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CD44 signaling in T cells leading to cell spreading and its regulation by CD45 Wong, Nelson Kwan Yin 2006

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CD44 SIGNALING IN T C E L L S L E A D I N G TO C E L L SPREADING A N D ITS R E G U L A T I O N B Y CD45 By NELSON K W A N YIN WONG B . S c , The University of Toronto, 1992 M . S c , The University of Western Ontario, 1998 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Microbiology & Immunology)  THE UNIVERSITY OF BRITISH C O L U M B I A February, 2006 © Nelson Kwan Y i n Wong, 2006  Abstract CD44 is a widely expressed adhesion molecule that has been implicated in mediating cellular signaling. In this dissertation, the signaling pathway initiated by CD44 that leads to actin rearrangement and cell spreading in T cells was studied. The results indicate that engagement of CD44 leads to actin-dependent clustering of this adhesion molecule. CD44 clustering then initiates the recruitment of signaling proteins, including the Src-family kinases (SFK) Lck and Fyn, phosphatidylinositol-3 kinase (PI3K), and non-receptor related focal adhesion kinase Pyk2. The outcome of actin rearrangement and cell spreading resultant of CD44 signaling was determined by CD45, a transmembrane tyrosine phosphatase. In the absence of CD45, elongated cell spreading and F-actin polymerization along the longitudinal axis of the cells were observed. This was accompanied by the accumulation of tyrosine phosphorylation at CD44 microclusters. