modulate the activity of Rac1 (Sastry et al. 2002), it is speculated that PTP-PEST regulates cell migration by interacting with and dephosphorylating other focal adhesion proteins, such as Cas and paxillin, to coordinate the activity of Rac1, a Rho GTPase that controls membrane ruffle formation.  Furthermore, PTP-PEST is also shown to differentially regulate Rho proteins (Rac1 and RhoA) at both the leading and retracting ends of a migrating cell by modulating their upstream regulators Vav2 and p190RhoGAP (Sastry et al. 2006).  A  35 recent study reported Src Kinase Associated Phosphoprotein 55 Homologue (SKAP-Hom) as a novel interacting protein, as well as substrate of PTP-PEST and demonstrated that the phosphorylation of SKAP-Hom regulates integrin-dependent cell migration (Ayoub et al. 2013), suggesting that PTP-PEST may modulate cell migration via regulation of SKAP-Hom phosphorylation.  PTP1B  PTP1B was the first PTP to be identified (Charbonneau et al. 1988; Brown-Shimer et al. 1990).  Mice that lack PTP1B are resistant to diabetes and obesity (Elchebly et al. 1999; Klaman et al. 2000).  The role of PTP1B in integrin signaling is unclear as PTP1B silencing and overexpression are both associated with decreased adhesion, motility and FA formation (Liu et al. 1998; Hassid et al. 1999).  Early studies suggested that PTP1B negatively regulates integrin signaling through its association and dephosphorylation of Cas (Liu et al. 1996; Liu et al. 1998).  However, in another study, PTP1B was proposed to positively regulate integrin signaling as fibroblasts overexpressing a catalytically inactive mutant PTP1B exhibit reduced fibronectin-induced tyrosine phosphorylation of FAK and paxillin (Arregui et al. 1998).  In support of this, a later study, which utilizes small molecule PTP1B inhibitors and a substrate-trapping method, also provides evidence showing that PTP1B positively regulates fibronectin-mediated integrin signaling by dephosphorylating the inhibitory pTyr527 of Src to facilitate Src activation (Liang et al. 2005).  Furthermore, Burdisso et al. (2013) have shown that PTP1B is required to promote integrin-mediated Rac1 activation, FA maturation, membrane protrusion and directional cell migration.   36 SHP2  Shp2 is a cytoplasmic classical PTP that is ubiquitously expressed.  It has two N-terminal SH2 domains, a catalytic domain and a C-terminal tail (Neel et al. 2003).  The role of Shp2 has been implicated in both growth factor receptor signaling and integrin signaling.  However, its precise mechanism of action in integrin-mediated signaling events is unclear due to conflicting data.  Nonetheless, Shp2 is required for integrin-induced cell spreading, migration and Erk activation as studied in cells lacking Shp2 or overexpressing a Shp2 mutant (Yu et al. 1998; Oh et al. 1999; Inagaki et al. 2000). Another study also shows increased stress fibers and enhanced activation of the small GTPases Rho, which controls stress-fiber formation, in Shp2 mutant cells (Schoenwaelder et al. 2000).  It has been proposed that integrin stimulation leads to Src-mediated SHP substrate 1 (SHPS-1) phosphorylation and recruits Shp2 to the membrane-bound SHPS-1 (Tsuda et al. 1998; Oh et al. 1999), suggesting that the effects of Shp2 on adhesion are mediated by Shps1-Shp2 complexes.  The effects of Shp2 on FAK tyrosine phosphorylation are also contradictory.  Some showed that the Shp2 triggers FAK dephosphorylation while others found little or no effect (Yu et al. 1998; Manes et al. 1999; Oh et al. 1999; Inagaki et al. 2000).  PTEN  PTEN is a well-known tumour suppressor that is frequently deleted or mutated in various cancers (Kong et al. 1997; Li et al. 1997; Steck et al. 1997).  It belongs to the DUSPs and also has the ability to dephosphorylate lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Yamada and Araki 2001).  It has an N-terminal region that is highly homologous to tensin, a focal adhesion protein that interacts with actin filaments.  Investigations of the role  37 of PTEN in integrin signaling showed that PTEN-depletion caused an increase in cell migration while the overexpression of PTEN led to reduced cell spreading and cell migration on fibronectin-coated dishes (Tamura et al. 1998).  In addition, PTEN also reduced integrin-mediated FAK phosphorylation concomitant with impaired focal adhesion formation and actin stress fiber formation.   Indeed, PTEN can dephosphorylate FAK both in vitro and in vivo and FAK phosphorylation/activation is associated with cell migration, this suggests that PTEN likely functions in integrin-mediated adhesion signaling through its regulation of FAK.  By co-transfecting cells with MEK1, a kinase that functions in the MAPK signaling cascade, and PTEN, impaired fibronectin-induced cell spreading resulting from PTEN expression can be partially rescued (Gu et al. 1998), indicating that PTEN plays a role in integrin-mediated MAP kinase pathway to control cell behavior.  1.6 PTP? and its roles in integrin signaling   PTP? was first discovered and cloned in 1990 by multiple groups (Kaplan et al. 1990; Krueger et al. 1990; Matthews et al. 1990; Sap et al. 1990).  It is a transmembrane receptor-like PTP that is ubiquitously expressed but is most abundant in the brain (Sap et al. 1990), particularly in the neocortex, hippocampus and cerebellum of postnatal rat brain (Sahin et al. 1995).  PTP? knockout mice are viable and display no apparent abnormalities (Ponniah et al. 1999; Su et al. 1999), suggesting that other PTP(s) may compensate for its function(s).  Despite their normal appearance, PTP?-deficient mice showed spatial learning deficits in the Morris water maze test, decreased levels of activity, reduced anxiety/fearfulness (Skelton et al. 2003) and ~50-70% less Src and Fyn activities in the brain (Ponniah et al. 1999; Su et al. 1999).  These mice lacking PTP? also had less myelinated  38 fibers in the brain, suggesting a role for PTP? in central nervous system (CNS) myelination (Wang et al. 2009).  Petrone et al. (2003) have found that PTP? is required for hippocampal neuronal migration and long-term potentiation as well.   Although these findings centers on the neuronal functions of PTP? in normal physiological setting, other reports have shown that PTP? plays a role in pathological contexts.  For example, the overexpression of PTP? has been associated with human cancers, including primary breast tumors, colorectal, head and neck and gastric cancer (Tabiti et al. 1995; Berndt et al. 1999; Ardini et al. 2000; Wu et al. 2006; Krndija et al. 2010).  This is in accord with an early study demonstrating that the overexpression of PTP? in rat embryo fibroblasts promotes anchorage-independent growth in soft agar and enhances tumor formation in nude mice (Zheng et al. 1992).  These findings reveal the oncogenic potential of PTP?.  ????? Structure and function of PTP??  PTP? is a receptor-like classical PTP characterized by a heavily glycosylated extracellular domain (ECD), a transmembrane (TM) region and two tandem catalytic domains (D1 and D2) (Fig. 1.7).  The mature 130 kDa PTP? protein contains both N- and O-linked carbohydrates whereas the 100 kDa precursor is exclusively N-glycosylated (Daum et al. 1994).  The ECD of PTP? is relatively short and has no known ligand.  It has been shown to bind in cis configuration with the neuronal GPI-anchored receptor contactin and neural cell adhesion molecule (NCAM) [Zeng, 1999; Bodrikov et al, 2005].  Other cell surface molecules including Close Homolog of L1 (CHL1) and NB3 can also interact with PTP??(Ye et al. 2008), yet it is unclear whether any of these cell surface molecules is functioning as a  39 ligand for PTP?.  Two splice variants of the ECD that differ by 9 amino acids were identified (Kaplan et al. 1990; Krueger et al. 1990; Matthews et al. 1990).  As conflicting results have been reported regarding Src-dependent focus formation of these two splice variants (Kapp et al. 2007; Tremper-Wells et al. 2010), it is unclear which one of these isoforms is more efficient in activating Src although it implies a role for the extracellular domain of PTP? in cellular transformation.  The TM region of PTP? was shown to be sufficient to drive PTP? dimerization using FRET analysis and chemical cross-linking (Jiang et al. 2000; Tertoolen et al. 2001).  Although some have suggested that PTP? dimerization inhibits PTP? activity (Bilwes et al. 1996), the existence and precise regulation and function of a PTP? dimer remain to be determined.  PTP? is one of 12 RPTPs that possesses two catalytic domains.  Like other RPTPs, the membrane proximal domain (D1) of PTP? is responsible for most of its catalytic activity (Wang and Pallen 1991) under physiological conditions (den Hertog et al. 1993) while the role of the low-activity membrane distal domain (D2) is elusive (Pallen 2003).  The catalytic domain D2 differs from D1 by two conserved amino acids which likely accounts for the differences in their catalytic activity (Lim et al. 1998; Buist et al. 1999).  The Tyr residue in the KNRY motif of D1 that is involved in substrate recognition is replaced by a Val (valine) residue in D2, and the Asp (aspartic acid) within the WPD loop in D1 (Zhang and Dixon 1994; Denu et al. 1996) which is responsible for supporting the acid-base reaction in substrate hydrolysis is substituted by a Glu (glutamic acid) residue.  Crystallography of PTP? also reveals that the D2 domain lacks the helix-loop-helix wedge motif (Sonnenburg et al. 2003) of D1 that can occlude the active site of one molecule within the PTP? D1 dimer (Bilwes et al. 1996).  The N-terminal region of D2 corresponding to the D1 helix-loop-helix  40 is highly flexible and is proposed to facilitate inter- or intra-molecular interactions (Sonnenburg et al. 2003).    ????? Phosphorylation of PTP??  PTP? can be phosphorylated at Ser180 and Ser204 in the juxtamembrane region, as well as at Tyr789 in the C-terminal tail.  Protein kinase C (PKC) can phosphorylate Ser180 and Ser204 in vivo in NIH 3T3 cells (den Hertog et al. 1995; Tracy et al. 1995) and regulated phosphorylation of these serine residues is associated with enhanced binding of Src to PTP? during mitosis (Zheng et al. 2002; Vacaru and den Hertog 2010).  Zheng et al. (2002) proposed that an intramolecular association between the C-terminal tail and the serine-phosphorylated membrane-proximal region of PTP? stabilizes an extended conformation of the C-terminal region of PTP?, favoring Src SH2 domain interaction with phosphoTyr789 of PTP? and Src activation during mitosis.  In contrast, Vacaru et al. (2010) showed that it is the dephosphorylation, rather than phosphorylation, of PTP? Ser204 catalyzed by a phosphatase PP2A that induces PTP?-Src association during mitosis.  They also showed that the interaction between PTP? and Src is not mediated by the binding of Src SH2 domain to PTP?-phosphoTyr789.    In vivo, about 20% of PTP? in NIH 3T3 cells is constitutively phosphorylated at Tyr789, the fifth amino acid from the C-terminus, and most of it is bound by Grb2 (den Hertog et al. 1994; Su et al. 1994).  However, the function of Grb2 in association with PTP? is not well defined.  Phosphorylation of PTP? Tyr789 does not affect the catalytic activity of PTP? (Su et al. 1996; Lammers et al. 1998; Zheng et al. 2000; Chen et al. 2006).  Studies  41 using an unphosphorylatable mutant of PTP? (Y789F) revealed different roles for PTP??Tyr789 in various processes including inhibition of neurite outgrowth, T-cell signaling, PTP? focal adhesion localization and integrin-induced or IGF-1-induced cell migration [Su, 1996; Maksumova, 2007; Lammers, 2000; Chen, 2006; Chen, 2009].  In integrin signaling, the phosphorylation of PTP? Tyr789 is mediated by activated Src in complex with FAK (Chen et al. 2006).  Maksumova et al. (2007) showed that in T-cell signaling, PTP? tyrosine phosphorylation also requires SFK activity and the presence of RPTP CD45 reduces PTP? tyrosine phosphorylation.  Other external stimuli, such as oxidative stress, also suppress tyrosine phosphorylation of PTP? (Hao et al. 2006).  1.6.3 Roles of PTP? in integrin signaling   As mentioned earlier, PTP? expression is often associated with neural development and cancer progression.  These physiological processes involve a wide range of cellular activities, such as cell growth, migration and survival, which are regulated by the transmembrane integrins.  A role for PTP? in cell adhesion was first established by Harder et al. in 1998.  They showed that the ectopic expression of PTP? in A431 epidermoid carcinoma cells results in increased cell-substratum adhesion and this is dependent on D1 of PTP?.  To further investigate the role of PTP? in integrin signaling, several groups, including our laboratory, have used mouse embryo fibroblasts (MEFs) derived from wild type and PTP? knockout mice.  Indeed, several signaling events downstream of integrin ligation are defective in the absence of PTP?.  In PTP?-null cells, Src and Fyn activities are reduced (Ponniah et al. 1999; Su et al. 1999), as is the phosphorylation of FAK Tyr397, Cas,  42 and ERK (Su et al. 1999; Zeng et al. 2003).  Concomitantly, cell spreading on fibronectin is impaired in PTP?-deficient cells (Su et al. 1999; Zeng et al. 2003).   Indeed, PTP? has been shown to colocalize and co-immunprecipitate with ?v-integrins in wild type MEFs upon cell spreading on fibronectin, and is proposed to act as a mechanical transducer of ?v/?3-integrin mediated signaling, stabilizing the integrin-cytoskeleton linkages (von Wichert et al. 2003).  Thus far, two roles of PTP? in integrin signaling have been established based on experimental data from our lab and other groups:  1) an integrin-proximal upstream role which requires the phosphatase activity of PTP? and 2) a downstream role which is independent of PTP? catalytic activity but dependent on the regulated phosphorylation of Tyr789 (Fig. 1.8).  Upon integrin stimulation, PTP? acts proximal to the integrins to dephosphorylate the inhibitory Src Tyr527 and activate Src (Ponniah et al. 1999; Su et al. 1999; Zeng et al. 2003).  Activated Src promotes FAK autophosphorylation at Tyr397 (Klinghoffer et al. 1999; Salazar and Rozengurt 2001).  The SH2 domain of Src then interacts with phosphoTyr397 of FAK (Schaller et al. 1994), enabling Src to phosphorylate Tyr576/577 in the activation loop of FAK.  The fully activated Src-FAK kinase complex can then phosphorylate other focal adhesion proteins, such as Cas and paxillin, to transduce downstream signals (Schlaepfer et al. 1999).  Ultimately, PTP? regulates Rac1-mediated FA formation and turnover, actin stress fiber and cytoskeletal organization, and cell migration (Herrera Abreu et al. 2008).  While the first role of PTP? is to function as a Src activator, PTP? also plays a distinct second role in integrin signaling downstream of the Src-FAK complex.  PTP? undergoes regulated phosphorylation at Tyr789 in response to integrin stimulation and this phosphorylation is not involved in the upstream signaling action of PTP? as it is not required for integrin-stimulated SFK or FAK activation, nor does it affect  43 PTP? activity (Chen et al. 2006).  In PTP?-null fibroblasts, delayed integrin-induced stress fiber and focal adhesion formation, and impaired migration are observed (Zeng et al. 2003).  Reintroduction of wild type (WT) PTP?, but not the unphosphorylatable (Y789F) PTP??mutant, into PTP?-null cells rescued the cells from these defective phenotypes (Chen et al. 2006).  This indicates that PTP? also plays a downstream role in integrin signaling and that this role depends on PTP??Tyr789 phosphorylation.  1.6.4 Binding partners of PTP?-phosphoTyr789:  Src and Grb2   The phosphorylation of PTP? at Tyr789 creates an SH2-domain binding site that can promote intermolecular interactions.  Two molecules, Grb2 and Src, have been shown to interact with PTP?-phosphoTyr789.  The binding of Grb2, via its SH2 domain, to PTP?-phosphoTyr789 is well established (den Hertog et al. 1994; Su et al. 1994), whereas the interaction between the Src SH2 domain and phosphoTyr789 of PTP? is controversial (Zheng et al. 2000; Vacaru and den Hertog 2010).    Src  As observed by den Hertog et al. (1994), the majority of PTP? phosphorylated at Tyr789 is bound by Grb2, however, Zheng et al. (2000) have shown that PTP? can interact with Src in vivo and that this interaction is blocked by the expression of GST-Src-SH2 domain or the unphosphorylatable mutant PTP?-Y789F.  Based on the consensus binding sequence (Songyang and Cantley 1995) and titration experiments (Zheng et al. 2000), PTP??Tyr789 within the neighbouring sequence (Y789ANK) provides a higher affinity  44 binding site for the SH2 domain of Grb2 than Src (Zheng et al. 2000).  Therefore, Src is proposed to displace Grb2 from tyrosine phosphorylated PTP? during mitosis when an increase in PTP?-Src association, concomitant with a decrease in PTP?-Grb2 association, was observed (Zheng and Shalloway 2001).  The displacement of Grb2 by Src from PTP?-phosphoTyr789 is thought to be mediated by serine phosphorylation of PTP? (Zheng et al. 2002)? which induces a conformational change in PTP? to favour Src binding.  This model suggested by Zheng et al. is controversial as another group (Vacaru and den Hertog 2010) has shown totally different results.  Vacaru et al. (2010) found that the interaction between Src and PTP? 1) is not induced upon PTP? serine phosphorylation but serine dephosphorylation, 2) is not mediated by Src SH2 domain binding to PTP?-phosphoTyr789, and 3) minimally affects PTP?-Grb2 interaction.  Based on these conflicting findings, the direct binding of the Src SH2 domain with PTP?-phoshpoTyr789 is highly questionable.    Grb2  The Grb2 SH2 domain interacting motif (YXNX) matches the C-terminal tail sequence of PTP? (Y789ANK) and only WT- but not the Y789F-mutant PTP? is able to interact with Grb2 in vivo (den Hertog et al. 1994).  Other reports have shown that the C-terminal SH3 (C-SH3) domain of Grb2 is also required for mediating tight association between Grb2 and PTP? (den Hertog and Hunter 1996; Su et al. 1996) (Fig. 1.9).  Using deletion mutants, the region of PTP? spanning residues 469-486 is found to interact with the C-SH3 domain of Grb2.  This stretch of amino acids lies in the C-terminal region of the membrane proximal PTP? D1 domain near the catalytic cleft, raising the possibility that Grb2 binding to PTP? is inhibitory. The N-terminal SH3 (N-SH3) domain of Grb2 is  45 commonly known to interact with the Ras guanine nucleotide exchange factor, Son of Sevenless (Sos).  However, PTP? does not co-immunoprecipitate with Sos nor is it found in the Grb2-Sos complex.  Furthermore, in vitro binding assays showed that PTP? and Sos exhibit mutually exclusive binding to Grb2, indicating that PTP?-bound Grb2 is not linked to Sos.  Since Grb2 that is coupled to Sos activates Ras and MAPK signaling, the interaction between PTP?-phosphoTyr789 and Grb2 has been proposed to sequester Grb2 from interacting with Sos, thereby diminishing Grb2-Sos-mediated signaling.  Although there are many speculative roles for Grb2 in PTP?-phosphoTyr789 mediated signaling, the exact function of Grb2 in this context remains unclear.  1.7 Rationale and hypothesis   Integrin-induced PTP? Tyr789 phosphorylation functions downstream of the Src-FAK kinase complex to regulate cell spreading and migration but the detailed phosphoPTP?-mediated molecular signaling events that link phosphoPTP? to cell motility are undefined.  As PTP?-phosphoTyr789 is not required for PTP? activity, it may be serving as a docking site to promote interactions with adaptor proteins or signaling molecules to mediate downstream events.  Thus, I hypothesize that integrin-induced PTP?-phosphoTyr789 functions as a docking site to recruit other focal adhesion proteins to regulate cytoskeletal rearrangement and cell migration.  The first aim of my study focuses on the identification of novel PTP?-phosphoTyr789 binding protein(s) and the associated downstream signaling mechanism.  Since Grb2 is the major protein that is bound to phosphoTyr789 of PTP? and the role of this PTP?-Grb2 complex in integrin signaling is  46 unclear, the second aim of my study is to delineate the role of phosphoPTP?-bound Grb2.  Having a precise understanding of PTP?-mediated signaling events may illuminate various pathways that modulate cell migration, which is an important process in normal development and in disease progression.                           Figure 1.1  Schematic diagram of the migration cycle. (A) Cell migration begins when the cell extends membrane protrusions to survey the surrounding microenvironment. (B) New adhesions form as the cell surface receptors interact with the substratum, anchoring the cytoskeleton to the extracellular matrix.  (C) Myosin-II-mediated stress fiber contraction pulls the cell body forward towards the direction of the stimulus. (D)  The trailing edge of the migrating cell retracts as a result of adhesion disassembly.   (Adopted from Larsen et al. 2003)  Protrusion Cell Body Nucleus Adhesion A B C D New adhesion Cell body contracts Tail releases 47 Figure 1.2  Integrin bi-directional signaling.  (A)  Inside-out signaling:  In resting state, the ? and ? subunits of integrins are in a closed ?bent ?conformation.  Intracellular signals, such as the binding of talin to the cytoplasmic tail of integrins, induces a conformational change in the integrins.  The extended position of integrins increases their ligand-binding affinity.  (B)  Outside-in signaling:  Integrin engagement with extracellular matrix (ECM) ligands trigger integrin clustering and intracellular signaling.  The cytoplasmic tail of integrins recruits signaling proteins to form focal adhesions that stabilize the linkage between the ECM and the cytoskeleton.  A B P P ????Cytoplasm Integrin clustering ECM Focal adhesion Cytoskeleton ????Talin Integrin Cytoplasm Inactive Active ????48 Figure 1.3  Structures of adhesions in a polarized/migrating cell.  As a cell moves toward extracellular stimuli (as indicated by the arrow), it begins to form finger-like protrusions called filopodia and sheet-like protrusions termed lamellipodia.  Small, short-lived nascent adhesions form to stabilize these structures at the leading edge of the lamellopodia.  These adhesions either dissemble at the lamellipodium-lamellum interface coinciding with disassembling actin filaments or they mature into focal complexes (FCs).  These slightly larger FCs then elongate and grow into mature focal adhesions (FAs) which are found at the ends of actin stress fibers or bundles. (Adapted from Parsons et al. 2010) Nucleus Lamellum Lamellipodium Filopodium Actin stress fibers Focal adhesion (FA) Nascent adhesion Disassembling adhesion Focal complex (FC) 49 Figure 1.4  Domain structures of FAK and Src.  The protein tyrosine kinase (PTK) FAK possesses an N-terminal FERM (protein 4.1, ezrin, radixin, and moesin homology) domain, a central kinase domain (KD), two proline-rich (PR) regions, and a focal adhesion targeting domain (FAT).  FAK has an autophosphorylation site at Tyr397, and other tyrosine phosphosites in its kinase domain (Tyr576/577) and at the C-terminal region (Tyr861 and Tyr925).  Like FAK, Src is a non-receptor PTK.  It differs from FAK in that it can associate with the plasma membrane through N-myristoylation.  Src contains an N-terminal SH3 domain, a central SH2 domain and a tyrosine kinase domain (KD).  Src Tyr527 phosphorylation is inhibitory while Src Tyr416 phosphorylation is indicative of Src activation.  FAK FERM KD FAT N C Tyr397 Tyr576/577 Tyr861 Tyr925 Src SH3 SH2 N C KD Tyr416 Tyr527 Myristoylation PR1 PR2 50 Figure 1.5  Domain structures of Cas and paxillin.  Cas and paxillin are scaffolding proteins with no enzymatic activity.  Cas possesses an N-terminal SH3 domain, a proline-rich region (PR), a substrate domain (SD) containing 15 tyrosine phosphorylation sites as indicated, a serine-rich region (SR), a Src binding domain (SBD) and a Cas family C-terminal homology domain (CCH).  The N-terminal half of paxillin is characterized by five leucine-rich (LD) motifs and a proline-rich (PR) region whereas its C-terminus contains four tandem LIM (lin-11, isl-1-mec-3) domains.  Upon integrin ligation, Tyr31 and Tyr118 of paxillin can be phosphorylated by FAK and Src while Ser188 and Ser190 can be phosphorylated by paxillin-interacting serine/threonine kinase(s).  Cas Tyr128 Tyr165 PR C SBD CCH Tyr372 Tyr410 Tyr387 Tyr362 Tyr327 Tyr306 SH3 N Substrate Domain (SD) SR Tyr192 Tyr222 Tyr224 Tyr234 Tyr249 Tyr267 Paxillin N C Lim4 Tyr31 PR Lim3 Lim2 Lim1 LD1 LD5 LD2 LD3 LD4 Tyr118 Ser188 Ser190 51 Figure 1.6  Domain structures of Crk, Nck and Grb2.  Crk, Nck and Grb2 are SH2/SH3-domain containing adaptor proteins that mediate protein-protein interactions.  Grb2 has a central SH2 domain flanked by two SH3 domains.  Its N-terminal SH3 contains a tyrosine residue (Tyr45) that can be phosphorylated.  Crk has an N-terminal SH2 domain followed by two SH3 domains. Tyrosine phosphorylation of Crk at Tyr221 forms an inhibitory intramolecular interaction with the SH2 domain.  It can also be phosphorylated at Tyr251 by Abl kinase to mediate non-canonical signaling pathways.  Nck consists of three SH3 domains followed by an SH2 domain.   N C SH3 SH3 SH2 Crk Tyr221 Nck SH3 SH3 SH3 SH2 N C N C SH3 SH3 SH2 Grb2 Tyr45 Tyr251 52 Figure 1.7  Domain structure of PTP?.  PTP? has a signal peptide (SP), a short but heavily glycosylated extracellular domain (ECD), a trans-membrane region (TM), two tandem phosphatase domains (D1 and D2).  It can be phosphorylated at two serine residues (Ser180 and Ser204) proximal to the transmembrane region and one tyrosine residue (Tyr789) near the C-terminus. PTP?? ECD D1 N C SP TM D2 N- and/or O-linked glycosylated Tyr789 Ser180 Ser204 DFSDY789ANFK 53 Figure 1.8  Two roles of PTP? in integrin signaling.  (A) The first role of PTP? is to activate Src.  PTP? acts proximal to activated integrins (not shown in diagram) to dephosphorylate the inhibitory phosphate (pTyr527) of Src to activate Src.  The SH2 domain of activated Src (auto-phosphorylated at Tyr416) interacts with autophosphorylated FAK and further phosphorylates FAK (B). The optimally activated Src-FAK complex in turn phosphorylates PTP? at the C-terminal Tyr789 residue which transduces signal downstream to regulate cytoskeletal dynamics and cell motility.  Src SH3 P Tyr-527 SH2 D1 D2 PTP? Src P PTP? P P Src P PTP? P P D1 D2 P A C B Cytoskeletal reorganization and cell migration 54 Figure 1.9  PTP?-Grb2 association. The central SH2 domain of Grb2 associates with Tyr789 phosphorylated PTP? while the C-terminal SH3 domain of Grb2 directly or indirectly interacts with the C-terminal region of the membrane proximal PTP? D1 catalytic domain.  PTP?-Grb2 association is induced upon integrin stimulation and this complex does not contain the well-known Grb2 binding partner, Sos, indicating that the interactions of Grb2 with PTP? and Sos are mutually exclusive.  Cytoplasm D1 D2 P Tyr-789 Extracellular N C SH2 Sos PTP? Grb2 55  56   Materials and Methods Chapter 2:  2.1 Cell lines and cell culture   Wild-type (PTP?+/+) and PTP?-null (PTP?-/-) mouse embryonic fibroblasts (MEFs) have been described previously (Zeng et al. 2003).  FAK-null (FAK-/-) MEFs and FAK-null MEFs re-constituted with GFP-tagged WT-, Y397F- or Y925F were gifts from Dr. David Schlaepfer.  All cells were cultured in Dulbecco?s modified Eagle?s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (HyClone, Thermo Fischer Scientific).  2.2 Cell stimulation and inhibition  2.2.1 Fibronectin (FN) stimulation  Adherent cells (~80% confluent) were starved in DMEM containing 0.5% FBS (Gibco) for 18h and trypsinized with 0.05% trypsin-EDTA (Invitrogen) to detach the cells.  Trypsinization was terminated by the addition of 0.5mg/ml soybean trypsin inhibitor (Invitrogen) in DMEM.  The cells were washed once more with 0.5mg/ml soybean trypsin inhibitor in DMEM, followed by another wash with serum-free DMEM.  They were then resuspended in DMEM containing 0.1% bovine serum albumin (BSA) and maintained in suspension by manually inverting the Falcon tube containing the resuspended cells every 10 min for 1 hour at 37?C (suspension, or 0 min).  Suspended cells (105 cells/ml) were plated onto fibronectin (FN)-coated dishes and incubated at 37?C for 15 min (or other times as specified) before harvesting.  The FN-coated dishes were prepared by incubating 10?g/ml  57 FN (Chemicon International, Inc.) in phosphate-buffered saline (PBS) overnight at 4 ?C.  They were then washed twice with PBS to remove excess FN and incubated with serum-free DMEM at 37?C for 1 h prior to use.  2.2.2 Serum stimulation   Wild type (PTP?+/+) MEFs treated with control or Grb2 siRNA using Lipofectamine RNAiMax were starved in 0.5% FBS DMEM for 18 h.  FBS was added to the adherent cells to a final concentration of 10%.  Cells were harvested at 30 seconds, 1 min, 2 min, and 5 min post addition of FBS.  2.2.3 Shp2 inhibitor treatment   Suspension cells (as described in section 2.2.1) were resuspended in DMEM containing 0.1% BSA and 100?M NSC 87877 (Shp1/Shp2 inhibitor, Santa Cruz) for 1 hour and then plated onto FN-coated dishes in the presence of the inhibitor.  2.3 Antibodies and immunological detection reagents  PTP? and PTP?-phosphoTyr789 antibodies have been described previously (Chen et al. 2006).  Antibodies to FAK (Cat no. 610088), Grb2 (Cat no. 610112), Cas (Cat no. 610272), Crk (Cat no. 610035), paxillin (Cat no. P13520), Arp3 (Cat no. 612135), dynamin (Cat no. D25520) and Shp2 (Cat no. 610621) were from BD Transduction Laboratories.  Anti-VSVG (Cat no. A5977), -actin (Cat no. A5060), and ?Flag (Cat no. F1804) antibodies were from Sigma-Aldrich.  Antibody to Src (v-Src) (Cat no. OP07) was from Calbiochem.  58 Antibody to BCAR3 (Cat no. A301-671A) was from Bethyl Laboratories, Inc. Antibody specific to the SH2 domain of Grb2 (Cat no. MAB38461) was from R&D Systems.  Anti-myc and phosphorylation site-specific antibodies to Myc Tag (9B11) (Cat no. 2276S), Shp2 Tyr542 (Cat no. 3751S), Cas Tyr410 (Cat no. 4011S), FAK Tyr925 (Cat no. 3284S) and Src Tyr416 (Cat no. 2101S) were from Cell Signaling and those to FAK Tyr397 (Cat no. 44624G) and Tyr576/577 (Cat no. 44652G) were from Invitrogen.  Anti-phosphotyrosine (4G10) (Cat no. 05-321), anti-Nck (Cat no. 06-288) and anti-Gab1 (Cat no. 06-579) antibodies were from Upstate Biotechnology.  Alexa Fluor 488-conjugated anti-rabbit IgG (Cat no. A11034), Alexa Fluor 488- (Cat no. A11029) or 594-conjugated anti-mouse IgG (Cat no. A11032), Alexa Fluor? 488 Phalloidin (Cat no. A12379) and Alexa Fluor? 594 Phalloidin (Cat no. A12381) were from Molecular Probes.  Antibodies to Grb2 (polyclonal rabbit) (Cat no. sc-255), N-wasp (D-15) (Cat no. sc-10122), Shc (PG-797) (Cat no. sc-967) and Fish (Cat no. sc-30122) were from Santa Cruz Biotechnology, Inc. and the Clean Blot IP detection reagent (HRP) (Cat no. 21230) from Thermo Scientific was specifically used for the detection of Grb2 in VSVG immunoprecipitates.   2.4 Grb2 expression constructs  The plasmid pSVE-Grb2-myc-P49L (Addgene, Cambridge, MA) containing full-length N-terminal SH3 mutant Grb2 with a C-terminal myc tag was used as the template in a PCR (primers F1, 5?-ATCATGGATCCGTCATGGAGGCGATTGCGAAATATG-3?, and R1, 5?-AGCGTAAGCTTTCACAAGTCTTCTTCAGAAATAAGCTTTTGTTCG-3?) to generate Grb2-myc-P49L with a 5? BamHI site and a 3? HindIII site.  The PCR product was cloned into pcDNA3.1(-) vector that has been digested with BamHI and HindIII to generate  59 the expression plasmid pcDNA3.1(-)-Grb2-myc-P49L.  The underlined bases in F1 were silent mutations that were created to generate a Grb2-siRNA resistant plasmid.   Sequences prior to the start codon (double underlined) also did not match with the Grb2 siRNA used.  Site-directed mutagenesis using overlap extension PCR of the pSVE-Grb2-myc-P49L template was performed to revert back the N-terminal SH3 mutant to WT-Grb2-myc (changing the mutated Lys to Pro).  Separate PCRs were performed to produce two pieces of the complete Grb2-myc that overlapped in sequence around the mutated region, using the primer pairs F1 (described above) with R2 (5?-TATGTAGTTCTGGGAATGAAGCCGTC-3?, mutated bases underlined) and F2 (5?-GACGGCTTCATTCCCAAGAACTACATA-3?, mutated bases underlined) and R1 (described above).  The resulting PCR products were gel purified and combined to use as templates in another PCR reaction using primers F1 and R1 to generate WT-Grb2-myc.  This was then cloned into BamHI- and HindIII-digested pcDNA3.1(-) to produce the expression plasmid pcDNA3.1(-)-WT-Grb2-myc.  The same procedure was used to generate the plasmids pcDNA3.1(-)-Grb2-myc-P206L (C-terminal SH3 mutant), and pcDNA3.1(-)-Grb2-myc-R86K (SH2 mutant) except that pcDNA3.1(-)-WT-Grb2-myc was used as the template in first-round PCR.  The primer pairs used for first-round PCRs were F1 and R3 (5?-GACATAATTGCGCAGAAACATGCCGGT-3?, mutated Pro ? Leu underlined) or R4 (5?-GCTCTCACTCTCTTTGATCAGGAAGGC-3?, mutated Arg ? Lys underlined) and F3 (5?-ACCGGCATGTTTCTGCGCAATTATGTC-3?, mutated Pro ? Leu underlined) or F4 (5?-GCCTTCCTGATCAAAGAGAGTGAGAGC-3?, mutated Arg ? Lys underlined) and R1, followed by second-round PCR on the mixed templates using primer pairs F1 and R1.  For pcDNA3.1(-)-Grb2-myc-P49L/P206L (N- and C-terminal  60 SH3 mutant), pcDNA3.1(-)-Grb2-myc-P49L was used as a template in first-round PCR with primer pairs F1 and R3 and F3 and R1, followed by second-round PCR as described above.  2.5 PTP?, paxillin and Grb2 expression and Grb2/Shp2 depletion  The pXJ41-neo-VSVG-PTP? plasmid (WT or Y789F), Grb2-expressing plasmids and pcDNA3.1-mCherry-paxillin plasmid (a gift from Dr. James Lim) were transfected into cells using Lipofectamine LTX reagent (Invitrogen).  Transfected cells were grown for 24h in antibiotic-free medium prior to further experimentation.  Control nontargeting and Grb2 targeting small interfering RNA (siRNA) were from Integrated DNA Technologies, Inc.  Sequences for the two Grb2 siRNA duplexes used are as follow:  duplex 1, 5'-GGUGUUC AUAAAUGGUAUUUGUACC-3' and 5'-GGUACAAAUACCAUUUAUGAACACCCU- 3' and duplex 2, 5'-CGCUCAGAAUGGAAGCCAUCGCCAA-3' and 5'-UUGGCGAUGG CUUCCAUUCUGAGCGCC-3'.  Shp2 targeting siRNA (ON-TARGET plus SMART pool, Mouse PTPN11) was from Dharmacon.  Cells were transfected with 20 nM siRNA using Lipofectamine RNAiMax reagent (Invitrogen) and grown for 48h before further experimentation.   2.6 Cell lysis, immunoprecipitation, and immunoblot analysis  Cells were harvested in RIPA (50 mM Tris [pH7.6], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 1mM phenylmethylsulfonyl fluoride (PMSF), 10 ug/ml leupeptin, and 10 ug/ml aprotinin) or 1% NP-40 lysis buffer (50 mM Tris [pH7.6], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 2  61 mM Na3VO4, 1mM phenylmethylsulfonyl fluoride (PMSF), 10 ug/ml leupeptin, and 10 ug/ml aprotinin).  RIPA buffer was used for immunoblotting while 1% NP-40 buffer was used for immuoprecipitation experiments as described previously (Zeng et al. 2003).  2.7 Immunofluorescence and TIRF microscopy  Cells were transiently co-transfected with pEGFP-Cas (a gift from Dr. Greg Longmore) and mCherry-Vinculin (a gift from Dr. James Lim), or in other experiments with mCherry-BCAR3 (Sun et al. 2012), using LipofectamineTM LTX (Invitrogen). The cells were placed on FN-coated glass coverslips for 15 min, washed twice with cold PBS and fixed for 30 min in 4% paraformaldehyde in PBS. Fixed cells were washed twice with PBS and imaged using the Olympus IX81 Cell^TIRF (total internal reflection fluorescent) system equipped with a CoolSnap HQ2 CCD camera. The corresponding fluorescent labelled proteins were visualized with a 60x TIRF objective lens (NA 1.49) using the 488nm and 561nm lasers set to achieve a calibrated penetration depth of 75nm and 80 nm, respectively. Images were analyzed using the software ImageJ and co-localization of Cas and vinculin was measured using Pearson?s correlation. In other experiments, cells transfected with control or Grb2 siRNA using Lipofectamine RNAiMax (Invitrogen) were stimulated, washed and fixed as described above.  Fixed cells were washed twice with PBS and permeabilized with 0.02% Triton X-100 in PBS for 15 min.  They were then washed twice with PBS and blocked with 3% BSA for 1 hour.  After blocking, the cells were incubated with anti-Src Tyr416 (1:100) or anti-FAK Tyr397 (1:250) and anti?vinculin (1:250) antibodies for 2 h at room temperature.  This was followed by incubation with Alexa Fluor 488-conjugated anti-rabbit IgG (1:250) and/or  62 Alexa Fluor 594-conjugated anti-mouse IgG (1:250 dilution). Cells were imaged as described above with the 488-nm and 561-nm lasers set to achieve a calibrated penetration depth of 85 nm for each laser.  Images were processed using the software ImageJ.  2.8 Kinase assay  Cell lysates (1 mg) were pre-cleared with 50 ?l of protein A/G plus agarose beads (Santa Cruz Biotechnology, Inc) at 4?C for 1 hour.  Pre-cleared lysates were incubated with anti-Src antibody for 1 hour and then with 50 ?l of protein A/G beads for 20 min.  Beads were washed 4 times for 5 min each with PBS and kinase reaction solution (Genway Universal Tyrosine Kinase Assay Kit) containing 1:1440 ?-mercaptoethanol was added to the beads.  Serial dilutions of these samples were prepared and transferred into a microplate in duplicates.  The samples were incubated with 40 mM ATP-2Na for 30 min and blocked for 30 min at 37?C.  They were then incubated with anti-phosphotyrosine (pY20)-HRP for 30 min at 37?C.  An HRP substrate, 3, 3', 5, 5'-tetramethylbenzidine (TMBZ), was added to the samples.  After 25 min, the absorbance of the samples was measured at 450 nm.  2.9 PTP? peptide affinity chromatography  2.9.1 Sample preparation   FN-stimulated (30 min) wild type (PTP?+/+) or PTP?-null MEFs were lysed in modified RIPA lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM Na3VO4, 1 mM NaF, 1 mM PMSF, 10 ?g/ml aprotinin, and 10 ?g/ml leupeptin). The sample was incubated on ice for 20 min to promote complete lysis and then  63 centrifuged at 13,000 rpm for 20 min to remove cell debris and collect the supernatant. After measuring the protein concentration, the supernatant was diluted to 1 mg/ml with detergent-free lysis buffer to bring the final Triton X-100 concentration of the lysate to 0.5%.   2.9.2 Affinity chromatography   Two affinity matrices were generated by 21st Century Biochemicals (Marlboro, MA, USA), comprising the PTP? peptide CYIDAFSDY789ANFK or the phosphotyrosyl 789 version of this peptide coupled to thiol-reactive beads. Approximately 1 ml bed volume of each peptide-coupled bead was transferred to a 15 ml tube and washed twice with modified RIPA lysis buffer containing 0.5% Triton X-100.  Then, 10 ml of the diluted protein lysate (equivalent to 10 mg of protein) was added to each tube and incubated overnight at 4oC on a rotator. Two chromatography columns (Econo-column) (Bio-Rad Laboratories) were equilibrated by washing with 15 ml of detergent-free lysis buffer.  Each slurry (beads and lysates) was transferred to a column and the beads were allowed to settle for 30 min before the flow-through was collected. The columns were washed with 10 ml of lysis buffer containing 0.05% Triton X-100, and then with 4 ml of detergent-free lysis buffer. Bound protein was eluted with 5 ml 0.1M glycine-HCl pH2.5. The first 4.5 ml of eluate was collected in a 15 mL tube containing 293.2 ?l of 1M Tris pH9.5 for neutralization. The eluate was concentrated to a final volume of ~270 ?l using an Amicon Ultra-15 Centrifugal Filter Device with a nominal 10 kD molecular mass limit (Millipore, Billerica, MA, USA).  Samples (20 ?l of the starting cell lysates, 30 ?l of the column flow-throughs, and 30 ?l of the column eluates) were diluted with an equal volume of 2X SDS sample buffer and boiled for 5 min. The samples were then loaded on a 10% SDS-PAGE. After resolution, the  64 proteins were transferred from the gel to a PVDF membrane at 110V for 80 min.  The membrane was subsequently immunoblotted for various proteins of interests.  2.10 Wound healing assay  Cells (5x106 cells/ml) were seeded into the microfluidic channels of the BioFlux 24-well plate (Fluxion Biosciences, Inc.) by shear flow.  The channels were pre-coated with 100?g/ml FN in PBS.  Cells were allowed to attach and form a monolayer in the channel overnight.  Then, half of the channel was trypsinized using 0.25% trypsin-EDTA under a flow rate of 20 dyn/cm2.  Trypsinization was terminated by the addition of DMEM containing 10% FBS.  Under sheer flow, an image of the ?wounded? edge was taken every 15 min for 24 hour using an Olympus IX81 microscope equipped with a CoolSnap HQ2 charge-coupled-device (CCD) camera.  A 20X objective lens was used to visualize cell movement in the wounded area.  Images were analyzed using the software Metamorph and ImageJ.  2.11 Focal adhesion enrichment assay   The protocol for the focal adhesion enrichment assay was as described (Kaplan KB et al. 1994; Sakai et al. 1994). Cells were placed on FN-coated dishes for 15 min as described (section 2.2.1), and the dishes were chilled on ice immediately. Cells were washed twice with ice-cold PBS and lysed with cytoskeleton stabilizing buffer (CSB) (0.3 M sucrose, 0.5% Triton X-100, 10 mM Pipes pH6.8, 100 mM KCl, 1 mM CaCl2, 2.5 mM MgCl2, 1 mM Na3VO4, 1mM PMSF, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin) for 5 min, and collected into tubes. This fraction is referred to as the detergent-soluble or soluble fraction (S). The dishes  65 were rinsed briefly (<5s) twice with ice-cold PBS.  After draining off excess PBS, solubilisation buffer (50 mM Hepes pH 7.4, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1mM PMSF, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin) was added to the dishes and kept on ice for 5 min. The dishes were scraped and the resulting solution was centrifuged at 16,000?g at 4?C for 15 min to clear cell debris. The supernatant is referred to as the insoluble or focal adhesion-enriched fraction (I).  Both the soluble and insoluble fractions were subjected to Western blotting and densitometric quantification.  The % of insoluble protein representing focal adhesion-localized protein was calculated as the amount of insoluble protein over the total amount of insoluble and soluble proteins multiplied by 100.  2.12 Statistical analyses  Densitometric quantification of immunoblots from at least three independent experiments was performed using Quantity One program (Bio-Rad Laboratories) and statistically analyzed using the Student?s t-test.   66  Identification of BCAR3 as a PTP?-phosphoTyr789 Binding Chapter 3:Partner that Mediates Integrin-Induced Cell Migration   3.1 Rationale   PTP? plays an upstream role in integrin signaling to disrupt Src auto-inhibition by dephosphorylating Src Tyr527, thereby activating Src (Ponniah et al. 1999; Su et al. 1999; Zeng et al. 2003).  Activated Src, together with FAK, in turn phosphorylates PTP? at its C-terminal Tyr789 residue (Chen et al. 2006).  In NIH 3T3 mouse fibroblasts, approximately 20% of cellular PTP? is constitutively phosphorylated on Tyr789 (den Hertog et al. 1994).  Our laboratory has previously found that disengagement of integrins from the ECM results in PTP? dephosphorylation and this can be restored by re-engaging integrins with their ECM ligands.  Like PTP?-null cells, PTP?-null cells expressing an unphosphorylatable mutant (Y789F) PTP? exhibit delayed cell spreading and migration (Chen et al. 2006).  However, these cells differ from PTP?-deficient cells in that they have normal Src and FAK activity, indicating that the Tyr789 residue, and likely its phosphorylation, plays a downstream role in integrin signaling to regulate cytoskeletal reorganization and cell motility independent of PTP?-mediated Src activation.  This suggests that PTP?-phosphoTyr789 acts as a potential docking site for SH2- or PTB-domain-containing proteins to mediate downstream signaling events that ultimately regulate cell migration.  So far, only the SH2 domain of Grb2 has been identified to directly interact with phosphoPTP? but the function of this association is unclear (den Hertog et al. 1994; Su et al. 1994; den Hertog and Hunter 1996; Su et al. 1996).  I hypothesized that either Grb2 or other unidentified phosphoPTP?-interacting partners may  67 be transmitting a signal from phosphorylated PTP? to the actin cytoskeleton to modulate cell motility.  The investigation of the role of PTP?-associated Grb2 will be discussed more extensively in the next two chapters.    In this study, the objective was to identify novel direct or indirect binding partner(s) of PTP?-phosphoTyr789 and to elucidate the molecular events that link PTP?-phosphoTyr789 to cell migration.  Two approaches were used to address this:  1) affinity column chromatography and 2) identification of downstream signaling events that are dependent on PTP??Tyr789 using adenovirus expressing PTP?-Y789F.  3.2 Detection of PTP?-phosphoTyr789 binding partners using phospho-peptide affinity chromatography   To identify novel PTP?-phosphoTyr789 binding partners, an affinity purification procedure involving a synthetic phosphotyrosine peptide corresponding to the PTP? sequence flanking Tyr789 was used (Fig. 3.1).  This phosphoTyr789-peptide and a peptide of identical sequence but in an unphosphorylated form were coupled to a solid matrix for use in affinity chromatography.  Lysate from fibronectin (FN)-stimulated (to engage integrins) PTP?-null fibroblasts was passed over these columns.  PTP?-deficient cells were used to prevent endogenous PTP? from competing with the phosphopeptide for binding to potential phosphoPTP?-associated proteins.  The eluted bound proteins were separated by SDS-PAGE and probed with antibodies against various proteins that are involved in integrin signaling.  The candidate binding proteins were selected based on one or more of the following criteria:  1) Involved in cytoskeletal reorganization, 2) contain an SH2-domain, or 3) can interact with the SH3 domain of Grb2.  Detected proteins that were bound specifically to the  68 phosphoTyr789-peptide column but not to the unphosphorylated form of the same peptide were then subjected to immunoprecipitation or PTP? co-immunoprecipitation to verify the interactions in vivo.  3.2.1 Identification of PTP??phosphopeptide-associated proteins   Fig. 3.2A shows that Arp2/3, N-Wasp, Gab1, Dynamin and Fish were all present in the flow through of the phospho- and non-phospho-peptide column but none of them were detected in the eluate, suggesting that none of these proteins are bound to the PTP? phospho- or unphosphorylated tail region.  Interestingly, when the same protein fractions were probed with various SH2-domain containing proteins (Grb2, Nck, Crk and Shc), which have been implicated in integrin-mediated signaling, these adaptor proteins were all detected in the eluate from the phospho-peptide column but not from the non-phospho-peptide column (Fig. 3.2B), indicating their ability to bind to phosphoPTP?.  Among these four adaptors, Nck appeared to be the least effective in binding to the phospho-peptide since most of it remained in the unbound fractions of the phospho-peptide.  On the other hand, Grb2, which was used as a positive control, and Shc were undetected in the unbound fractions, suggesting that they were saturated by the phospho-peptide.  As mentioned earlier, the reason for using PTP?-null fibroblast lysate was to eliminate competitive binding of phosphoPTP?-interacting proteins with endogenous PTP?, however, it is also conceivable that in PTP?-null fibroblasts, there might be alterations in protein-protein interactions or conformation that affect the availability or affinity of the PTP?-interacting proteins.  Therefore, lysates of wild type MEFs were also incubated with  69 the phospho- and non-phospho-peptide columns.  Overall, the results were similar between wild type and PTP?-null fibroblasts.  None of the cytoskeletal or Grb2-interacting proteins was bound to the phosphoPTP?-peptide except dynamin yet it was also detected in the non-phospho-eluate (Fig. 3.3A).  This suggests that dynamin is capable of interacting with the C-terminal sequence of PTP? but it is not dependent on PTP? Tyr789 phosphorylation, or that this may represent a non-specific interaction.  The SH2-domain containing adaptors examined were all capable of binding to the phospho-peptide with Nck being the least effective as shown in Fig. 3.3B.  Unlike the previous experiment with PTP?-null cells where most of Crk and Shc were bound to the phospho-peptide, these experiments with wild type cells showed that the amount of Crk and Shc in the bound versus unbound fractions from the phospho-column were comparable.  This indicates that the presence of PTP? affects Crk and Shc binding to the phosphoPTP?-peptide.  Whether this is due to a competitive effect or other molecular interventions are unclear.  The latter seems more probable since the binding of Grb2 to the phospho-peptide was unaffected by the presence of PTP?.  Altogether, these experiments revealed three potential phosphoPTP?-interacting proteins: Nck, Crk and Shc.  To verify that these proteins interact with integrin-mediated phosphoPTP? in vivo, WT- and Y789F-PTP? were expressed in PTP?-null cells and immunoprecipitated with anti-PTP? antibody.  The presence of these proteins in the PTP? immunoprecipitates was then examined.      70 3.3 In vivo validation of PTP?-phosphoTyr789 candidate binding partners   In Fig. 3.4A and 3.4B (upper panels), PTP? tyrosine phosphorylation is concomitant with Grb2 association in WT-PTP? expressing cells but is not detected in immunoprecipitates of unphosphorylatable Y789F-PTP?.  However, neither Shc nor Crk were detected in immunoprecipitates of WT- or mutant PTP?? although they are present in the cell lysates (Fig. 3.4A and 3.4B, lower panels).  The detection of Nck in the immunoprecipitates was difficult as the heavy chain of the PTP? antibody used for immunoprecipitation interfered with the detection of this 47 kDa protein (indicated by the arrow in Fig. 3.4B).   Thus, a reciprocal experiment was performed where Nck was immunoprecipitated and probed for the presence of PTP?, but PTP? was not detected in the Nck immunoprecipitates (Fig. 3.4C).  These results showed that only Grb2 was able to complex with phosphoPTP?.  All the other SH2-domain containing adaptors were able to interact with the phosphoPTP?-peptide but not with endogenous integrin-stimulated phosphoPTP?.  This indicates that the use of a PTP? phospho-peptide, which likely differs from the tertiary structure of the whole PTP? protein, might not be the most effective method to screen for potential phosphoPTP?-interacting proteins since it may be prone to yield false positives.  3.4 PTP? Tyr789 is required for integrin-induced Cas tyrosine phosphorylation   In a collaborative effort with my colleague, Dr. Guobin Sun, we carried out experiments to identify defective integrin signaling events in Y789F-PTP? expressing cells (i.e. dependent on PTP??Tyr789), with the rationale that such defects could be traced to  71 reveal potential phosphoPTP?-interacting candidates.  Integrin-induced activation of the Src-FAK kinase complex results in tyrosine phosphorylation of its downstream substrates, paxillin and Cas (Bellis et al. 1995; Vuori et al. 1996; Hanks and Polte 1997; Tachibana et al. 1997).  Since paxillin phosphorylation is independent of PTP? Tyr789 (Chen et al. 2006), we examined Cas tyrosine phosphorylation in Y789F-PTP? expressing cells.  Cas is a major scaffolding protein that plays a key role in integrin-mediated cell migration (Cary et al. 1998; Honda et al. 1999).  Tyrosine phosphorylation of the Cas substrate domain (SD) leads to the recruitment of Crk to transduce downstream signals that regulate cell membrane protrusion and motility (Klemke et al. 1998).  In PTP?-null cells, cell migration is delayed concomitant with impaired Cas phosphorylation (Su et al. 1999).  To determine whether Cas tyrosine phosphorylation is dependent on PTP??Tyr789, WT- and Y789F-PTP? were expressed in PTP?-null cells and integrin signaling was stimulated by plating the cells on fibronectin-coated culture dishes.  Fig. 3.5A and 3.5B show that Cas tyrosine phosphorylation is reduced by ~60% (? 3.4%) in PTP?-null cells, consistent with the report published by Su et al. (Su et al. 1999).  Re-expression of WT-PTP? but not the unphosphorylatable mutant Y789F could restore ~95% (? 3.3%) of Cas tyrosine phosphorylation, suggesting that PTP??Tyr789 phosphorylation promotes Cas tyrosine phosphorylation.  To confirm that PTP??Tyr789 enhances Cas SD tyrosine phosphorylation, we examined the tyrosine phosphorylation of Cas Tyr410, which represents one of the major tyrosine residues residing in the SD of Cas.   As shown in Fig. 3.5C, integrin-induced Cas Tyr410 phosphorylation was rescued by WT-PTP? but not Y789F-PTP?, indicating that PTP??Tyr789 is necessary for optimal Cas SD phosphorylation.     72 3.5 Cas localization to focal adhesions is defective in PTP?-Y789F expressing cells   Src is the kinase that is responsible for the phosphorylation of the SD of Cas (Ruest et al. 2001).  Since Src is properly activated in PTP?-Y789F expressing cells, this does not account for the reduction in Cas phosphorylation in these cells.  However, in addition to Src activity, its direct association with the Src-binding domain (SBD) of Cas or indirect association with Cas through FAK may also affect Src-mediated Cas phosphorylation (Ruest et al. 2001; Fonseca et al. 2004).  Immunoprecipitation of Cas from FN-stimulated wild type MEFs, PTP?-null MEFs and PTP?-null MEFs re-expressing WT- or Y789F-PTP? revealed that the association between Src and Cas is disrupted in PTP?-null cells and this could be restored by exogenous WT- but not Y789F-PTP? (Fig 3.6), indicating that PTP??Tyr789 is required for Src-Cas complex formation.  To determine whether the failure of Cas to associate with Src corresponds to the improper or impaired localization of Cas to focal adhesions in Y789F-PTP? expressing cells, fluorescent microscopy was used to visualize the subcellular distribution of Cas.  GFP-labeled Cas and mCherry-labeled vinculin were co-transfected into wild type and PTP?-deficient cells, and into PTP?-deficient cells reconstituted with WT- or Y789F-PTP????Vinculin was used as a focal adhesion marker.  As shown in Fig. 3.7, fewer Cas-containing focal adhesions are observed in PTP?-null and PTP?-Y789F expressing cells concomitant with decreased Cas co-localization with vinculin as indicated by the merged signal (yellow) at the protrusive edges.  Quantitative analysis of the co-localization of Cas and vinculin was performed using Pearson?s correlation, where a coefficient of 0 indicates no correlation and +1 represents complete co-localization.  A total of 20 randomly chosen fields,  73 all at the spreading edge, from 10 different cells in each cell group were analyzed.  Wild type and PTP?-null MEFs expressing WT-PTP? had higher Pearson?s values (0.83 ? 0.07 and 0.79 ? 0.08, respectively) compared to PTP?-deficient or Y789F-PTP? expressing cells (~0.62 ? 0.12 and ~0.48 ? 0.10, respectively) (Fig. 3.8).  This suggests that PTP??Tyr789 mediates Cas focal adhesion localization.  3.6 BCAR3 SH2 and GEF domains physically link integrin-induced phosphoPTP? to Cas   PTP?-Y789F expressing cells exhibit defects in multiple integrin-mediated signaling events including Cas association with Src, tyrosine phosphorylation and membrane targeting of Cas, Rac activation (Sun et al. 2012), cell spreading and cell migration (Chen et al. 2006).  Interestingly, all of these defects are also reported for BCAR3 (Breast Cancer Antiestrogen Resistance-3)-depleted cells (Schrecengost et al. 2007; Schuh et al. 2010; Wilson et al. 2013).    Since BCAR3 can bind to the C-terminus of Cas through its GEF-like domain and possesses an SH2 domain in its N-terminal region (Gotoh et al. 2000), we postulated that BCAR3 acts as an intermediate between PTP? and Cas to mediate Cas focal adhesion localization.  To test this notion, the expression of BCAR3 in MEFs was first confirmed by Western blot and its association with Cas and PTP? was then examined by co-immunoprecipitation.  In PTP?-null cells transfected with VSVG-tagged PTP?, Cas and Src were detected in the VSVG immunoprecipitates in control but not BCAR3 siRNA-treated cells (Fig. 3.9A), suggesting that the presence of BCAR3 is important for PTP? to complex with Cas and Src.  Furthermore, Cas and Src were not detected in the VSVG  74 immunoprecipitates of VSVG-tagged PTP?-Y789F expressing cells (Fig. 3.9B), indicating that PTP? Tyr789 is required for the binding of BCAR3, Cas and Src to PTP?.    To confirm that the SH2 and GEF domains of BCAR3 are mediating its interaction with phosphoPTP? and Cas, respectively, FLAG-tagged WT- or R177K (SH2 domain mutant)- or R748A (GEF domain mutant)-BCAR3 and VSVG-tagged PTP? were co-expressed in PTP?-null cells.  VSVG-PTP? and FLAG-BCAR3 were immunoprecipitated from these cell lysates separately and probed for Cas, Src, VSVG-PTP? and FLAG-BCAR3.  As predicted, BCAR3, Cas and Src all failed to associate with PTP? in the SH2 mutant BCAR3 expressings cells (Fig. 3.9C, upper panel) while BCAR3 and Cas association was intact (Fig. 3.9C, middle panel).  On the other hand, the BCAR3 GEF-mutant failed to interact with Cas and Src (Fig. 3.9C, middle panel) but exhibited partial interaction with PTP? (Fig. 3.9, top and middle panels).  These findings strongly suggest that BCAR3 is acting as a physical bridge between PTP? and Cas-Src where its SH2 domain is interacting with phosphoPTP? and its GEF domain is binding to the Cas-Src complex.  3.7 BCAR3 localization to focal adhesions is dependent on the BCAR3 SH2 domain and PTP? Tyr789   Next, to test whether the SH2 domain of BCAR3 is required to induce integrin-mediated BCAR3 translocation to the cell membrane, mCherry-labeled WT- or R177K-BCAR3 was expressed in wild type MEFs and the cells were plated on FN-coated coverslips for 15 min.  Cells were fixed and immunostained for vinculin.  Using TIRF microscopy, the subcellular localization of BCAR3 (red) and vinculin (green) were visualized.  WT-BCAR3 was present in vinculin-containing focal adhesions as shown by the overlapping signals in  75 the merged image (yellow) in Fig. 3.10 (upper panel).  Conversely, the R177K-BCAR3 mutant was not localized to the periphery of the spreading cells and did not co-localize with vinculin (Fig. 3.10, bottom panel), suggesting that the SH2 domain of BCAR3 is required for its focal adhesion localization.  To verify that Tyr789 of PTP? is mediating the recruitment of BCAR3 to focal adhesions, PTP?-null cells infected with adenovirus expressing WT- or Y789F-PTP? were transfected with mCherry-WT-BCAR3, and subjected to FN-stimulation and immunostaining as described above.  BCAR3 co-localized with vinculin in the presence of WT-PTP? but not Y789F-PTP? (Fig. 3.11).  These findings demonstrate that integrin-induced BCAR3 translocation to the protruding cell edge and to focal adhesions is dependent on its SH2 domain, as well as on an intact Tyr789 residue of PTP?, substantiating the notion that BCAR3 interacts with phosphoPTP? via its SH2 domain to recruit Cas to focal adhesions.    3.8 Discussion   Integrin-induced PTP??Tyr789 phosphorylation regulates cell migration (Chen et al. 2006) yet the underlying molecular mechanisms remain unclear.  I postulated that phosphorylated Tyr789 serves as a docking site to mediate protein-protein interactions to transduce downstream signals.  Using affinity column chromatography, I have identified three adaptor proteins, Nck, Shc and Crk, that can bind to the synthetic phosphotyrosine peptide corresponding to the C-terminal region of PTP? flanking Tyr789.  However, none of these molecules interacted with integrin-induced PTP?-phosphoTyr789 in vivo.  This is not surprising since the SH2- and PTB-domain binding motifs of Nck, Crk and Shc respectively  76 recognize pYXXP, pYXXI/L or NPXpY (Songyang et al. 1993; Songyang et al. 1994; Prigent et al. 1995), none of which correspond to the amino acid sequence flanking PTP??Tyr789 (AFSDpY789ANFK).  These findings reveal that the interactions between the phosphoPTP? peptide and Nck, Crk or Shc were either non-specific or indirect, thus these interactions were not detected in co-immunoprecipitations due to their non-existence or loss of associations during the immunoprecipitation process.    Aside from phospho-peptide affinity chromatography, my colleague and I were able to identify a novel PTP?-phosphoTyr789 interacting partner, BCAR3, which directly links PTP? to Cas to promote cell migration.  By re-introducing WT-PTP? or mutant Y789F-PTP? into PTP?-null MEFs, we pinpointed the tyrosine phosphorylation of Cas as an early integrin-mediated event that is dependent on an intact PTP??Tyr789 residue.  Cas is primarily phosphorylated by integrin-stimulated Src kinase and this is dependent on its localization to newly forming focal contacts (Vuori et al. 1996; Fonseca et al. 2004).  In PTP?-Y789F expressing cells, Src activity and its movement into focal adhesion-enriched fractions upon integrin engagement are unaltered while Cas movement into the focal adhesion-enriched fractions (Sun et al. 2012) and co-localization with vinculin-containing adhesions are diminished, indicating that impaired Cas focal adhesion targeting rather than defective Src activity is the major cause of reduced Cas phosphorylation.   Furthermore, our lab and others (Lammers et al. 2000; Sun et al. 2012) have shown that Y789F-PTP? fails to properly translocate to focal adhesions.  These results indicate that PTP?-phosphoTyr789 is important for the subcellular localization of PTP?, as well as Cas, to integrin-containing focal adhesions where Cas can interact with and be effectively phosphorylated by Src.   77  Since Cas does not possess any SH2- or PTB-domain that can directly interact with PTP?-phosphoTyr789, it is likely being recruited to focal adhesions by phosphoPTP? via one or more intermediary protein(s).  BCAR3, first identified in a screen for breast cancer anti-estrogen resistance genes, belongs to the novel SH2 domain-containing protein (NSP) family (van Agthoven et al. 1998).  It has an N-terminal SH2 domain and a C-terminal GEF-like domain.  The SH2 domain has no known ligand while the GEF domain associates with the C-terminal homology (CCH) region of Cas (Gotoh et al. 2000).  BCAR3 localizes to membrane ruffles and lamellipodia in REFs ectopically expressing BCAR3 (Riggins et al. 2003).  In breast cancer cells, the depletion of BCAR3 results in impaired cell migration and invasion, reduced adhesion-mediated Cas tyrosine phosphorylation and Cas-Src association, as well as the loss of Cas at the plasma membrane (Schrecengost et al. 2007; Schuh et al. 2010).  On the other hand, the overexpression of BCAR3 results in increased membrane ruffling, loss of F-actin stress fibres, enhanced cell migration and co-localization with Cas at the cell membrane (Cai et al. 2003; Schrecengost et al. 2007).  The parallel effects of BCAR3 depletion and expression of mutant Y789F-PTP? in the regulation of Cas and Cas-associated signaling indicated that PTP? and BCAR3 might function in the same signaling cascade.  Furthermore, the structural features of BCAR3 make it an appealing candidate as the molecular linker between phosphoPTP? and Cas.  In wild type MEFs, we found that PTP? forms a complex with BCAR3, Cas and Src.  The formation of this complex is dependent on the presence of BCAR3, as the association between PTP? and Cas or Src is disrupted in BCAR3-depleted cells, supporting our hypothesis that BCAR3 mediates PTP? and Cas-Src complex formation.  Furthermore, we showed that PTP??Tyr789 and the SH2 domain of BCAR3 are required for PTP? to complex with Cas-Src.  These findings  78 demonstrate that BCAR3 interacts with phosphoPTP? to facilitate Cas recruitment to focal adhesions.   The N-terminal SH3 and CCH domains of Cas are responsible for its focal adhesion targeting and both are required to achieve optimal focal adhesion recruitment and SD tyrosine phosphorylation of Cas (Donato et al. 2010).  FAK has been shown to interact with the N-terminal SH3 of Cas to recruit it to focal adhesions and promote its tyrosine phosphorylation through FAK-bound Src.  The CCH domain of Cas resembles the helical structure of the FAK FAT domain (Arold et al. 2002) but is ineffective in localizing Cas to focal adhesions on its own (Donato et al. 2010).  Indeed, the molecular basis for CCH domain-mediated Cas focal adhesion targeting was undetermined.  Two proteins, BCAR3 and Ajuba, have been shown to interact with the CCH domain of Cas and regulate Cas localization to focal adhesions (Pratt et al. 2005; Schrecengost et al. 2007).  BCAR3-depleted cells and Ajuba-null cells both display decreased levels of Cas at membrane ruffles and lamellipodia, concomitant with reduced cell migration. We have demonstrated that BCAR3 localizes Cas to focal adhesions by interacting with PTP?-phosphoTyr789.  Ajuba might also play a role in PTP??Tyr789-mediated cell migration but unlike BCAR3, Ajuba does not have any SH2- or PTB-domain.  Instead, it can interact with the SH3 domain of Grb2 (Goyal et al. 1999), suggesting that it could link PTP? to Cas through a PTP?-Grb2-Ajuba-Cas complex.  Further investigations will be required to test this model.    Our current results support a model where integrin activation induces Src-mediated PTP? phosphorylation at Tyr789, creating an SH2 binding site for the BCAR3-Cas complex at nascent adhesion sites.  This complex could account for the known C-terminal-dependent focal adhesion localization of Cas.  The phosphoPTP?-mediated recruitment of the BCAR3- 79 Cas complex to focal adhesions brings Cas into proximity with Src, thus promoting Src-mediated tyrosine phosphorylation of Cas.  While the C-terminal region of Cas interacts with PTP?-bound BCAR3, the N-terminal SH3 domain of Cas may interact with focal adhesion-localized FAK.  In this way, dual Cas anchorage points could mediate opposing mechanical forces that lead to physical stretching of the Cas molecule to optimally expose the central SD domain and enhance Src-mediated SD tyrosine phosphorylation (Sawada et al. 2006).  Since PTP? also regulates Src-mediated FAK phosphorylation and activation (Zeng et al. 2003), our model suggests that PTP? functions as a master co-ordinator to precisely control the spatial two-pronged anchoring of Cas in focal adhesions.  In this study, we have discovered a novel molecular complex of PTP?-BCAR3-Cas-Src that is involved in PTP?-phosphoTyr789-mediated cell migration.  Given that Cas is known to promote metastasis in various cancer cells (Tikhmyanova et al. 2010) and that both Cas (also known as BCAR1) and BCAR3 are often highly expressed in breast cancer cells (van der Flier et al. 2000; Schrecengost et al. 2007), the likelihood of the PTP?-BCAR3-Cas module functioning in Cas-mediated cell spreading and invasion within a malignant context is very high and will be of great interest for further investigation and potential molecular targeting against cancer.          Collect flow throughs (FTs) Cell lysates Add wash buffer and collect washes Add elution buffer and collect eluates Figure 3.1  Schematic diagram of affinity column chromatography with PTP? non-phosphopeptide and phosphopeptide.  Fibronectin- stimulated wild type or PTP?-null (?-/-) cell lysates were incubated either with a non-phospho- (blue) or phospho-PTP?? (yellow) peptide affinity column. The sequences of the peptides are shown.  The flow through (FT), containing the unbound proteins, was collected from each column.  Then the columns were washed and the bound proteins (eluate) were eluted.  Protein contents of the FTs and eluates from each column were analyzed using SDS-PAGE and Western blot. CYIDAFSDY789ANFK CYIDAFSDpY789ANFK SDS-PAGE & Western blot 80 A B Figure 3.2  SH2-domain containing proteins in PTP?-/- MEFs are able to bind to PTP?-phosphopeptide. PTP?-null (?-/-) MEFs were serum starved overnight.  The next day, cells were trypsinized, kept in suspension for 1 h and replated on FN-coated dishes for 30 min.  Cell lysates were incubated either with a non-phospho- (pep) or phospho-PTP??(pY-pep) peptide affinity column. The flow through (FT), containing the unbound proteins, was collected from each column.  Then the columns were washed and the bound proteins (eluate) were eluted.  (A) The whole cell lysate, FT and eluate from each column were probed for Arp3, N-Wasp, Gab1, dynamin and Fish. (B) Same as (A) except the samples were probed for the SH2-domain containing adaptor proteins, Grb2, Nck, Crk and Shc. All blots are representative of two independent experiments. Arp Dynamin Fish Gab1 N-Wasp Cytoskeletal Grb2-binding ?-/-   pep        pY-pep         pep        pY-pep Affinity Matrix: Lysate Eluate Eluate unbound bound FT FT Crk Nck Shc Grb2 SH2-containing adaptor proteins ?-/-   pep        pY-pep         pep       pY-pep Affinity Matrix: Lysate Eluate Eluate unbound bound FT FT 81 A B Crk Nck Shc Grb2 SH2-containing adaptor proteins ?+/+   pep        pY-pep       pep        pY-pep Affinity Matrix: Lysate Eluate Eluate unbound bound FT FT Figure 3.3  SH2-domain containing proteins in wild type MEFs are able to bind to PTP?-phosphopeptide. Wild type MEFs (?+/+) were serum starved overnight.  The next day, cells were trypsinized, kept in suspension for 1 h and replated on FN-coated dishes for 30 min.  Cell lysates were incubated either with a non-phospho- (pep) or phospho-PTP?? (pY-pep) peptide affinity column. The flow through (FT), containing the unbound proteins, was collected from each column.  Then the columns were washed and the bound proteins (eluate) were eluted.  (A) The whole cell lysate, FT and eluate from each column were probed for Arp3, N-Wasp, Gab1, dynamin and Fish. (B) Same as (A) except theh samples were probed for the SH2-domain containing adaptor proteins, Grb2, Nck, Crk and Shc. All blots are representative of two independent experiments. Arp Dynamin Fish Gab1 N-Wasp Cytoskeletal Grb2-binding ?+/+   pep        pY-pep         pep        pY-pep Affinity Matrix: Lysate Eluate Eluate unbound bound FT FT 82 A Figure 3.4  Shc, Crk and Nck do not co-immunoprecipitate with PTP?. PTP?-null MEFs were infected with adenovirus (AdV) expressing WT- or Y789F-PTP? for 24 h, then starved in reduced serum medium overnight. The next day, cells were trypsinized, kept in suspension for 1 h and replated on FN-coated dishes for 30 min. (A) PTP? immunoprecipitates were probed for phosphotyrosine (PY20), PTP?, Grb2 and Shc.  Lysates were immunoblotted with PTP?, Grb2 and Shc antibodies (n=2). (B) PTP? immunoprecipitates were probed for phosphotyrosine (PY20), PTP?, Grb2, Crk and Nck.  Lysates were immunoblotted with PTP?, Grb2, Crk and Nck antibodies (n=2). (C) Nck immunoprecipitates were probed for Nck and PTP??(n=2). Lysate?Shc Grb2 PTP??PY20 Shc Grb2 PTP??IP: PTP??AdV: WT Y789F B Lysate?Crk Grb2 PTP??Nck Crk Grb2 PTP??PY20 IP: PTP??AdV: WT Y789F Nck C PTP??Nck IP: Nck?AdV: WT Y789F 83 A Figure 3.5  PTP? Tyr789 is required for optimal integrin-induced Cas tyrosine phosphorylation. Wild type (+/+), PTP?-null (-/-) and PTP?-null fibroblasts infected with adenovirus (AdV) expressing wild-type (WT) or unphosphorylatable PTP? (Y789F) were plated on fibronectin-coated plates for 30 min. (A) Cas immunoprecipitates were probed for phosphotyrosine (pY4G10) and Cas. (B) Results from three independent experiments were quantified by densitometry and the tyrosine phosphorylation per unit of Cas is shown on the graphs as mean ? S.D.  Asterisks indicate significant differences as compared to wild type cells  (*P = 0.001, **P = 0.02).  (C)  Lysates were probed with Cas-pTyr410 (pY410), Cas, PTP?-pTyr789 (pY789), PTP? and actin antibodies. B pY4G10 Cas IP: Cas +/+ -/- -/- -/- AdV: PTP?:?WT Y789F pTyr Per Unit Cas * ** +/+ -/- -/- -/- AdV: PTP?:?WT Y789F 0.8 0.6 0.4 0.2 1.0 C Cas Cas-pY410 Actin PTP??PTP?-pY789 +/+ -/- -/- -/- AdV: PTP?:?WT Y789F 84 Figure 3.6  Integrin-induced Cas-Src association is impaired in PTP?-Y789F expressing cells.  Wild type (+/+), PTP?-null (-/-) and PTP?-null fibroblasts infected with adenovirus (AdV) expressing wild-type (WT) or unphosphorylatable mutant PTP? (Y789F) were plated on fibronectin-coated plates for 30 min.  Cas immunoprecipitates were probed for Src and Cas while the cell lysates were probed for PTP?-pTyr789 (pY789), PTP?, Cas, Src and actin. (n=2) Actin PTP??PTP?-pY789 Cas Src +/+ -/- -/- -/- AdV: PTP?:?WT Y789F Src Cas Lysate?IP: Cas?85 mCherry-vinculin EGFP-Cas 0.463 mCherry-Vinculin pEGFP-Cas 0.630 pEGFP-Cas 0.903 mCherry-Vinculin pEGFP-Cas PTP?+/+ PTP?-/- PTP?-/-  + WT PTP?-/-  + Y789F 0.816 mCherry-Vinculin pEGFP-Cas mCherry-Vinculin merge Figure 3.7 PTP? Tyr789 promotes Cas localization to focal adhesions (1). Wild-type (PTP?+/+) and PTP?-/- MEFs, or PTP?-/- MEFs infected with adenovirus (AdV) expressing WT- or Y789F-PTP? were co-transfected with mCherry-vinculin and EGFP-Cas. After 24 h, the cells were suspended for 1 h and then placed on fibronectin-coated dishes for 15 min. Vinculin and Cas signals were imaged using TIRF microscopy. Scale bar (in upper ?merge? panel) = 30?m. The extent of co-localization between the EGFP and mCherry signals from one area of the cell (inset) was scatter plotted (far right panels) and calculated using Pearson?s correlation coefficient (fraction number within plot).  86 n.s. * ** *** 0.2 1.0 0.8 0.6 0.4 Pearson?s co-efficient PTP?: -/-  -/-   -/- WT AdV: Y789F +/+  Figure 3.8  PTP? Tyr789 promotes Cas localization to focal adhesions (2). Wild-type (+/+) and PTP?-/- (-/-) MEFs, or PTP?-/- MEFs infected with adenovirus (AdV) expressing WT- or Y789F-PTP? were co-transfected with mCherry-vinculin and EGFP-Cas. After 24 h, the cells were suspended for 1 h and then placed on fibronectin-coated dishes for 15 min. Pearson?s correlation coefficient was calculated from two areas per cell [representative images shown in Fig. 3.7] and from 10 cells of each type and is shown as the mean ? S.D. for the 20 areas analyzed. Asterisks indicate significant differences (*, p=2x10-4; **, p=4x10-7; ***, p=5x10-8) and n.s. indicates no significant difference (p=0.210).  87 Figure 3.9  PTP?? Tyr789 and BCAR3 SH2 domain are required for integrin-induced formation of a PTP?-BCAR3-Cas-Src complex.  (A) PTP?-/- (-/-) cells and PTP?-/- cells expressing VSVG-tagged WT-PTP? were treated with control or BCAR3 siRNA. VSVG immunoprecipitates were probed for BCAR, Cas, Src and VSVG (VSVG-PTP?).  Lysates were probed for BCAR3 and actin. (B) Lysates from adherent wild-type MEFs (+/+) and PTP?-/- MEFs (-/-) and from PTP?-/- MEFs transfected with VSVG-tagged WT- or Y789F-PTP? were used to prepare VSVG immuno-precipitates. These and the lysates were probed as indicated. (C) PTP?-/- MEFs were untreated or co-transfected with VSVG-WT-PTP? and FLAG-tagged WT- or mutant (R177K or R748A) BCAR3.  Anti-VSVG and anti-FLAG immunoprecipitates and cell lysates were probed as indicated. -/- -/- -/- PTP?: VSVG-PTP?: WT WT Con BCAR3 siRNA: IP-VSVG  VSVG- PTP? Src Cas BCAR3 BCAR3 actin Lysate A IP-VSVG  B actin PTP?  pY789  VSVG- PTP? BCAR3 Cas Src  PTP?:  +/+  WT Y789F VSVG-PTP?:  -/-  -/-  -/- Lysate IP-VSVG PTP?-/- VSVG-PTP?: WT GEF VSVG Src FLAG-BCAR3: WT WT WT FLAG BCAR3 Cas Src VSVG IP-FLAG actin Lysate PTP?  BCAR3 Src  Cas  FLAG VSVG PTP?  pY789  - SH2 - C *Courtesy of Dr. Guobin Sun (Sun et al. 2012) Cas 88 Vinculin mCherry Merge BCAR3-SH2 BCAR3-WT Fig. 3.10  The BCAR3 SH2 domain is required for its localization to focal adhesions.  Wild-type MEFs were transfected with mCherry-BCAR3-WT or mCherry-BCAR3-SH2 (R177K) mutant. Cells were starved, placed in suspension for 1 h and then placed on fibronectin-coated coverslips for 15 min. Cells were fixed and immunostained with vinculin. Both vinculin and BCAR3 signals were imaged using TIRF microscopy. Scale bar = 30 ?m.  Insets (top right) displaying the corresponding boxed areas are expanded 2.5 times. 89 PTP?-WT BCAR3-WT PTP?-Y789F BCAR3-WT Vinculin mCherry Merge Fig. 3.11  BCAR3 focal adhesion localization is defective in PTP?-Y789F expressing cells.  PTP?-/- MEFs infected with adenovirus (AdV) expressing WT- or Y789F-PTP? were transfected with mCherry-BCAR3-WT. Cells were starved, placed in suspension for 1 h and then placed on fibronectin-coated dishes for 15 min. Cells were fixed, immunostained with vinculin and imaged using TIRF microscopy. Scale bar = 30 ?m.  Insets (top right) displaying the corresponding boxed areas are expanded 2.5 times.  90  91  The Role of Grb2 in Integrin-Mediated PTP? Tyr789 Chapter 4:Phosphorylation   4.1 Rationale   BCAR3, Src and Grb2 are three SH2-domain containing proteins that have been identified as binding partners of PTP?-phosphoTyr789 (den Hertog et al. 1994; Su et al. 1994; Zheng et al. 2000; Sun et al. 2012).  As discussed in Chapter 3, BCAR3 physically links integrin-induced phosphoPTP??to the Cas-Crk signaling axis to regulate Cas-mediated cell migration (Sun et al. 2012).  Inactive Src is phosphorylated at Tyr527 and this phosphoresidue forms an inhibitory intramolecular interaction with the SH2 domain to block access to the kinase domain (Liu et al. 1993).  PTP? activates Src by dephosphorylating Src Tyr527 (Zheng et al. 1992; den Hertog et al. 1993; Ponniah et al. 1999; Su et al. 1999).  Active Src in association with FAK then phosphorylates PTP? at the C-terminal tail Tyr789 (Chen et al. 2006).  Approximately 20% of PTP? is constitutively phosphorylated at Tyr789 and most of it is bound to Grb2 (den Hertog et al. 1994).  Grb2 is an adaptor protein with a central SH2 domain and two flanking SH3 domains.  Although Grb2 interacts with PTP? via its SH2 domain and C-terminal SH3 domain binding to PTP?-phosphoTyr789 and a small region within the PTP? D1 catalytic domain, respectively (den Hertog et al. 1994; Su et al. 1994; den Hertog and Hunter 1996; Su et al. 1996), the function of the PTP?-Grb2 interaction remains unclear.  It has been proposed that Grb2 functions to protect phosphoPTP? from dephosphorylation, or that Grb2 may negatively regulate PTP? activity by blocking substrate binding to the D1 catalytic pocket of  92 PTP? (den Hertog et al. 1994; den Hertog and Hunter 1996; Su et al. 1996).  Furthermore, since the well-known Grb2 N-terminal SH3 domain binding partner, Sos, a GEF that activates Ras to mediate MAPK activation, is not found in the phosphoPTP?-bound Grb2 complex (den Hertog et al. 1994), PTP? has also been proposed to sequester Grb2 and preclude its binding to Sos thereby attenuating Grb2/Sos-mediated signaling.  To investigate the role of Grb2 in integrin-induced PTP?-mediated signaling, Grb2-targeting siRNA was used to silence Grb2 expression in MEFs to identify PTP?-mediated signaling events that are affected by the loss of Grb2.      4.2 Integrin stimulation induces PTP?-phosphoTyr789 association with Grb2   The association between PTP?-phosphoTyr789 and Grb2 has been established in cells proliferating in culture but not within the context of integrin signaling.  To determine whether integrin-mediated PTP? Tyr789 phosphorylation induces its association with Grb2, wild type MEFs were serum starved overnight, trypsinized and kept in suspension for 1 h, and then replated on fibronectin (FN)-coated dishes for 30 min.  PTP? was immunoprecipitated from lysates of adherent, suspended and FN-stimulated cells and probed for Grb2, total phosphotyrosine (PY20) and PTP?.  In adherent cells, where integrin signaling is turned ?on?, PTP? is highly phosphorylated and found in association with Grb2, whereas in suspended cells, where integrin signaling is terminated, PTP? phosphorylation and its concomitant association with Grb2 are reduced (Fig. 4.1).  Upon FN stimulation, both PTP? tyrosine phosphorylation and PTP?-Grb2 association are restored, indicating that Grb2 interacts with phosphoPTP? in an integrin-dependent fashion.  This is in accord with  93 Fig. 3.3 which shows that Grb2 only associates with WT- but not the Y789F-PTP? mutant upon FN stimulation, confirming that PTP??Tyr789 is required for integrin-induced Grb2 binding to PTP?.    4.3 Integrin-induced PTP? Tyr789 phosphorylation is defective in Grb2 knockdown cells   To investigate the role of Grb2 in coordinating PTP?-mediated signaling events, Grb2 expression was silenced in wild type MEFs using Grb2-specific siRNA.  Two commercially available siRNA duplexes (designated 1 and 2) were tested for their knockdown efficiency.  Over a period of 24, 48 and 72 hours post transfection, duplex 2, which targets the N-terminus of Grb2, consistently showed a >90% knockdown efficiency while duplex 1, which targets the 5?-untranslated exon of Grb2, showed <55% efficiency compared to the non-targeting control siRNA (Fig. 4.2A, upper panel).  To determine whether Grb2 knockdown exerts any effects on PTP? expression and/or Tyr789 phosphorylation, control and Grb2-siRNA treated cell lysates were probed for PTP?-pY789 and PTP?.  Fig. 4.2A (lower panel) shows that Grb2 depletion had no effect on PTP? expression but proportionally reduced PTP??Tyr789 phosphorylation in accord with the amount of Grb2 expressed, demonstrating that Grb2 modulates PTP? phosphorylation in a dosage-dependent manner.  Since duplex 2 is more effective in depleting endogenous Grb2, it was used in subsequent experiments to silence Grb2 expression.  Next, to examine whether Grb2 specifically regulates integrin-induced PTP??Tyr789 phosphorylation, cells treated with control or Grb2 siRNA were trypsinized, kept in suspension for 1 h and replated on FN-coated dishes to stimulate integrin signaling for 15  94 min.  (Note: A 15 min-stimulation as opposed to 30 min, which was shown in Fig. 4.1, was used from here on because optimal differences in the amount of integrin-induced protein tyrosine phosphorylation could be observed between control and treatment groups.)  As expected, integrin-stimulation induced PTP??Tyr789 phosphorylation in control cells, and in the absence of Grb2, integrin-induced PTP?-phosphoTyr789 was undetectable (Fig. 4.2B).  Indeed, the loss of Grb2 also abolished the residual phosphoPTP? in the suspended cells.  To verify that the effect of Grb2 on PTP??Tyr789 phosphorylation is not due to off-target effects of the siRNA, siRNA-resistant myc-tagged Grb2 was re-expressed in Grb2-depleted cells.  SiRNA resistance was achieved by generating several silent mutations within the sequences where the Grb2 siRNA targets.  Re-expression of Grb2 rescued defective phosphoPTP? observed in Grb2 knockdown cells (Fig. 4.2C).  This validates that the reduction of PTP??Tyr789 phosphorylation mediated by the loss of Grb2 is likely not an off target effect.  Grb2 might be functioning to protect phosphoPTP?-Tyr789 from dephosphorylation or promote PTP?-phosphorylating kinase activity.  4.4 Cas phosphorylation and cell migration are impaired in the absence of Grb2   The phosphorylation of PTP? at Tyr789 is important for Cas-mediated cell migration (Sun et al. 2012).  PTP?-Y789F expressing cells display defective Cas phosphorylation and delayed cell migration (Chen et al. 2006; Sun et al. 2012).  Since Grb2-depleted cells also display defective PTP??Tyr789 phosphorylation, it is probable that they mimic the phenotype of PTP?-Y789F expressing cells.  Immunoblotting cell lysates from FN-stimulated control and Grb2-siRNA treated cells with the phospho-specific antibody, Cas- 95 pY410, which recognizes one of the phosphorylated tyrosine residues within Cas substrate domain, shows that Cas Tyr410 phosphorylation is indeed reduced in the absence of Grb2 (Fig. 4.3).      To determine if Grb2 is also important for cell migration, control and Grb2-depleted cells were seeded as a monolayer in a microfluidic channel and half of the monolayer was trypsinized under controlled shear flow to simulate wounding.  Cell movement was tracked in real time over 24 hours using live cell microscopy.  The tracts of 10 randomly chosen cells from the leading edge of the control and Grb2-depleted cell group were manually tracked, and the tracks are depicted in Fig. 4.4A and 4.4B respectively.  It is apparent that the control cells are able to polarize and migrate more readily into the empty ?wound? area while cells that lack Grb2, despite exhibiting dynamic protrusions at the cell edges, appeared to meander aimlessly in proximity to the wounded edge.  The total distance travelled by Grb2-depleted cells was ~30% less than control cells, and the net distance (linear distance measured between the original and the final positions) was ~50% less (Fig. 4.4C, D), suggesting that the loss of Grb2 not only reduces cell motility but also inhibits directional cell movement, hindering the cells from effectively moving into the wounded area.  Grb2 is known to interact with a multitude of proteins involved in integrin signaling (Bisson et al. 2011), therefore whether this migration defect solely or primarily results from impaired Grb2-dependent PTP? phosphorylation or other Grb2-mediated signaling processes is unknown.      96 4.5 PTP? translocates to focal adhesions in the absence of Grb2-mediated PTP? Tyr789 phosphorylation    In addition to cell migration, PTP??Tyr789 phosphorylation has been implicated in the integrin-induced focal adhesion (FA) localization of PTP?.  Our lab and others have shown that PTP? is not properly localized to focal adhesions in PTP?-Y789F expressing cells (Lammers et al. 2000; Sun et al. 2012), yet the mechanism of PTP? FA localization is unknown.  To determine whether Grb2 is involved in the recruitment of PTP? to FAs, a focal adhesion enrichment and isolation assay (Kaplan et al. 1994; Sakai et al. 1994) was used to separate focal adhesion proteins (insoluble fraction, I) from cytoplasmic proteins (soluble fraction, S) in suspended and FN-stimulated control and Grb2-deficient cells.  As PTP? phosphorylation is virtually abolished in Grb2-depleted cells, its localization to FAs was expected to be impaired.  Surprisingly, defects in PTP??Tyr789 phosphorylation in Grb2-deficient cells did not prevent PTP? translocation to focal adhesions upon integrin stimulation (Fig. 4.5A).  In both control and Grb2-depleted cells, very little (~15%) PTP? is present in the insoluble fraction from suspended cells but upon FN stimulation, ~75% of PTP? is found in the insoluble fraction.  The increased amount of PTP? in the insoluble fraction post integrin stimulation is indicative of PTP? movement from the cytosol to focal adhesions.  Results from three independent experiments show an average of ~61% and ~71% of total PTP? in the focal adhesion-enriched fraction of the control and Grb2 knockdown cells respectively with no significant difference observed between the two cell groups (Fig. 4.5B).  These findings clearly indicate that Grb2 is not required to recruit PTP? to the FAs.  On the other hand, they raise the question of whether it is an intact Tyr789 residue or its  97 phosphorylation that is required for PTP? translocation to focal adhesions.  The difference between Grb2-depleted cells and PTP?-Y789F expressing cells lies in the fact that endogenous PTP? is present in Grb2-depleted cells, meaning that either perhaps a very low amount of or transiently phosphorylated PTP? is sufficient to localize PTP? to the FAs or maybe the biochemical structure of the tyrosine residue is necessary to put PTP??in a favorable conformation for this particular cellular process to occur.  4.6 Detection of transient PTP? phosphorylation in Grb2-depleted cells   As mentioned above (section 4.1), Grb2 binding to PTP?-phosphoTyr789 has been proposed to protect PTP? from dephosphorylation.  This infers that without Grb2, phosphoPTP? would only be detected transiently as it is more susceptible to dephosphorylation.  To attempt to detect transient phosphorylation of PTP? at Tyr789, control and Grb2-siRNA treated cells were harvested at an earlier time point (5 min) of integrin-stimulation.  However, even at this early time point, no phosphoPTP? was detected (Fig. 4.6A).  Since it is technically unfeasible to stimulate cells on FN for less than 5 min as most of the cells would either be loosely attached or unattached, serum was directly added to adherent cells to stimulate cell signaling (i.e. via growth factor receptors).  Serum was used in place of FN to attempt to detect potentially short-lived PTP??Tyr789 phosphorylation at earlier time points.     98 4.6.1 Serum stimulation induces PTP? Tyr789 phosphorylation   Wild type MEFs were serum starved overnight and stimulated with 10% FBS over a time course of 5 min.  Figs. 4.6B and C show that in unstimulated serum-starved cells (time 0), the amount of PTP?-phosphoTyr789 was ~4-fold lower in Grb2-depleted cells.  Upon serum stimulation, the amount of phosphoPTP? continuously decreased in control cells but in Grb2-depleted cells phosphoPTP? peaked (~2-fold increase) at 30 seconds post stimulation and gradually decreased thereafter.  There was no further induction of PTP? phosphorylation in control cells upon serum stimulation, suggesting that PTP? was already at its maximal phosphorylated state prior to stimulation.  Plotting the amount of phosphoPTP? per unit PTP? over time reveals that, aside from the initial 30 sec post stimulation, the rate of PTP? dephosphorylation is similar with or without Grb2.  These findings reveal that PTP? can be transiently phosphorylated in Grb2-deficient cells but appears not to undergo hyperdephosphorylation in the absence of Grb2.    4.6.2 Overexpression of BCAR3 in the absence of Grb2 fails to rescue PTP? phosphorylation   In addition to Grb2, BCAR3 can also interact with phosphoPTP?-Tyr789 via its SH2 domain in an integrin-dependent manner (Sun et al. 2012).  To verify that PTP?-BCAR3 complex formation is impaired in Grb2-depleted cells where phosphoPTP??is defective, VSVG-tagged PTP? and control or Grb2 siRNA were co-transfected into PTP?-/- cells.  The presence of BCAR3 and Grb2 was detected in VSVG immunoprecipitates from cells treated with non-targeting siRNA but not in those treated with Grb2 siRNA where phosphoPTP? is  99 impaired (Fig. 4.7A).  This confirms that PTP??Tyr789 phosphorylation is required for BCAR3 binding to PTP? and indicates that endogenous BCAR3, which has the ability to bind to phosphoPTP? directly, was unable to protect phosphoPTP?.  The latter could either be due to a relatively low expression of endogenous BCAR3 in MEFs that is insufficient to confer protection or to the presence of unphosphorylated PTP? that cannot recruit BCAR3.  To test whether BCAR3 overexpression can rescue phosphoPTP? in the absence of Grb2, FLAG-tagged wild type or SH2 mutant (R177K) BCAR3, which is unable to interact with PTP? (Sun et al. 2012), were overexpressed in wild type MEFs treated with Grb2 siRNA.  Myc-tagged wild type or SH2 mutant (R86K) Grb2, which does not interact with PTP? (den Hertog and Hunter 1996), were also expressed in Grb2-depleted cells as experimental controls.  As expected, both of the SH2 mutants that cannot bind to PTP? failed to rescue PTP? Tyr789 phosphorylation (Fig. 4.7B).  However, while WT-Grb2 was able to restore PTP?-phosphoTyr789, WT-BCAR3 was unable to rescue defective phosphoPTP? in the absence of Grb2.  This shows that excess BCAR3 cannot replace the function of Grb2 in mediating PTP??Tyr789 phosphorylation and suggests that re-expressed Grb2 may be rescuing PTP? phosphorylation through a mechanism that is independent of its SH2-domain-mediated binding to PTP?.  It appears that Grb2, rather than having a protective effect on phosphoPTP?, may be important for the activity of the kinase(s) that phosphorylate PTP?.     100 4.7 Investigation of FAK-Src complex activity in cells lacking Grb2    FAK and SFKs are major kinases that are involved in integrin signaling.  Their activation is among the early events downstream of integrin engagement.  In FAK-null and SYF (Src-/-Yes-/-Fyn-/-) cells, PTP? Tyr789 phosphorylation is impaired (Chen et al. 2006).  However, in Src+/+ cells that are deficient in Yes and Fyn, PTP? Tyr789 phosphorylation is unaltered (Chen et al. 2006).  These findings suggest that FAK and Src, likely acting in the well characterized Src-FAK complex (Schaller et al. 1999; Mitra and Schlaepfer 2006), are essential for PTP? phosphorylation.  To investigate whether Grb2 is regulating PTP??Tyr789 phosphorylation via these upstream kinases, I examined the phosphorylation and activation of Src and FAK in Grb2-deficient cells.   4.7.1 Integrin-induced Src phosphorylation and activity are unaffected by Grb2   Grb2 plays an upstream regulatory role in the activation of Lck, a member of the SFK family, in thymocyte development (Jang et al. 2010).  To investigate whether Grb2 plays a parallel role in the integrin-induced activation of Src in MEFs, lysates from FN-stimulated control and Grb2 siRNA-treated cells were probed with anti-Src-phosphoTyr416 (Src-pY416) antibody.  As this antibody recognizes multiple SFK family members, Src was specifically immunoprecipitated from the lysates and probed for Src-pTyr416 as well.  Results show that silencing Grb2 expression had no effect on integrin-induced Src phosphorylation at Tyr416, an event associated with Src activation (Figs. 4.8A, B).  To verify that Src activity is unaltered in Grb2-depleted cells, Src immunoprecipitates were prepared from lysates of integrin-induced PTP?-/- cells and from PTP?+/+ cells treated with  101 control or Grb2 siRNA and subjected to in vitro kinase assays.  PTP?-/- cells were included as a negative control as they have reduced Src activity (Ponniah et al. 1999; Su et al. 1999).  As shown in Fig. 4.8C, Src kinase activity was reduced by ~25% (? 5%) in Grb2-depleted cells as compared to the control.  This reduction is relatively small in comparison to the 85% decrease observed in PTP?-/- cells, suggesting that it is likely not the primary cause of the loss of PTP??Tyr789 phosphorylation observed upon Grb2 depletion.  To examine the subcellular distribution of phosphorylated Src, control and Grb2 siRNA-treated cells were allowed to attach and spread on FN-coated dishes for 15 min, and were then fixed and immunostained with anti-Src-pY416 antibody.  Spreading cells were visualized using TIRF imaging technology.  As shown in Fig. 4.8D, phosphoSrc signal intensity and residence at the periphery of the cells were independent of the presence of Grb2 but the morphologies of the spreading cells were quite different in the absence of Grb2.  While control cells displayed a relatively uniform and rounded morphology, Grb2-depleted cells exhibited a distinctive spreading phenotype characterized by multiple random pointed protrusions at the cell edge.  Cells lacking Grb2 appeared to lose their ability to form the broad, sheet-like lamellipodia.  Coincidentally, a similar spreading phenotype has been reported where FAK-depleted rat embryo fibroblasts (REFs) exhibited abnormal spike-like protrusions on FN and this defect could only be rescued by WT- but not the Y397F-FAK mutant (Tilghman et al. 2005).        102 4.7.2 Integrin-induced FAK phosphorylation and FAK-Src association are impaired in Grb2-depleted cells   The resemblance between Grb2-depleted MEFs and the unphosphorylatable FAK-Y397F expressing REFs suggests that the autophosphorylation of FAK at Tyr397 triggered by integrin engagement may be defective in the absence of Grb2, yielding a similar spreading phenotype.  To examine FAK Tyr397 phosphorylation in the absence of Grb2, control and Grb2-deficient cells were co-immunostained with FAK-pY397 and vinculin antibodies.  Figure 4.9A shows that FAK-phosphoTyr397 localized to the periphery of the control cells and co-localized with vinculin (shown in yellow), a cytoskeletal protein that is commonly used as a focal adhesion marker.  In contrast, the intensity of the FAK-phosphoTyr397 signal is relatively weak in Grb2-depleted cells and its co-localization with vinculin is hardly observed, suggesting that FAK phosphorylation at Tyr397 is impaired in these cells.  To verify that integrin-induced FAK autophosphorylation is defective in Grb2-depleted cells, cell lysates from suspended and FN-stimulated control and Grb2 knockdown cells were probed for FAK-pTyr397 and FAK (Fig. 4.9B).  Integrin engagement stimulates FAK phosphorylation at Tyr397 in control cells but this occurs to a much lesser extent in Grb2-depleted cells.  FAK-phosphoTyr397 mediates the SH2-domain binding of Src which is necessary for Src to phosphorylate FAK Tyr576/577 in the catalytic loop to promote FAK activation (Xing et al. 1994; Owen et al. 1999; Hanks et al. 2003).  Acting in concert with each other, the active Src-FAK complex phosphorylates downstream substrates to mediate downstream signaling.  To confirm that FAK-Src complex formation is impaired in Grb2-depleted cells, Src was immunoprecipitated from suspended and FN-stimulated control and  103 Grb2-depleted cell lysates.  Probing the immunoprecipitates with anti-Src-pY416 antibody showed that Grb2 silencing does not affect Src phosphorylation but significantly reduces FAK-Src association upon integrin stimulation (Fig. 4.9C). This indicates that the defect in FAK phosphorylation at Tyr397 leads to impaired FAK-Src complex formation, and the defect in FAK-Src association then affects Src-mediated FAK Tyr576/577 phosphorylation as shown in Fig. 4.9B.  Taken together, these experiments indicate that the phosphorylation and activation of FAK is dependent on the presence of Grb2.  Re-introduction of siRNA-resistant WT-Grb2 into Grb2-depleted cells rescued defective FAK Tyr397 phosphorylation (Figs. 4.10A, B), confirming that the effect of Grb2 on FAK phosphorylation is specific and not due to off-target effects.  These findings identify an upstream role of Grb2 in regulating PTP? phosphorylation through FAK activation and FAK-Src complex formation.  4.7.3 Similarities between Grb2 knockdown cells and FAK-Y397F expressing cells   To demonstrate that FAK-phosphoTyr397 is indeed required for PTP? phosphorylation, PTP? phosphorylation was examined in FAK-null cells and FAK-null cells re-expressing GFP-tagged wild type and Y397F mutant FAK.  Fig. 4.11 shows that PTP??Tyr789 phosphorylation was barely detectable in FAK-null cells compared to normal MEFs (wt) expressing endogenous FAK.  In the FAK-expressing cells, although similar levels of GFP-labeled FAK WT- and Y397F were expressed, only the WT but not the Y397F mutant was able to rescue PTP?-phosphoTyr789, demonstrating that the phosphorylation of FAK at Tyr397 precedes and is required for proper PTP??Tyr789 phosphorylation.    104 4.8 Discussion   Grb2 was identified as a binding partner of PTP?-phosphoTyr789 almost twenty years ago (den Hertog et al. 1994; Su et al. 1994), yet its role in complex with PTP? remains a mystery.  In this study, I demonstrated that PTP? and Grb2 associate in an integrin-dependent manner. Using a siRNA-mediated silencing approach, I further investigated the role of Grb2 in phosphoPTP?-mediated signaling.  Interestingly, I found that in the absence of Grb2, PTP? Tyr789 phosphorylation was abolished, indicating that Grb2 plays a role in the regulation of PTP? phosphorylation.  The phosphorylation of PTP? at Tyr789 has been shown to promote Cas-mediated cell migration and FA localization of PTP? as PTP?-Y789F expressing cells showed defective cytoskeletal rearrangement, delayed cell migration and impaired PTP? focal adhesion localization (Lammers et al. 2000; Chen et al. 2006; Sun et al. 2012).  However, integrin-stimulated Grb2-depleted cells, which contain mostly unphosphorylated PTP?, display normal PTP? translocation to FAs.  This could be attributed to an extremely low amount of phosphorylated endogenous PTP? that might be present but undetectable by Western blotting and that is sufficient to support the translocation of PTP? to FAs.  However, studies with a phospho-mimic PTP?-Y789E mutant suggest otherwise.  Replacement of the tyrosine residue with glutamic acid creates a constitutive negative charge that mimics a phosphorylated tyrosine but does not support the binding of SH2-domain containing proteins to PTP?.  In PTP?-null cells re-expressing VSVG-tagged PTP?-Y789E, PTP? is unable to effectively localize to FAs (Appendix Figs. A.1A-C), suggesting that either an interacting protein or an intact tyrosine residue is required to bring PTP? to FAs.   105 Since Grb2-depleted cells have undetectable Src-FAK complex-mediated PTP?-Tyr789 phosphorylation, the significantly reduced levels of PTP? phosphorylation is unlikely to mediate an interaction with another protein.  Thus, the translocation of PTP? to FAs upon integrin engagement is probably facilitated by an intact tyrosine residue rather than a phosphoPTP?-interacting protein.  The mechanism by which Tyr789 modulates PTP? localization to FAs remains to be determined.    Grb2 may regulate PTP??Tyr789 phosphorylation by two different but not exclusive mechanisms.  One is to protect phosphoPTP? from dephosphorylation and the other is to promote PTP? phosphorylation catalyzed by its upstream kinases.  My results, while not eliminating the possibility of a protective role of Grb2, support a model where Grb2, through an unknown mechanism, mediates FAK autophosphorylation at Tyr397.  This enables subsequent Src binding and Src-mediated FAK phosphorylation.  The resulting active Src-FAK complex then phosphorylates the C-terminal tail of PTP? at Tyr789.  This is in accord with defective FAK phosphorylation and complex formation with Src observed in Grb2 knockdown cells, and the abnormal spreading phenotype and loss of PTP? phosphorylation of these cells resembles that of FAK-Y397F expressing cells (Tilghman et al. 2005).  The detailed molecular mechanism(s) that controls FAK autophosphorylation at Tyr397 downstream of integrin ligation is unclear.  It is however known that in order for FAK Tyr397 phosphorylation to occur, it must be released from its auto-inhibitory conformation and be localized to focal adhesions.  Grb2 may play a role in either or both of these events that lead up to FAK autophosphorylation.  Alternatively, FAK Tyr397 phosphorylation can also be regulated by phosphatases that specifically target this phosphosite and Grb2 may participate in the inhibition of one or more of these phosphatases.  106  The FERM domain of FAK directly binds to and inhibits its kinase domain (Cooper et al. 2003; Jacamo and Rozengurt 2005).  Disruption of this intramolecular interaction is required for the autophosphorylation of FAK Tyr397, which lies in the linker region between the FERM domain and the kinase domain, to occur (Frame et al. 2010).  FERM domain interacting partners, such as phosphatidylinositol-4,5-bisphosphate (PIP2) and ?1 integrins, can bind and trigger a conformational change in FAK to dissociate the FERM-kinase domain interaction, enabling FAK phosphorylation and subsequent activation (Schaller et al. 1995; Chen et al. 2000; Cai et al. 2008; Frame et al. 2010).  It is possible that Grb2 promotes FAK Tyr397 autophosphorylation by facilitating the binding of FERM domain binding partners with FAK through direct or indirect protein-protein interactions, or it might be coordinating the production of local concentrations of PIP2 at sites where integrins cluster to enhance FAK recruitment (Fig. 4.12A).  In addition to the FERM domain, a FAK family interacting protein, FIP200, is also known to bind to the kinase domain of FAK and inhibit FAK autophosphorylation and kinase activity.  The FAK-FIP200 complex dissociates upon integrin-mediated cell adhesion concomitant with FAK activation (Abbi et al. 2002).  Thus, Grb2 may also play a role in regulating the FAK-FIP200 interaction.  FAK can be recruited to sites of adhesion through its C-terminal FAT domain interaction with paxillin or talin (Scheswohl et al. 2008; Lawson et al. 2012).  While these interactions may contribute to FAK localization to FAs, they are not essential.  FAK is able to localize to FAs both in paxillin-null cells and in FAK-E1015A mutant expressing cells that have impaired FAK-talin interaction (Lawson and Schlaepfer 2012).  Indeed, whether FAK is recruited by paxillin and talin to FAs or vice versa is controversial.  Nevertheless, cells lacking either paxillin or talin exhibit defective FAK-phosphoTyr397 (Hagel et al. 2002;  107 Zhang et al. 2008).  Indeed, the LIM 1, 2, 3 and one of the LD domains of paxillin have been shown to be required for FAK autophosphorylation (Wade and Vande Pol 2006).  These findings suggest that Grb2 may not necessarily be involved in mediating the direct association between paxillin or talin with FAK but it might be coordinating the spatio-temporal localization of paxillin and talin with other focal adhesion components that are required for FAK autophosphorylation (Fig. 4.12B).  Several integrin-related PTPs, including PTP1B, PTEN, Shp2, PTP-PEST and LMW-PTP, have been shown to regulate FAK phosphorylation (Liu et al. 1998; Tamura et al. 1998; Yu et al. 1998; Angers-Loustau et al. 1999; Rigacci et al. 2002; Hartman et al. 2013).  However, whether PTP1B and PTP-PEST associate with FAK is unknown; it is likely that their effects on FAK are indirect and are mediated through Cas, which is a known substrate of these two phosphatases.  On the other hand, PTEN, Shp2 and LMW-PTP have been shown to complex with FAK.  Indeed, Shp2 and LMW-PTP have specifically been shown to dephosphorylate Tyr397 of FAK (Tamura et al. 1998; Rigacci et al. 2002; Hartman et al. 2013).  This makes Shp2 and LMW-PTP the most probable candidates to be targeted by Grb2 to inhibit their activity and increase FAK autophosphorylation.  Given that Grb2 inhibits Shp2 activity in FGFR2-mediated signaling (Ahmed et al. 2013), it is plausible to predict that a parallel model might exist in MEFs where Grb2 interacts with and inhibits Shp2 to prevent the dephosphorylation of FAK Tyr397 (Fig. 4.12C).  Being a versatile adaptor protein with a large number of known interacting partners, Grb2 is highly likely to regulate FAK Tyr397, as well as PTP??Tyr789, phosphorylation through multiple mechanisms that are yet to be defined.   PY20 PTP??IP: PTP?     Adh    0     30 Grb2 PTP?+/+ Figure 4.1  Grb2 associates with phosphoPTP? in an integrin-dependent manner. Wild-type (PTP?+/+) MEFs were starved overnight (adherent cells, Adh) and then trypsinized and kept in suspension for 1 h (0) to terminate integrin signaling.  Cells were then plated on dishes coated with the integrin ligand fibronectin (FN) for 30 min (30).  PTP? immunoprecipitates were probed with Grb2, phosphotyrosine (PY20) and PTP? antibodies.  108 A actin Grb2 C    1     2 C    1     2 C     1     2 Grb2 siRNA: 24h 48h 72h PTP?-pY789 PTP? B PTP? PTP?-pY789 0      15      0     15  C             Grb2 siRNA: Grb2 actin C Grb2 siRNA: Grb2-myc: actin Grb2 myc PTP??PTP?-pY789 C      +       +  -       -       + Figure 4.2  Grb2 knockdown reduces PTP??Tyr789 phosphorylation. (A) Wild type MEFs were treated with control (C) or Grb2 siRNA duplex 1 or 2 for 24, 48 and 72 h. Cell lysates were probed for Grb2, actin, PTP?-pY789 and PTP??(n=1). (B) Wild type MEFs were treated with control (C) or Grb2 siRNA for 48 h and then starved overnight. Cells were trypsinized and held in suspension for 1 h (0), and then a portion of the suspended cells was plated on fibronectin (FN)-coated plates for 15 min (15). Lysates were probed as shown.  Blots are representative of at least three independent experiments.  (C) Wild type MEFs treated with either control (C) or Grb2 siRNA or co-transfected with Grb2 siRNA and myc-tagged Grb2-WT were stimulated on FN-coated plates for 15 min. Lysates were immunoblotted with Grb2, myc, actin, PTP?-pY789, and PTP? antibodies.  All blots are representative of at least two independent experiments.  Blots are representative of at least three independent experiments. FN: 109 Figure 4.3  Grb2 depletion reduces Cas SD domain phosphorylation at Tyr410. Wild type MEFs were treated with control (C) or Grb2 siRNA for 48 h and then starved overnight. Cells were trypsinized and held in suspension for 1 h (0), and then a portion of the suspended cells was plated on fibronectin (FN)-coated plates for 15 min (15). Lysates were probed for Grb2, actin, Cas-phosphoTyr410 (Cas-pY410) and Cas (n=3).  0      15      0     15  C             Grb2 siRNA: Grb2 actin Cas Cas-pY410 FN: 110 A B -500-300-100100300500-500 -300 -100 100 300 500cDistance (?m) -500-300-100100300500-500 -300 -100 100 300 500Distance (?m) Control Grb2 kd D C Total Migration Distance (?m) Grb2 kd 200 C 300 400 500  600 700 100 Net Migration Distance (?m) C            Grb2 kd 200 250  150  100 50  Figure 4.4  Cell migration is reduced in the absence of Grb2. The migration of control and Grb2 siRNA-treated wild type MEFs into the cleared area of a ?wounded? cell monolayer was monitored for 24 h by time lapse live-cell microscopy. Cell movement was tracked using ImageJ  MtrackJ  plugin. The 2D random migration of 10 cells from each of the (A) control and (B) Grb2 siRNA-treated cell group was measured and graphed. (C) The total distance of each track was measured for 10 cells in each group. The means are shown as horizontal bars and are significantly different (P = 0.001). (D) The net (linear) distance travelled was measured from the starting point of a track to the end. Results from 10 cell tracks are shown for each group. The means are shown as horizontal bars and are significantly different (P = 0.023). 111 % of insoluble PTP? C FN: 0         15          0          15  Grb2 siRNA: 80  60  40  20 PTP? Grb2 S      I      S      I      S      I      S      I   PTP?-pY789 C         Grb2          C          Grb2   Sus                        FN15  siRNA: A B Figure 4.5. PTP? localizes to focal adhesions in the absence of Tyr789 phosphorylation.  (A) Wild type MEFs were treated with control (C) or Grb2 siRNA for 48h and starved in 0.5%  FBS DMEM overnight.  Cells were then trypsinized and put into suspension (Sus, 0) for 1 h.  A portion of the suspensded cells were plated on fibronectin (FN)-coated plates for 15 min.  Both suspension and FN-stimulated cells were harvested in a mild buffer to extract the cytosolic proteins as the soluble fraction (S) and then in RIPA buffer to extract cytoskeletal proteins as the insoluble fraction (I).   Lysates were then separated on SDS-PAGE and immunoblotted with antibodies against PTP?-pY789, PTP? and Grb2. (B) The percentage of PTP? in the insoluble fraction from suspended and FN stimulated cell lysates were graphed (n=3).  No significant difference was observed between the control (C) and Grb2 knockdown cells.  112 B PTP?-pY789 actin C                                     Grb2 Grb2 10% FBS:  PTP? siRNA:  0.5  0 1 2 5  0.5  0 1 2 5  C Time (min) PTP?-pY789 / PTP? 2.0  1.5   1.0  0.5  1 2 3 4 5  PTP? Actin Grb2 0     5     15     0     5     15   PTP?-pY789 C                  Grb2 FN:  siRNA:  A Figure 4.6  Serum stimulation induces reduced and transient PTP? Tyr789 phosphorylation in Grb2 knockdown cells. (A) MEFs were treated with control (C) or Grb2 siRNA for 48 h and then starved overnight. Cells were trypsinized and held in suspension for 1 h (0), and then a portion of the suspended cells was plated on fibronectin (FN)-coated plates for 5 min (5) and 15 min (15). Lysates were probed as shown. (B) MEFs were treated with control or Grb2 siRNA, and 48 h later they were serum starved overnight and then stimulated with 10%  FBS for 0.5, 1, 2, and 5 min. Lysates were immunoblotted with PTP?-pY789, PTP?, Grb2 and actin antibodies. (C) PTP? phosphorylation per unit PTP? (arbitrary units) in control siRNA- (closed circles) and Grb2 siRNA-treated (open circles) cells was calculated from densitometric quantification of the blots in (B) (n=1). 113 A BCAR3 Grb2 siRNA: VSVG-PTP?: VSVG?PTP?-/-  -          C        +?-        WT     WT?Grb2?PTP? Grb2 actin PTP?-pY789 BCAR3 VSVG IP: VSVG Lysate B PTP?-pY789 PTP? Grb2 actin Grb2 siRNA: Grb2-myc:    -          -      WT   R86K     -          -?   C        +        +        +        +         +?BCAR3 FLAG-BCAR3:    -          -        -         -        WT    R177K?myc FLAG Figure 4.7 BCAR3 overexpression fails to rescue phosphoPTP?-Tyr789.  (A) PTP?-/- MEFs were untreated or transfected with control (C) or Grb2 siRNA and VSVG-tagged WT PTP?. Anti-VSVG immunoprecipitates from adherent cell lysates were probed for BCAR3, Grb2 and VSVG (VSVG-PTP?). Lysates were probed for PTP?-phosphoTyr789 (PTP?-pY789), PTP?, VSVG (VSVG-PTP?), BCAR3, Grb2 and actin (n=3). (B) Wild-type MEFs were transfected with either control (C) or Grb2 siRNA, or co-transfected with Grb2 siRNA and myc-Grb2-WT, myc-Grb2-R86K, FLAG-BCAR3-WT or FLAG-BCAR3-R177K. Lysates were probed for PTP?-pY789, PTP?, myc, Grb2, FLAG, BCAR3 and actin (n=2). 114 A 0     15      0     15  C             Grb2 siRNA: Src Src-pY416 C Relative Kinase Activity  C Grb2 siRNA 1.0  0.5   PTP?-/- Control Grb2 Src-pY416 D Figure 4.8  Grb2 knockdown does not prevent integrin-induced Src activation. Wild type MEFs treated with control (C) or Grb2 siRNA were kept in suspension for 1 h (0) and re-plated on fibronectin (FN)-coated coverslips or dishes for 15 min (15). (A) Cell lysates were probed for Src-phosphoTyr416 (Src-pY416) and Src. (B) Src immunoprecipitates from lysates of FN stimulated cells were probed for Src-pY416 and Src. (C) Src immunoprecipitates were prepared from lysates of PTP?-/- MEFs and from control (C) and Grb2 siRNA-treated wild-type MEFs after FN stimulation for 15 min, and used in in vitro kinase assays. Kinase activity is shown relative to that in the control cell immunoprecipitate (PTP?-/- cells, n=1; control and Grb2 siRNA-treated cells, n=2). (D) Cells plated on coverslips were washed, fixed and immunostained with Src-pY416 antibody. Scale bar = 20 ?m. FN: 115 B IP: Src Src-pY416 Src C   Grb2 siRNA: FN15  Actin Grb2 B FAK-pY397 0      15      0     15  C              Grb2 siRNA: FAK FAK-pY576/577  C IP: Src 0      15      0     15  C             Grb2 siRNA: FAK Src Src-pY416 C Grb2 kd FAK-pY397 Vinculin Merge A Figure 4.9  Integrin-induced FAK Tyr397 phosphorylation and FAK-Src complex formation are dependent on Grb2. Wild type MEFs treated with control (C) or Grb2 siRNA were kept in suspension (0) for 1 h and replated on FN-coated coverslips or dishes for 15 min (15).  (A) Cells on coverslips were washed, fixed and immunostained with FAK-phosphoTyr397 (FAK-pY397, green) and vinculin (red) antibodies. Scale bar = 20?m.  (B) Cell lysates were collected from the plates and probed for FAK-pY397, FAK-phosphoY576/577 (FAK-pY576/577), and FAK. (C) Anti-Src immunoprecipitates were immunoblotted with Src-pY416, Src and FAK.  All blots are representative of three independent experiments. FN: 116 B A      C      +        +  Grb2 siRNA: Grb2-myc: -       -        +   actin Grb2 myc FAK-pY397 FAK       -           -          +  * Grb2 C Grb2 siRNA: Grb2-myc: 100 80 60 40 20 FAK-pTyr397 / FAK ** Figure 4.10  Re-expression of WT-Grb2 rescues integrin-stimulated FAK Tyr397 phosphorylation. Wild type MEFs transfected with either control (C) or Grb2 siRNA or co-transfected with Grb2 siRNA and myc-tagged Grb2 were kept in suspension for 1 h and plated on fibronectin (FN)-coated  dishes for 15 min. (A) Lysates were immunoblotted with Grb2, myc, actin, FAK-pY397 and FAK antibodies.  (B) The relative amount of FAK-pY397 per unit FAK from 4 experiments as described above was quantified by densitometry and is shown in the graph.  Asterisks indicate significant differences (*P=0.00003, **P=0.001).  117 -    WT  Y397F  FAK -/- actin FAK-pY397 PTP?-pY789 PTP? FAK wt Figure 4.11  FAK-Y397F expressing cells exhibit defective PTP??Tyr789 phosphorylation. Wild type MEFs (wt), FAK-null MEFs (FAK-/-) and FAK-null MEFs stably re-expressing GFP-tagged wild type (FAK-WT) or mutant (FAK-Y397F) FAK were immunoblotted with FAK-pY397, FAK, PTP?-pY789, PTP? and actin antibodies (n=1).   118 Figure 4.12  Potential actions of Grb2 in the activation of FAK.  FAK autophosphorylation at Tyr397, hence activation, is governed by various mechanisms.  (A) In the resting state, the FERM domain of FAK interacts with and blocks the kinase domain (KD) to inhibit FAK activity.  Integrin stimulation triggers a localized increase of phosphatidylinositol-4,5-bisphosphate (PIP2) at adhesion sites.  PIP2 then binds to the FERM domain and induce a conformational change in FAK to dissociate the FERM-kinase domain interaction, enabling the autophosphorylation of FAK.  Grb2 may function to coordinate the production of PIP2 or regulate FERM domain interaction with PIP2 or other proteins (X)  to promote FAK activation.  (B) Several domains of Paxillin, but not necessarily Paxillin-FAK interaction, is associated with FAK autophosphorylation.  Thus, Grb2 may activate FAK via the regulation of paxillin.  (C) Shp2 can dephosphorylate FAK at Tyr397.  While Grb2 can inhibit Shp2 activity by binding directly to Shp2, Grb2 may protect phosphoFAK-Tyr397 from Shp2-mediated dephosphorylation. B A C Grb2 ECM??? ??FAK?PIP2 PIP2 FERM?P Tyr397?ECM??? ??X  OR ?Grb2 ECM??? ??Shp2 FAK?P Tyr397?Tyr397?119  120  Identification of a Second Role of Grb2 in the Regulation of Chapter 5:Integrin-Stimulated PTP? Tyr789 Phosphorylation   5.1 Rationale   My work described in the previous chapter demonstrated that Grb2 plays an upstream role in the activation of FAK to promote Src-FAK-mediated PTP? Tyr789 phosphorylation.  Several possibilities of how Grb2 might regulate FAK autophosphorylation were discussed.  Based on a recent report demonstrating that Grb2 can bind and inhibit Shp2 activity to up-regulate FGFR2 phosphorylation upon ligand binding (Ahmed et al. 2013), Shp2 appears to be an attractive candidate that could be regulated by Grb2 to control FAK phosphorylation at Tyr397.  Shp2 is a widely expressed intracellular classical PTP that contains two tandem SH2 domains in the N-terminal region and a phosphatase domain in the C-terminal region.  Previous studies have shown that Shp2-/- cells display reduced FAK dephosphorylation upon detachment from the ECM and mimick the spreading phenotype of FAK-/- cells, establishing a role for Shp2 in FAK-mediated integrin signaling (Yu et al. 1998).  Correspondingly, the depletion of Shp2 by siRNA increased FAK Tyr397 phosphorylation in cardiomyocytes cultured on collagen-coated plates (Marin et al. 2008).  Furthermore, Far Western blotting and in vitro phosphatase assays have confirmed that FAK-phosphoTyr397 is necessary for its interaction with the catalytic domain of Shp2 and is preferentially dephosphorylated by Shp2 over FAK-phosphoTyr576 (Hartman et al. 2013).  However, integrin-mediated FAK phosphorylation has also been shown to be downregulated in Shp2-/- fibroblasts (Oh et al. 1999), suggesting that Shp2 may play dual roles in the regulation of FAK phosphorylation depending on the cell type and/or external stimuli.  To investigate whether Shp2 is involved  121 in Grb2-mediated FAK phosphorylation, Shp2 expression or its activity was suppressed in Grb2-depleted cells.      5.2 Knockdown or inhibition of Shp2 cannot rescue defective FAK-phosphoTyr397 in Grb2 knockdown cells   MEFs were transfected with Grb2 or Shp2 or simultaneously co-transfected with Shp2 and Grb2 siRNA to determine whether silencing the expression of Shp2 could rescue integrin-induced FAK Tyr397 phosphorylation.  Fig. 5.1A shows that while Grb2 depletion concomitantly reduced integrin-mediated FAK and PTP? phosphorylation, the loss of Shp2 had no effect on any of these signaling events.   Concurrently silencing both Grb2 and Shp2 showed similar effects to knocking down Grb2 alone, demonstrating that Shp2 could not rescue defective FAK-phosphoTyr397 in the absence of Grb2.  Likewise, when control and Grb2-deficient cells were treated with the Shp2 inhibitor, NSC 87877, prior to plating onto FN-coated dishes, FAK and PTP? phosphorylation were unaltered by the presence of the inhibitor in control siRNA or Grb2 siRNA-treated cells (Fig. 5.1B).  Although a slight increase was observed in PTP?-phosphoTyr789 in NSC 87877-treated wild type MEFs, it was not consistently seen.  Taken together, these results strongly suggest that Shp2 is not involved in Grb2-dependent FAK phosphorylation.  5.3 Effects of paxillin overexpression on integrin-induced FAK and PTP? phosphorylation in Grb2-depleted cells   The recruitment of FAK to focal adhesions is essential for its activation in response to adhesion stimuli (Schaller et al. 1999) and requires its FAT domain (Hildebrand et al. 1993).   122 Though the binding of paxillin to the FAT helical bundle of FAK is not absolutely required, it is the major mechanism that promotes FAK localization to focal adhesions.  Two domains of paxillin (LD2 and LD4) can interact with FAT (Brown et al. 1996); while binding of either one is sufficient to localize FAK to focal adhesions, interactions with both are necessary for optimal activation of FAK (Scheswohl et al. 2008).  LIM domains 1, 2, 3, and a single LD motif on paxillin have also been shown to be important for FAK-Tyr397 phosphorylation in embryonic stem cells (Wade and Vande Pol 2006).  I thus investigate whether Grb2 may activate FAK by regulating paxillin.  5.3.1 Paxillin expression and association with FAK are impaired in the absence of Grb2   To examine the association between paxillin and FAK in Grb2-depleted cells, FAK was immunoprecipitated from the lysates of suspended and FN-stimulated control or Grb2 siRNA-treated cells.  Figs. 5.2A (upper panel) and 5.2B show that paxillin association with FAK was increased by ~1.5-fold ? 0.23 (n=3) upon integrin stimulation in control cells but not in Grb2-depleted cells (0.71-fold ? 0.29, n=3).  Probing lysates from the same experiments with paxillin consistently revealed a lower amount of paxillin in Grb2 knockdown cells (Fig. 5.2A, lower panel), which either could be due to reduced paxillin expression or decreased paxillin solubility in the lysis buffer.  These findings suggest that Grb2 can mediate paxillin expression or availability to regulate paxillin-FAK interaction and perhaps FAK activation.  Immunoprecipitating paxillin from integrin-stimulated control and Grb2-depleted cells and probing for total phosphotyrosine reveal that integrin-induced paxillin phosphorylation is reduced, though not totally abrogated, in cells lacking Grb2 (Fig.  123 5.2C).  This is in accord with findings demonstrating that paxillin phosphorylation on Tyr31 and Tyr118 enhances its interaction with FAK and recruits it to focal adhesions (Zaidel-Bar et al. 2007).  5.3.2 Overexpression of paxillin in the absence of Grb2 rescues FAK Tyr397 but not PTP? Tyr789 phosphorylation   To determine if paxillin is regulated by Grb2 upstream of FAK activation, mCherry-labeled paxillin was overexpressed in Grb2-deficient cells.  Figs. 5.3A (top and middle panels) and 5.3B show that in the absence of Grb2, integrin-induced FAK Tyr397 phosphorylation is decreased by ~54% ? 7.7% (n=4) while exogenous paxillin restores this defect to ~89% ? 2.8% (n=4) relative to the control.  To determine whether the association of Src with FAK is rescued by the restoration of FAK-phosphoTyr397, Src was immunoprecipitated from the FN-stimulated lysates and probed for associated FAK.  It is evident that FAK is present in the Src immunoprecipitates from control and paxillin-overexpressing cells whereas in Grb2-deficient cells, FAK is only faintly detected (Fig. 5.3C), suggesting that Src binding to FAK is dependent on paxillin-mediated FAK autophosphorylation.  Interestingly, the restoration of FAK activation by exogenous paxillin did not rescue PTP??Tyr789 phosphorylation (Fig. 5.3A, bottom panel), an event downstream of activation of the Src-FAK kinase complex.  This indicates that Grb2 regulates PTP??Tyr789 phosphorylation through two separate mechanisms; one that is dependent on FAK activation and association with Src, and one that is not.     124 5.4 Different domains of Grb2 are required for PTP? and FAK tyrosine phosphorylation   To determine which domains of Grb2 are required for PTP??Tyr789 and FAK Tyr397 phosphorylation, various Grb2 functional mutants were expressed concurrently with Grb2 siRNA treatment.  A panel of Grb2 siRNA-resistant point mutants was generated using overlap extension PCR.  Each of the point mutations disrupts the binding ability of a particular functional domain of Grb2.  Fig. 5.4A depicts myc-tagged WT-Grb2, the N-terminal SH3 mutant (P49L), the C-terminal SH3 mutant (P206L), the double SH3 mutant (P49L/P206L) and the SH2 mutant (R86K).  Re-introduction of these mutants into Grb2-depleted cells (Fig. 5.4B, upper panel) shows that while the Grb2-P49L mutant was able to partially rescue 50% or more of PTP??Tyr789 phosphorylation, Grb2-P206L, -P49L/P206L and -R86K mutants maintained a ~90% reduction in phosphoPTP??that was comparable to that in Grb2-depleted cells (Fig. 5.4B, middle panel).  This suggests that the C-SH3 and SH2 domains of Grb2 are essential for PTP??Tyr789 phosphorylation.  On the other hand, all Grb2 mutants except the P49L mutant, which best rescued phosphoPTP?, appeared to effectively rescue FAK-phosphoTyr397 (Fig. 5.4B, bottom panel).  Some of these mutants (P206L, R86K) even promoted FAK Tyr397 phosphorylation to levels greater than re-expressed WT Grb2 or the control.  The corresponding amounts of phosphoFAK per unit FAK are indicated above the blots and are representative of three independent experiments.  These findings present a paradox where the N-SH3 domain is important for FAK Tyr397 phosphorylation, yet it seems to affect phosphoFAK only when there is an intact C-SH3 domain as even the double mutant was able to restore FAK Tyr397 phosphorylation.  Future investigations will be required to provide an explanation for this perplexing observation.   125 Nevertheless, it is possible to conclude from this experiment that distinct domains of Grb2 are required to promote PTP??Tyr789 and FAK Tyr397 phosphorylation, supporting the notion that FAK and PTP? phosphorylation are differentially regulated by Grb2.  To examine the effects of the Grb2 mutants on integrin-mediated cell spreading, myc-tagged WT and mutant Grb2 were expressed in Grb2-depleted cells.  The cells were then plated on FN-coated coverslips, fixed and immunostained with anti-myc antibody as well as phalloidin to identify cells that express WT- or mutant Grb2 and to probe for F-actin, respectively.  By staining the actin cytoskeleton, the shape of the spreading cells, particularly the edges, could be clearly observed.  In Fig. 5.5, WT-Grb2 and P49L-Grb2 expression restored the defective phenotype of Grb2-depleted cells.  They displayed normal appearing lamellipodia ruffles upon spreading on FN.  On the other hand, the P206L-, P49L/P206L- and R86K-Grb2 mutants exhibited fewer random pointed protrusions, but were less well-spread compared to the WT-Grb2 expressing cells.  In accord with PTP?-Y789F expressing cells demonstrating delayed cell spreading, the P206L-, P49L/P206L- and R86K-Grb2 mutant-expressing cells, which lack phosphorylated PTP?, also exhibited a similar spreading phenotype.  These findings support the idea that PTP? phosphorylation plays a key role in the regulation of cell spreading.  The tyrosine phosphorylation of PTP? is sufficient to promote a normal spreading phenotype even when FAK Tyr397 phosphorylation is down-regulated as shown by the P49L-Grb2 mutant.  Since the C-SH3 and SH2 domains of Grb2 that are required for PTP??Tyr789 phosphorylation have been shown to mediate the association between PTP? and Grb2 (den Hertog and Hunter 1996; Su et al. 1996), I speculated that the association of Grb2 with PTP? through these domains is critical for PTP??Tyr789 phosphorylation.  To verify that Grb2  126 association with PTP? is dependent on Grb2 C-SH3 and SH2 domains in MEFs, myc-tagged WT or mutant Grb2 were transfected into MEFs and the introduced Grb2 was immunoprecipitated using anti-myc antibody.  Probing myc-Grb2 immunoprecipitates for PTP? confirmed that the interaction of PTP? and Grb2 is indeed dependent on the C-SH3 and SH2 domains of Grb2 (Fig. 5.6), indicating that Grb2-dependent PTP??Tyr789 phosphorylation correlates with the physical interaction between Grb2 and PTP?.  5.5 Enhanced BCAR3 expression fails to restore PTP? Tyr789 phosphorylation under normal FAK phosphorylation   In most of the Grb2 mutant expressing cells and paxillin-overexpressing cells, PTP??Tyr789 phosphorylation was defective despite normal FAK activation, raising the possibility that PTP? may be phosphorylated in the presence of active kinase and then be rapidly dephosphorylated in the absence of Grb2 binding to protect PTP?-phosphoTyr789.  To re-examine the ability of overexpressed BCAR3 to substitute for Grb2 in such a protective role, WT- or R177K-BCAR3 and R86K-Grb2 were co-expressed in Grb2 knockdown cells.  As expected, in cells depleted of endogenous Grb2 and re-expressing mutant Grb2-R86K alone or together with BCAR3-R177K mutant, FAK-phosphoTyr397, but not PTP??Tyr789 phosphorylation, was rescued (Fig. 5.7).  In the same way, in Grb2-depleted cells re-expressing mutant Grb2 and WT-BCAR3, mutant Grb2 rescued phosphoFAK but exogenous WT-BCAR3 was unable to restore PTP?-phosphoTyr789.  This reveals that defective PTP? phosphorylation is not due to hyperdephosphorylation yet represents a continued failure, despite activated FAK, to achieve PTP? Tyr789 phosphorylation.  127 5.6 Association of FAK with PTP? is dependent on Grb2 C-terminal SH3 and SH2 domains   In order for the Src-FAK kinase complex to phosphorylate PTP? Tyr789, its activation and spatial positioning with access to PTP? are essential.  To investigate whether PTP?-associated Grb2 might be required to bring activated FAK into proximity with PTP? so as to phosphorylate Tyr789, VSVG-tagged WT-PTP? was co-transfected with non-targeting control or Grb2 siRNA into PTP?-null cells and PTP? was immunoprecipitated from the cell lysates using anti-VSVG conjugated beads.  FAK and Grb2 were detected in the immunoprecipitates from the control but not the Grb2-depleted cell lysates (Fig. 5.8), suggesting that the interaction of FAK with PTP? is dependent on Grb2.  Indeed re-expressing WT-Grb2 in the presence of Grb2 siRNA restored the interactions between PTP?, FAK, and Grb2 (Fig. 5.8), confirming that Grb2 mediates PTP?-FAK association.  By examining PTP?-FAK complex formation in Grb2 mutant expressing cells, it was further observed that the P206L-, P49L/P206L- and R86K-Grb2 mutants failed to restore PTP?-FAK association in the absence of endogenous Grb2 (Fig. 5.9A), indicating that the PTP?-binding SH2 and C-SH3 domains of Grb2 are required for FAK to complex with PTP?. This supports a model whereby PTP?-bound Grb2 functions as an anchor for FAK to complex with PTP? and suggests that this is permissive for PTP? phosphorylation by FAK.  The interaction of Grb2 with PTP? is reported to depend on Grb2-SH2 binding to PTP?-phosphoTyr789, posing the conundrum that Tyr789 phosphorylation appears to be a prerequisite for recruiting Grb2 and for Grb2-dependent FAK association that then promotes PTP??Tyr789 phosphorylation.  To confirm that PTP??Tyr789 phosphorylation is critical for  128 PTP? complex formation with Grb2 and FAK, VSVG-tagged PTP?-WT and -Y789F mutant were expressed in PTP?-null cells.  PTP? was immunoprecipitated from the cell lysates with anti-VSVG conjugated beads and probed for FAK and Grb2.  Results show that PTP?-Y789F was unable to associate with FAK or Grb2 (Fig. 5.9B), indicating that the phosphorylation of PTP? precedes Grb2 binding and the recruitment of FAK to the complex.  These findings suggest that perhaps a basal level of phosphorylated PTP? at Tyr789 is required to recruit Grb2 and Grb2-dependent FAK, where the activated FAK may then phosphorylate other molecules of unphosphorylated PTP? in the proximity, perhaps those nearby due to integrin-mediated clustering.  5.7 FAK Tyr925 is required for integrin-induced PTP? Tyr789 phosphorylation   Grb2 is known to interact with FAK-phosphoTyr925 (a site phosphorylated by FAK-bound Src) via its SH2 domain (Schlaepfer et al. 1994).  To investigate whether this phosphosite is required for FAK to complex with PTP? in a Grb2-dependent manner to promote PTP? phosphorylation, FN-stimulated cell lysates from FAK-null cells re-expressing the unphosphorylatable mutant FAK (FAK-Y925F) were probed for PTP?-phosphoTyr789.  Fig. 5.10 clearly shows that unlike FAK-Y397F expressing cells, FAK-Y925F expressing cells did not exhibit impaired integrin-induced FAK-Tyr397 phosphorylation as compared to cells reconstituted with WT-FAK, but were unable to induce PTP??Tyr789 phosphorylation.  This indicates that FAK Tyr925 is required for PTP? phosphorylation which is likely mediated through FAK Tyr925 phosphorylation-dependent association with Grb2.  Since both PTP?-phosphoTyr789 and FAK-phosphoTyr925 bind to  129 the SH2 domain of Grb2, this mechanism would require two molecules of Grb2 to link PTP? and FAK, as discussed further in section 5.8 below.    5.8 Discussion   In chapter 4, I described the identification of a novel upstream regulatory role of Grb2 in the promotion of PTP??Tyr789 phosphorylation via the Src-FAK kinase complex.  To follow up, I investigated the mechanism of Grb2-mediated FAK activation and discovered that Grb2 plays distinguishable roles in the regulation of FAK Tyr397 and PTP? Tyr789 phosphorylation.  By re-expressing various Grb2 mutants in Grb2-depleted cells, I found that the C-SH3 and SH2 domains of Grb2 required for PTP?-binding are also required for PTP??Tyr789 phosphorylation.  While these domains are dispensable for FAK autophosphorylation, and presumably its activation, they are responsible for PTP?-FAK association which is dependent on phosphoPTP?.  These findings point to a second role of Grb2 in the regulation of PTP? phosphorylation that relies on FAK interaction with PTP? that is distinct from, and likely follows, FAK activation.  This presents a paradox where phosphoPTP? is required for PTP? phosphorylation, discussed later in this section.  FAK is a known substrate of Shp2, a non-receptor PTP.  In EGF-stimulated, but not non-stimulated, breast cancer cells, Shp2 has been reported to dephosphorylate FAK at Tyr397 and promote cell migration (Hartman et al. 2013).  In Shp2-/- fibroblasts, integrin-stimulated FAK phosphorylation is reduced, rather than increased (Oh et al. 1999).  Nonetheless a delay is observed in FAK Tyr397 dephosphorylation in these cells once they are detached from the ECM (Yu et al. 1998).  All of these reports indicate that Shp2- 130 mediated dephosphorylation of FAK may be cell type- (i.e. cardiomyocytes) or stimulus-specific (i.e. EGF, IGF-1, FGFR2) (Manes et al. 1999; Rafiq et al. 2006; Marin et al. 2008; Ahmed et al. 2013).  Thus, it is not surprising that the depletion or inhibition of Shp2 in MEFs had no effect on integrin-induced FAK Tyr397 phosphorylation regardless of the presence of Grb2, suggesting that Shp2 is likely not involved in integrin-induced FAK activation.  Whether or not other FAK-targeted phosphatases, such as PTEN and LMW-PTP, are involved in the regulation of integrin-mediated FAK phosphorylation remains to be determined.  Paxillin lacks enzymatic activity yet several of its functional domains have been associated with FAK Tyr397 phosphorylation, including the focal adhesion localizing LIM2 and LIM3 domains (Brown et al. 1996), the non-focal adhesion localizing LIM1 domain, and a single LD motif that does not necessarily need to be the FAK binding LD2 or LD4 motif (Thomas et al. 1999; Hoellerer et al. 2003).  In Grb2-depleted cells, the expression and integrin-induced phosphorylation of paxillin are reduced concomitant with decreased paxillin-FAK association.  All of these factors may affect paxillin-mediated FAK focal adhesion localization which promotes FAK Tyr397 autophosphorylation.  By overexpressing paxillin in the absence of Grb2, FAK Tyr397 phosphorylation and its association with Src are restored, suggesting the likelihood that Grb2 may be modulating paxillin stability, availability or coordinating protein associations to facilitate FAK Tyr397 phosphorylation.  However, it is also possible that the restoration of integrin-stimulated FAK Tyr397 phosphorylation resulting from the excess, above normal paxillin expression may be compensating for defective paxillin-independent actions of Grb2.  131  Although heterologously expressed paxillin was able to restore integrin-induced FAK Tyr397 phosphorylation and FAK-Src complex formation in Grb2-depleted cells, it failed to rescue PTP??Tyr789 phosphorylation. This suggested that Grb2 plays a role downstream of Src-FAK complex activation to either promote Tyr789 phosphorylation or prevent PTP? dephosphorylation. The inability of overexpressed BCAR3, which like Grb2 is a PTP?-phosphoTyr789-binding protein (Sun et al. 2012), to restore detectable PTP? Tyr789 phosphorylation in Grb2-deficient cells with paxillin-remediated Src-FAK activation supports the notion that reduced PTP? phosphorylation is not due to enhanced susceptibility to phosphatase(s).  Distinguishing the functional domains of Grb2 required for PTP? and FAK phosphorylation provides further insights for the mechanisms that govern their differential regulation.  The N-SH3 domain of Grb2 is important for FAK phosphorylation under the condition of an intact C-SH3 domain.  When the binding abilities of both N- and C-SH3 domains are disrupted simultaneously, FAK phosphorylation at Tyr397 is unaffected.  However, when only the N-SH3 domain is mutated, FAK-phosphoTyr397 is impaired.  One possible explanation for this interesting observation is that N-SH3 domain mediated signaling might be required to counterbalance that mediated by the C-SH3-domain in order to promote FAK autophosphorylation.  While functional Grb2 SH2 and C-SH3 domains are dispensable for FAK Tyr397 phosphorylation, they are essential for PTP? Tyr789 phosphorylation, PTP?-Grb2 association, as well as PTP?-FAK association.  Since Grb2-SH2 interaction with PTP? is dependent upon phosphorylation of Tyr789, my results support a model whereby basal or low level of Tyr789-phosphorylated PTP? recruits Grb2 and promotes a second site of direct or indirect interaction with Grb2-C-SH3, and the PTP?- 132 bound Grb2 mediates complex formation with Src-FAK to bring active Src-FAK into proximity with other nearby, perhaps integrin-clustered, PTP? molecules to allow their phosphorylation. This prospective model is further substantiated by the requirement of FAK Tyr925, which when phosphorylated serves as a binding site for the Grb2 SH2 domain, for integrin-induced PTP? phosphorylation at Tyr789.  Grb2 can dimerize in a head-to-tail fashion through its C-SH3 and SH2 domains without conferring any self-inhibitory effects but forming a more stabilized structure (Maignan et al. 1995; McDonald et al. 2008).  As a dimer, Grb2 may connect PTP? to FAK through their respective phosphosites, Tyr789 and Tyr397, binding to the two available Grb2 SH2 domains (Fig. 5.11A).  It is possible that Grb2 links PTP? to FAK by functioning as a dimer, or that another unidentified Grb2-binding molecule anchors and positions PTP?-Grb2 and FAK-Grb2 (Fig. 5.11B).  Furthermore, it is plausible that the C-SH3 domain interaction with PTP? might enhance Grb2 dimerization which in turn stabilizes PTP? association with Grb2.  This could explain why the Grb2 C-SH3 mutant fails to interact with phosphoPTP? despite having a functional SH2 domain.  Taken together, I have identified a second role of Grb2 as a mediator of PTP? interaction with the Src-FAK kinase complex that permits PTP? Tyr789 phosphorylation. This interaction is proposed to be mediated by the actions of a Grb2 dimer or two otherwise closely linked molecules of Grb2, as such a spatial focus is envisioned to facilitate rapid signal transduction from the integrins to promote PTP? phosphorylation and its associated downstream signaling events.    actin Grb2    C           Grb2       SHP2   Grb2/Shp2   siRNA: PTP?-pY789 PTP? Shp2 FAK-pY397 FAK 0     15      0    15      0    15      0    15   FN: A actin Grb2    -             C          Grb2  siRNA: PTP?-pY789 PTP?  -      +      -      +     -      + FAK-pY397 FAK NSC 87877: B Figure 5.1  Shp2 silencing or inhibition does not rescue the effects of Grb2 depletion.  (A) MEFs  treated with control (C), Grb2 and/or Shp2 siRNA were kept in suspension (0) for 1 hour and replated on fibronectin (FN)-coated dishes for 15 min (15). Lysates were probed for Grb2, Shp2, actin, PTP?-pY789, PTP?, FAK-pY397 and FAK (n=2). (B) MEFs untreated or treated with control (C) or Grb2 siRNA were kept in suspension (0) for 1 h with or without 100 mM NSC 87877 (Shp2 inhibitor) and replated on FN-coated dishes for 15 min. Lysates were probed for Grb2, actin, PTP?-pY789, PTP?, FAK-pY397, and FAK (n=2).   133 A 0      15      0     15  C            Grb2 IP: FAK siRNA: FAK paxillin FAK?Lysate paxillin?actin?FN: B Relative Paxillin Association with FAK  C Grb2 siRNA: 2.0  1.5   1.0  0.5   0       15  * n.s. 0       15  FN: Figure 5.2  Paxillin expression and association with FAK are reduced in Grb2-depleted cells. MEFs treated with control (C) or Grb2 siRNA were kept in suspension (0) for 1 h and replated on fibronectin (FN)-coated dishes for 15 min (15). (A) Immunoprecipitates of FAK were probed with FAK and paxillin (upper panels). Cell lysates were immunoblotted for FAK, paxillin and actin (lower panels). (B) The relative amount of paxillin associated with FAK in suspended and FN-stimulated lysates from at least 3 experiments as in (A) was quantified by densitometry and is shown in the graph. Asterisks indicate significant differences (*P=0.014) and n.s. denotes non-significance.  (C) Immunoprecipitates of paxillin were probed with phosphotyrosine (pY4G10) and paxillin. Lysates were immunoblotted with paxillin and actin antibodies.   C 0      15      0     15  C            Grb2 IP: Paxillin siRNA: pY4G10 Paxillin Paxillin?actin?Lysate FN: 134 A C 0      15      0     15      0      15         C             Grb2           Grb2 siRNA: mCh-paxillin:         -                 -                 +  siRNA: mCh-paxillin: -        -       +  Src FAK IP: Src Grb2 C Grb2 B 100 80 60 40 20 siRNA: C Grb2 Grb2 mCh-paxillin:        -          -          +  ** * FAK-pTyr397 / FAK Figure 5.3  Exogenous  paxillin restores FAK activation but not PTP??Tyr789 phosphorylation in Grb2-deficient cells.  (A) MEFs were treated with control (C) or Grb2 siRNA or co-transfected with Grb2 siRNA and mCherry (mCh)-paxillin for 48 h, then starved in reduced serum medium overnight. The next day, cells were trypsinized, kept in suspension (0) for 1 h and replated on fibronectin-coated dishes for 15 min (15). Lysates were immunoblotted with Grb2, paxillin, actin, PTP?-pY789, PTP?, FAK-pY397 and FAK antibodies. Lanes 1-6 in each panel are from a single gel but the samples in lanes 3 and 5 were inadvertently exchanged on the original gel, and these lane images were spliced out and repositioned correctly in the final image shown here (indicated by the white lines). (B) The relative amount of FAK-pY397 per unit FAK in FN-stimulated lysates from at least 3 experiments as in (A) was quantified by densitometry and is shown in the graph. Asterisks indicate significant differences (*P=0.0003; **P=0.0008).  (C) Src immunoprecipitates from FN-stimulated lysates from experiments as in (A) were probed for FAK and Src. exogenous paxillin 135 actin Grb2 paxillin PTP? PTP?-pY789 FAK-pY397 FAK 1 2 3 4 5  6 A Grb2 siRNA Grb2-WT-myc Grb2-R86K-myc Grb2-P49L-myc Grb2-P206L-myc Grb2-P49L/P206L-myc SH3 SH2 SH3 myc SH2 SH3 myc SH3 SH2 myc SH2 myc SH3 SH3 myc     C      +       +      +      +       +      + Grb2 siRNA:WT:    -       -       +       -       -       -       - P49L: P206L:    -       -       -       +       -       -       -    -       -       -       -       +       -       - P49L/P206L: R86K:    -       -       -       -       -       +       -    -       -       -       -       -       -       + FAK-pY397 FAK PTP??PTP?-pY789 actin Grb2-SH2 myc B 1.0 0.6 0.9 0.7 1.3 1.0 1.4 Grb2-myc- FAK-pY397/FAK Figure 5.4  Grb2-SH 2 and -C-terminal SH 3 domains are required  for PTP??Tyr789 phosphorylation.  (A) Schematic diagram of siRNA resistant myc-tagged Grb2-WT/mutants. Silent mutations were made in the depicted region targeted by the Grb2 siRNA. The closed circles depict the domain with the functional point mutation. (B) MEFs treated with either control (C) or Grb2 siRNA duplex or co-transfected with Grb2 siRNA and Grb2-myc-WT or -mutants were kept in suspension for 1 h and replated on fibronectin-coated dishes for 15 min. Lysates were probed with Grb2-SH2, myc, actin, PTP?, PTP?-pY789, FAK-pY397 and FAK antibodies. The relative amount of FAK-pY397 per unit FAK was calculated from densitometric analysis and is shown above the FAK blots. Results are representative of three independent experiments.  136 Figure 5.5  Grb2-SH 2 and -C-terminal SH 3 domains promote cell spreading. MEFs treated with either control (C) or Grb2 siRNA duplex or co-transfected with Grb2 siRNA and Grb2-myc-WT or -mutants were kept in suspension for 1 h and replated on fibronectin (FN)-coated coverslips for 15 min. Cells were fixed and immunostained with myc (green) and phalloidin (red).  Images were taken using TIRF microscopy (n=2).  Scale bar (bottom right panel) = 20?m.  Control Grb2 siRNA phalloidin myc WT-Grb2 P49L-Grb2 P206L-Grb2 P49L/P206L-Grb2 R86K-Grb2 137 R86K: IP: Myc Lysate myc PTP? myc actin PTP??Grb2 WT:    -       -       -       -       -       +        P49L: P206L: P49L/P206L:    -      +       -       -       -       -           -       -      +       -       -       -           -       -       -      +       -       -           -       -       -       -      +       -        Grb2-myc- Figure 5.6  PTP?-Grb2 association is dependent on Grb2-SH 2 and ?C-terminal SH 3 domains. MEFs were transfected with Grb2-myc-WT or -mutants. Anti-myc immunoprecipitates from adherent cell lysates were probed for PTP? and myc. Lysates were immunoblotted with PTP?, myc, Grb2 and actin antibodies (n=3). 138    -          -    R86K  R86K   R86K?   C         +        +        +         +?   -          -        -       WT    R177K?PTP? Grb2 siRNA: Grb2-myc: PTP?-pY789 FLAG-BCAR3: Grb2 actin BCAR3 myc FAK FAK-pY397 1.00 0.10 0.22 0.25  0.23 1.00 0.57  0.91 1.05  0.88 Figure 5.7  Excess  BCAR3 is unable to rescue PTP? phosphorylation under normal FAK activation. MEFs were treated with control (C) or Grb2 siRNA or transfected with Grb2 siRNA, Grb2-R86K-myc and FLAG-BCAR3 for 48 h, then starved in reduced serum medium overnight. The next day, cells were trypsinized, kept in suspension for 1 h and replated on fibronectin-coated dishes for 15 min. Lysates were immunoblotted with PTP?-pY789, PTP?, FAK-pY397, FAK, myc, Grb2, BCAR3, and actin antibodies. The relative amounts of PTP?-pY789 per unit PTP?, and FAK-pY397 per unit FAK, were calculated from densitometric analysis and are shown below the blots.  All blots are representative of two independent experiments. 139 FAK FAK -         -       WT?Grb2-myc: VSVG-PTP?: Grb2 siRNA: C        +        +?+        +        +?VSVG?Grb2?PTP? myc actin PTP?-pY789 Grb2 VSVG IP: VSVG Lysate Figure 5.8  Grb2 is required  for PTP?-FAK association. PTP?-/- MEFs were untreated or transfected as shown with control (C) or Grb2 siRNA, myc-tagged Grb2-WT, and VSVG-WT PTP?. VSVG immunoprecipitates from adherent cell lysates were probed for FAK, Grb2 and VSVG (VSVG-PTP?). Lysates were probed for PTP?-pY789, PTP?, VSVG (VSVG-PTP?), FAK, myc, Grb2 and actin. Blots are representative of two independent experiments. Grb2-myc Grb2-myc 140 PTP? Grb2 actin PTP?-pY789 VSVG FAK Src FAK VSVG-PTP?: Src?WT  Y789F?Grb2?VSVG?IP: VSVG Lysate IP: VSVG FAK  -       WT   P49L P206L P49L/ R86K?Grb2-myc: VSVG?VSVG-WT?: Grb2 siRNA: +        +        +         +        +        +?+        +        +         +        +        +?P206L Lysate PTP? myc actin PTP?-pY789 FAK VSVG Grb2?A B Figure 5.9  PTP?-FAK association is mediated by Grb2 associaton with phosphoPTP?. (A) PTP?-/- MEFs were transfected with Grb2 siRNA, myc-tagged Grb2-WT/mutants, and VSVG-WT PTP?. VSVG immunoprecipitates from adherent cell lysates were probed for FAK, Grb2 and VSVG (VSVG-PTP?). Lysates were probed for PTP?-pY789, PTP?, VSVG (VSVG-PTP?), FAK, myc and actin (n=3). (B) PTP?-/- MEFs were transfected with VSVG-WT- and -Y789F-PTP?. VSVG immunoprecipitates from adherent cell lysates were probed for FAK, Src, Grb2 and VSVG (VSVG-PTP?). Lysates were probed for PTP?-pY789, PTP?, VSVG (VSVG-PTP?), FAK, Src, Grb2 and actin (n=1).   141 Fig. 5.10  Integrin-induced PTP? Tyr789 phosphorylation is defective in FAK-Y925F expressing cells.  FAK-null (FAK-/-) MEFs and FAK-null MEFs re-expressing wild type (WT-) or mutant (Y397F- or Y925F-) FAK were starved in 0.5%  FBS DMEM overnight.  Cells were then trypsinized, put into suspension (0) for 1 h and replated on fibronectin (FN)-coated plates for 15 min. Lysates were separated on SDS-PAGE and immunoblotted with FAK-pY397, FAK, PTP?-pY789, PTP?, Grb2 and actin antibodies (n=2). PTP? PTP?-pY789 FAK-pY397   0      15  FAK: FAK Y397F WT -/- Y925F  Actin Grb2   0     15    0     15    0     15  142 FN: Fig. 5.11  Models  of Grb2 interactions in the regulation of integrin-induced PTP? Tyr789 phosphorylation. Upon  integrin stimulation, basal level of  phosphorylated PTP? Tyr789 serves as a binding site for the SH2 domain of Grb2.  Grb2 may either be recruited as a monomer or (A) as a dimer which can link PTP? to FAK through their respective phosphosites, Tyr789 and Tyr397. (B) Grb2 may also connect PTP? to FAK through an unidentified protein (X)  as shown.  The linkage between PTP? and FAK brings the Src-FAK complex into proximity with other nearby unphosphorylated PTP? molecules, thus facilitating Src-FAK complex-mediated PTP? phosphorylation at Tyr789.  ?? ??PTP??FAK?P P Src P P FN??? ??P ?? ??PTP??FAK?P P Src P P FN??? ??P A B X  143  144  Discussion and Future Directions Chapter 6:  6.1 The interplay between BCAR3, Grb2 and PTP? in integrin signaling   In the present study, I have described the functional roles of two PTP?-phosphoTyr789 interacting partners, BCAR3 and Grb2, in integrin signaling.  BCAR3 associates with phosphoPTP? to recruit Cas to focal adhesions and promote Cas-mediated downstream signaling.  Grb2, on the other hand, links phosphorylated PTP? to the Src-FAK complex to generate maximal PTP? phosphorylation.  I also demonstrated that this small 25 kDa adaptor protein plays an early upstream role in the activation of FAK, likely through paxillin.  While both BCAR3 and Grb2 can associate with phosphoPTP? within the first 15 min of fibronectin stimulation and their interactions have been shown to be mutually exclusive (Sun et al. 2012), the mechanism(s) that governs their differential interactions with phosphoPTP? is unclear.  In this chapter, I will discuss the potential dynamic interplay between BCAR3, Grb2 and phosphoPTP? and propose a model whereby these molecules may co-operate with other focal adhesion proteins to mediate integrin-induced cytoskeletal re-organization and cell motility.  6.1.1 Regulation of integrin-induced PTP? Tyr789 phosphorylation by BCAR3 and Grb2   PTP? is a receptor-like tyrosine phosphatase that is best known for its role as a SFK activator.  Dephosphorylation of Src Tyr527 by PTP? disrupts the autoinhibitory conformation and exposes the kinase domain of Src, enabling the phosphorylation of other  145 focal adhesion proteins by this kinase (Ponniah et al. 1999; Su et al. 1999).  Active Src associates with autophosphorylated FAK at Tyr397 and phosphorylates tyrosine residues in the catalytic domain of FAK to fully activate it (Hanks et al. 2003).  Together, the optimally activated Src-FAK kinase complex phosphorylates Tyr789 of PTP??(Chen et al. 2006) which is four amino acids away from the C-terminus.  Several years ago, our laboratory identified a requirement for this phosphosite in integrin-mediated cell migration downstream of active Src-FAK (Chen et al. 2006).    Normally, approximately 20% of PTP? is constitutively phosphorylated at Tyr789 and most of it is bound to the SH2 domain of Grb2 (den Hertog et al. 1994).  The phosphorylation of Tyr789 and Grb2-PTP? association can be induced by integrin-ECM ligation.  Based on my results, the binding of Grb2 to PTP? is not required for the protection of phosphoTyr789 from dephosphorylation but is essential for the promotion of Src-FAK-mediated PTP? Tyr789 phosphorylation.  Both the SH2 domain and the C-SH3 domain of Grb2, which interacts with the C-terminal region of the D1 catalytic domain of PTP?, are required for PTP?-FAK complex formation, suggesting that these two interacting domains of Grb2 may function to link FAK, likely in complex with Src, to phosphorylated PTP?? This in turn is postulated to initiate the phosphorylation of other proximal, perhaps integrin-clustered, molecules of PTP??to rapidly increase the population of phosphorylated PTP??(Fig. 6.1D).  Aside from the direct interaction of Grb2 with phosphoPTP?, I also showed that Grb2 can regulate paxillin-mediated activation of FAK, which is important for PTP? Tyr789 phosphorylation (Fig. 6.1B).  FAK-null cells or FAK-Y397F cells, in which FAK cannot be autophosphorylated and is thus unable to interact with Src, display defective phosphorylation of PTP? Tyr789.  146  While Grb2 mediates PTP? Tyr789 phosphorylation through the regulation of FAK, BCAR3 alters PTP? Tyr789 phosphorylation via its regulation of Src. In BCAR3-depleted cells, Src activity is reduced (Schuh et al. 2010), however, the detailed mechanism of BCAR3-regulated Src activation is unclear as BCAR3 lacks the catalytic property of a kinase or a phosphatase to directly activate Src.  In accord with Schuh et al. (2010) we observed a reduction in Src Tyr416 phosphorylation in BCAR3-depleted MEFs (Sun et al. 2012), indicative of impaired Src activation.  Furthermore, we found that these BCAR3-deficient cells exhibit a ~50% reduction in the phosphorylation of PTP? Tyr789 upon integrin stimulation (Sun et al. 2012).  Since active Src, in complex with FAK, phosphorylates PTP??Tyr789 (den Hertog et al. 1994; Chen et al. 2006), reduced Src activity compromises the phosphorylation of Tyr789.  Nevertheless, the loss of BCAR3 does not totally abrogate Src activity, suggesting that another mechanism (i.e. PTP?-mediated Src activation) might be functioning in concert with BCAR3 to promote Src activation and subsequent PTP? phosphorylation (Figs. 6.1A, C).  Since the dephosphorylation of Src inhibitory phosphoTyr527 and phosphorylation of Src Tyr416 are associated with Src activation, it is possible that Src requires both PTP?-dependent dephosphorylation of phosphorylated Src at Tyr527 and BCAR3-dependent phosphorylation of Src Tyr416 to achieve optimal Src activity.  In contrast to the depletion of BCAR3, the depletion of Grb2 almost completely abrogates PTP? Tyr789 phosphorylation.  Indeed, PTP??Tyr789 phosphorylation is ~90% reduced in Grb2 SH2 or C-SH3 mutant expressing cells where PTP?-Grb2 association is undetectable, suggesting that Grb2-mediated positioning of the Src-FAK complex is also essential for PTP??phosphorylation.     147 6.1.2 Roles of PTP?, BCAR3 and Grb2 in integrin-mediated cell migration   Cell migration involves a sophisticated network of tightly controlled signaling events to produce a highly coordinated response.  The spatiotemporal regulation of intracellular molecules generates a directed movement which requires the forward protrusion of the cell membrane, and tail retraction via focal adhesion disassembly.  Unlike the simplified model where signaling events are perceived to proceed through a linear axis, more comprehensive models depict an intricate signaling network which highlights the interdependence of multiple signaling processes (Zaidel-Bar et al. 2007).  Results presented in this thesis dissertation clearly demonstrate the complexity of protein-protein interactions and post-translational modification within the context of integrin-induced cell migration.  I have only focused on elucidating a fraction of the PTP?-dependent integrin-proximal events that govern cell motility.  Here, I will discuss how these upstream signaling events may contribute to cytoskeletal re-organization and ultimately cell migration.  One of the many early events that regulates integrin-induced cell migration is the phosphorylation of receptor PTP? at Tyr789.  In PTP?-null cells expressing PTP?-Y789F, cell migration is delayed concomitant with impaired actin stress fiber assembly and focal adhesion formation (Chen et al. 2006), suggesting that PTP??Tyr789 phosphorylation, while not absolutely required, functions to promote cell motility.  PTP?-Y789F-expressing cells display reduced Cas tyrosine phosphorylation, and in many instances, the latter defect has been shown to inhibit Crk-coupled cell migration [Shin, 2004; Chodniewicz, 2004; Klemke, 1998]. Crk is an adaptor protein that links Cas to the downstream small GTPases Rac1 and Rap1 to regulate cell adhesion and actin cytoskeleton organization [Cho, 2002; Sakakibara, 2002].  148  BCAR3 is an intermediate that recruits Cas to phosphoPTP? to facilitate Cas-mediated signaling.  Interestingly, as discussed above, BCAR3 also functions to promote Src activation which is necessary for PTP??Tyr789 phosphorylation.  These findings reveal the interdependence between PTP?, BCAR3 and Src.  Both PTP? and BCAR3 are required for the full activation of Src (Ponniah et al. 1999; Su et al. 1999; Schuh et al. 2010).  Active Src then functions in conjunction with FAK to phosphorylate PTP? Tyr789 (Chen et al. 2006), creating a binding site for the SH2 domain of BCAR3 (Sun et al. 2012).  The interaction between BCAR3 and PTP?-phosphoTyr789 recruits Cas to focal adhesions where Cas can interact with membrane-bound Src, enabling Src-mediated Cas SD phosphorylation and downstream signaling events.    In 2006, Sawada et al. proposed that the N-terminal SH3 domain and the C-terminal region of Cas provide two distinct sites that anchor Cas in cytoskeleton-adhesion complexes, and the two anchorage points facilitate the physical stretching of the Cas SD domain and thus increase its susceptibility to phosphorylation by Src.  Since the GEF domain of BCAR3 is known to constitutively interact with the CCH domain of Cas (Riggins et al. 2003) and the SH2 domain of BCAR3 binds to PTP?-phosphoTyr789, recruiting Cas to the plasma membrane, we hypothesized that BCAR3 acts as the C-terminal anchor of Cas at focal adhesions by docking onto PTP??(Sun et al. 2012).  On the other hand, FAK can interact with the SH3 domain of Cas (Polte and Hanks 1995; Hanks and Polte 1997) and may act as the N-terminal anchor of Cas.  This dual anchorage facilitates force-induced Cas SD stretching and phosphorylation.  The interaction between FAK and Cas has been shown to promote phosphorylation of the SD of Cas (Fonseca et al. 2004).  Since FAK exhibits relatively weak catalytic activity toward Cas as compared to Src while a Src-FAK complex  149 effectively phosphorylates the SD of Cas (Ruest et al. 2001), this indicates that FAK, rather than directly phosphorylating Cas, is recruiting Src through its autophosphorylated Tyr397 to promote Cas SD phosphorylation.  As discussed above, PTP?-BCAR3 promotes Cas recruitment to the focal adhesions where Cas can also directly interact with Src to facilitate Cas SD phosphorylation.  Indeed, the phosphorylation of Cas SD via direct Src binding to Cas might play a more important role than indirect FAK-linked association of Src with Cas. A schematic diagram shown in Fig. 6.2B depicts the mechanism by which PTP? phosphoTyr789 coordinates the positioning and SD phosphorylation of Cas upon integrin stimulation.  Like BCAR3, Grb2 also regulates PTP? Tyr789 phosphorylation and interacts directly with this phosphosite.  Indeed, the binding of Grb2 to phosphoTyr789 of PTP? likely precedes the binding of BCAR3 to PTP? because in the absence of Grb2, PTP? Tyr789 phosphorylation is abolished and without this SH2 domain binding site, BCAR3 is unable to interact with PTP?.  This suggests that BCAR3 binding to phosphoTyr789 of PTP? is dependent on Grb2-mediated PTP? Tyr789 phosphorylation.  As shown in Fig. 6.2A, Grb2-mediated PTP?-FAK complex formation is proposed to initiate subsequent phosphorylation of additional molecules of PTP? that are not bound to Grb2 and are therefore available to bind BCAR3 once phosphorylated.  Future studies will be required to understand the underlying factors and conditions that govern the interactions between PTP? and BCAR3 or Grb2.  Since PTP? Tyr789 plays a role in cell migration and Grb2 is the predominant species (den Hertog et al. 1994) that is bound to this phosphosite, it is logical to hypothesize that Grb2 plays a part in PTP? Tyr789-mediated cell migration.  However, the role of Grb2  150 in cell migration is debatable.  Fusing a focal adhesion targeting (FAT) sequence to Grb2 shows that Grb2 localization to focal adhesions does not stimulate cell migration on fibronectin but promotes cell cycle progression (Shen and Guan 2001).  This is in accord with the role of Grb2 binding to Src-phosphorylated FAK Tyr925 to activate Ras and the ERK/MAPK signaling pathway (Schlaepfer et al. 1998) that is linked to cell proliferation.  Conversely, several reports have suggested a role for FAK Tyr925 in FA turnover and cell migration as Y925F-FAK expressing cells exhibited larger FAs, decreased FA disassembly rate and reduced cell migration (Katz et al. 2003; Deramaudt et al. 2011).  Tyr925 of FAK lies within the FAK FAT domain which also contains the binding site for paxillin (Liu et al. 2002).  Upon Tyr925 phosphorylation, Grb2 interacts with FAK while paxillin dissociates from FAK (Tachibana et al. 1995; Hayashi et al. 2002; Gao et al. 2004).  This switch between FAK association with paxillin to FAK association with Grb2 is thought to promote FAK dissociation from the focal adhesions and FA turnover.  This is in accord with the atypical spreading phenotype of Grb2-deficient cells which show abnormal pointed membrane protrusions, demonstrating defective FA turnover (Fig. 5.5).    The FAK-Grb2 interaction may also regulate Cas-mediated cell migration as the mutation of FAK Tyr925 to Phe reduces Cas tyrosine phosphorylation and Rac1-Dock180 association, suggesting a role for FAK Tyr925 in the Cas-Dock180-Rac1 signaling axis (Deramaudt et al. 2011).  In view of my results and the experimental model proposed in Chapter 5, the reduction in Cas tyrosine phosphorylation caused by the FAK-Y925F mutation could be explained by the loss of Grb2-linked PTP?-FAK association that reduces FAK-Src-catalyzed PTP? phosphorylation at Tyr789.  The reduction in PTP? Tyr789 phosphorylation means that there is less PTP? phosphoTyr789 available for BCAR3 binding,  151 resulting in defective BCAR3-mediated localization of Cas to focal adhesions and Cas phosphorylation.  The different roles of FAK-associated Grb2 suggest that there are at least two distinct pools of FAK-Grb2 complexes, one that is bound to Sos to mediate Ras-dependent signaling and another that is bound to phosphoPTP? via a Grb2 dimer or a Grb2-linked complex to promote PTP? Tyr789 phosphorylation that is upstream of Cas phosphorylation.    Overall, my work supports the notion that BCAR3 and Grb2 play important roles in the upstream regulation of integrin-mediated PTP? Tyr789 phosphorylation and PTP? Tyr789-dependent cell migration via their interplay with FAK, Cas and Src. The schematic diagram in Fig. 6.3 illustrates the web of connections between these major proteins of interest, PTP?, BCAR3 and Grb2, and the key focal adhesion proteins, FAK, Src and Cas, in integrin-mediated signaling.  While some of the binding interactions, and the phosphorylation or activation links shown in the diagram are previously known, some are newly identified through this study.  My results highlight the cyclical interdependence of PTP?, BCAR3, Grb2, FAK, Cas and Src in integrin signaling, showing that these proteins act both upstream and downstream of each other.  This is in line with current signaling models that feature high levels of connectivity between adhesion components, forming an intricate and growing protein signaling network (Zaidel-Bar et al. 2007; Zaidel-Bar and Geiger 2010).    6.1.3 A potential PTP?-Grb2-FAK complex mediator   In Chapter 5, I showed that PTP?-FAK complex formation is dependent on Grb2.  Since the binding of PTP? or FAK to Grb2 each requires the SH2 domain of Grb2 and since   152 each Grb2 molecule only has a single SH2 domain, I proposed a model whereby PTP? and FAK are linked by a Grb2 dimer or via an unidentified protein that clusters two Grb2 molecules to bring Grb2-bound PTP? and Grb2-bound FAK into close proximity.  The recruitment of FAK to PTP? can then facilitate Src-FAK mediated-PTP? tyrosine phosphorylation.  Grb2 can form a dimer in solution [Maignan, 1995; McDonald, 2008] and the existence of a Grb2 dimer in a physiological context has recently been shown in human embryonic kidney 293T (HEK293T) cells (Lin et al. 2012).  While this is supportive of the model that I proposed, it does not eliminate the possibility that the two molecules of Grb2 needed to interact with PTP? and Grb2 are themselves linked by an intermediate protein.  In this case, the unknown protein should possess properties that will allow it to interact with the C-terminal SH3 (C-SH3) domain of Grb2 since the SH2 domain of each of the two Grb2 molecules is occupied by PTP?-phosphoTyr789 or FAK-phosphoTyr925 and the N-terminal SH3 domain of Grb2 is not required for PTP?-FAK association as shown in Fig. 5.9A.  Two proteins, Gab1 (a scaffolding protein) and N-Wasp (a cytoskeletal protein), can interact with the C-SH3 domain of Grb2 (Carlier et al. 2000; Lewitzky et al. 2001).  Although both of these proteins were undetected in the eluent from the PTP? phosphopeptide column as discussed in Chapter 3, this does not rule out the possibility that they associate with PTP? via Grb2 because the conditions or phosphopeptide matrix used in the affinity chromatography might not be ideal for detecting interactions that occur in vivo.  Thus, it might be worthwhile to investigate complex formation between PTP?, Grb2 and N-Wasp or Gab1 upon integrin stimulation using co-immunoprecipitation.  Indeed, McDonald et al. (2011) have recently shown that the C-SH3 domain of Grb2 can bind to two distinct RXXK motifs within the Gab1 proline-rich (PR) region.  In a follow-up study, they further  153 demonstrated that Gab1 interacts with Grb2 in a 1:2 stoichiometric ratio, raising the possibility that two molecules of Grb2 could indeed be linked by a Gab1 intermediate (McDonald et al. 2012).  Formation of the Gab1-Grb2 signaling complex is mediated by the binding of the C-SH3 domain within each of two molecules of Grb2 to two distinct regions in the PR region of Gab1.  Thus, Gab1 is an attractive candidate for further investigation as an important component of the PTP?-Grb2-FAK signaling complex.  Alternatively, a screen can be performed to identify other potential PTP?-Grb2-bound proteins using a combination of chemical cross-linking and mass spectrometry (MS) (Sinz 2006; Leitner et al. 2010).  For example, tagged PTP? (i.e. VSVG-PTP?) can be expressed in PTP?-null cells and chemically cross-linked prior to cell lysis to capture stable and transient interactions.  Tagged PTP? can then be purified and its associated components can be identified using MS.  The list of proteins generated from MS can be compared to the list of Grb2-interacting proteins recorded in public protein-protein interaction databases.  The proteins that are found in both of these lists represent potential candidates for further testing in order to validate their association with PTP?-bound Grb2 and FAK-bound Grb2.  6.2 PTP?, BCAR3 and Grb2 in cancer signaling   PTP? was first considered oncogenic when its overexpression in rat embryo fibroblasts led to persistent Src activation concomitant with cellular transformation and tumorigenesis (Zheng et al. 1992; Pallen 1993).  Later, elevated levels of PTP? were found to be associated with late stage colon carcinomas, gastric cancer, and low tumor grade, estrogen receptor-positive (ER+) breast cancers (Tabiti et al. 1995; Ardini et al. 2000; Wu et  154 al. 2006).  Most of these studies have attributed the tumorigenicity of PTP? to its ability to dephosphorylate and activate Src.  However, this is conceptually contradictory since high expression of PTP?, indicative of elevated Src activity, is correlated with low tumor grade rather than high tumor grade breast cancer.  Transfecting PTP? into the breast cancer cell line MCF-7 ER+ and subcutaneously injecting these PTP?-overexpressing cells into immune-deficient mice reduces cell proliferation and delays metastasis, respectively (Ardini et al. 2000), suggesting that PTP? can reduce tumor aggressiveness.  On the other hand, the treatment of ER-negative (ER-) breast cancer and colon cancer cells, but not ER+ breast cancer cells, with PTP?-targeting siRNA resulted in reduced Src kinase activity, suppressed anchorage-independent growth and induction of apoptosis (Zheng et al. 2008).  These findings indicate that PTP? may contribute to tumor progression through different mechanisms depending on the cellular context.  Indeed, a group has examined several PTPs, including SH-PTP1, SH-PTP2, PTP1B, LAR, Cdc25B and PTP?? and found that none of these PTPs are responsible for activating Src in breast cancer cells with remarkably elevated Src kinase activity (Egan et al. 1999).  This suggests that PTP? is not always the main Src activator in all breast cancers.  Another recent study shows that PTP? can regulate the Akt/PI3K pathway to mediate tumor initiation and maintenance in human epidermal growth factor receptor 2 (HER2)-positive breast cancers (Meyer et al. 2013).  PTP?-dependent activation of the Akt/PI3K pathway may or may not be dependent on Src activation, thus there might be multiple mechanisms by which PTP? modulates cancer cell signaling.     Cancer cell adhesion plays an important role in tumor progression and metastasis.  While PTP? might be promoting anchorage-independent growth through the activation of Src in some cancer cells, it might be promoting cell adhesion in other cancer types.  PTP?  155 overexpression results in Src dephosphorylation in the human epidermoid carcinoma cell line A431 and increased cell-substratum adhesion (Harder et al. 1998).  This is in accord with another report showing that the expression of PTP? regulates focal adhesion and stress fiber formation in colon cancer cells through Src activation to promote cytoskeletal contractility (Krndija et al. 2010).  Additionally, this study confirms a role for PTP? in response to extracellular forces (i.e. substrate stiffness) to regulate cancer cell signaling.  This is in accord with this role of PTP? in untransformed fibroblasts (von Wichert et al. 2003; Jiang et al. 2006). Since our results support a physical connection between PTP? and another mechanotransduction protein, Cas, via BCAR3, and both Cas and BCAR3 are highly expressed in breast cancers, we hypothesized that the PTP?-BCAR3-Cas signaling module may participate in cancer cell signaling.  Preliminary results from our laboratory have revealed a role for this signaling complex in the migration and invasion of a rhabdomyosarcoma cell line that also expresses relatively high levels of Cas and BCAR3.  Further investigation of the role of this complex in various breast cancer cell lines, including the low tumor grade ER+ MCF-7 line and the more aggressive ER- BT-549 and MDA-MB-231 lines, is underway.  As the expression and interaction of BCAR3 and Cas have been associated with anti-estrogen resistance (Brinkman et al. 2000; Cai et al. 2003), the PTP?-BCAR3-Cas signaling complex may also function in conferring anti-estrogen resistance in breast cancer cells.  Not only BCAR3, but also Grb2 can interact with PTP?-phosphoTyr789.  Huang et al. (2011) have identified a splice mutant of PTP? termed RPTP?245 that is present in some colon, breast, and liver tumors.  RPTP?245 is a truncation mutant that only encodes the first 245 residues of the full length PTP? protein.  It therefore has extracellular and  156 transmembrane domains, but lacks both D1 and D2 catalytic domains and hence has no enzymatic activity.  However, its expression increases PTP?-mediated Src activation and induces formation of tumors in vivo.  Ectopically expressed RPTP?245 heterodimerizes with endogenous PTP? and the expression of RPTP?245 correlates with reduced Grb2 binding to phosphoPTP?-Tyr789, suggesting that RPTP?245 binding to PTP? diminishes PTP? C-terminal tail accessibility for Grb2 binding.  The inverse correlation between RPTP?245-modulated Grb2-PTP? association and Src activation also implies a negative regulatory role for PTP?-associated Grb2 in tumor cell signaling.  Future work is necessary to focus on the functionality of PTP?-phosphoTyr789 in different cancer models and determination of how its association with different binding partners affects tumor formation and progression.  6.3 Conclusion   In summary, I have identified a novel role of Grb2 as an upstream regulator of integrin-induced PTP??Tyr789 phosphorylation through its two actions in paxillin-mediated FAK activation and as an adaptor linking FAK to phosphoPTP?.  In collaboration with my colleague Guobin Sun, we have also discovered a novel signaling complex, PTP?-BCAR3-Cas, that is formed downstream of PTP? Tyr789 to modulate integrin-mediated cell migration.  It would be of great interest to dissect the molecular mechanisms governing the specific binding of phosphoPTP?-interacting proteins that may result in differential regulation of cell migration in normal tissue.  The signal transduction mechanisms in a normal cell model can then be compared to a metastatic cancer cell model to better understand the molecular defects underlying aberrant migration that contributes to cancer  157 progression.  This invaluable knowledge may ultimately provide insights for the development of effective molecular treatments to combat cancer.                                Figure 6.1  Regulation  of PTP??Tyr789 phosphorylation by BCAR 3 and Grb2.  A schematic diagram showing the different pathways that regulate PTP? Tyr789 phosphorylation. (A) represents the direct dephosphorylation of Src Tyr527 catalyzed by PTP?, resulting in Src activation as indicated by Tyr416 phosphorylation.  (B) shows Grb2-mediated activation of FAK via paxillin.  The exact molecular events in this pathway are unclear, thus they are represented by a dotted line, meaning that their interactions/relations may be indirect.  (C) depicts the activation of Src by BCAR3 through an unknown mechanism.  Activated FAK as represented in (B) may interact with active Src (from (A) and/or (B) and/or other pathways) to form the Src-FAK complex that phosphorylates PTP? at the C-terminal tail Tyr789, creating an SH2-domain binding site for Grb2. (D) PTP?-bound Grb2 is proposed to form a dimer (or Grb2 molecules are potentially juxtaposed via an unknown (X) protein), wherein one molecule of Grb2 interacts with FAK-phosphoTyr925, and another Grb2 molecule binds to PTP?-phosphoTyr789, thus positioning FAK close to other integrin-clustered PTP? molecules to promote PTP??phosphorylation.  Paxillin?FAK?FAK?Src Src BCAR3  PTP??Src P A B C D P P P P P Tyr416 Tyr397 Tyr416 Grb2  Grb2  Tyr925 Tyr789 Tyr416 Tyr397 Grb2  PTP??P X  PTP??PTP??PTP??P 158 Figure 6.2  Model  of phosphoPTP?-associated-Grb2- and -BCAR 3-mediated cell migration.  (A) Grb2 interacts with phosphoTyr789 of PTP? via its SH2 domain, and via a secondary binding through its SH3 domain.  Grb2 binds to phosphoPTP? as a dimer or as two independent monomers linked by an unknown protein (X), bringing activated FAK (through phosphoTyr925) and Src into proximity with PTP? where the Src-FAK kinase complex phosphorylates other nearby integrin-clustered PTP? molecules.  (B) The newly phosphorylated Tyr789 residues of PTP? then serve as docking sites for SH2-domain containing proteins, such as BCAR3. The GEF domain of BCAR3 is constitutively bound to the C-terminal homology domain of Cas, thus recruiting Cas to focal adhesions (FAs).  FAK may also interact with the N-terminal SH3 domain of Cas. These N- and C-terminal Cas interations form a two-pronged anchor for Cas at FAs. This provides an opposing traction force that facilitates Cas substrate domain (SD) stretching and phosphorylation by Src.  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Wild type (PTP?+/+), PTP?-null (PTP?-/-), and PTP?-null MEFs transfected with VSVG-tagged WT-, Y789F- or Y789E-PTP? were kept in suspension for 1 h and replated on fibronectin (FN)-coated plates for 15 min. (A) Lysates were immunoblotted with PTP?-pY789, PTP?, VSVG and actin antibodies. (B) FN-stimulated cells were harvested in a mild buffer to extract the cytosolic proteins as the soluble fraction (S) and subsequently in RIPA buffer to extract the cytoskeletal proteins as the insoluble fraction (I).   Lysates were probed with PTP? and RhoGDI? antibodies. (C) The percentage of total PTP? in the insoluble fraction from 3 experiments was quantified by densitometry.  Asterisks indicate significant differences (*P=0.016; **P=0.006). Appendix 194 

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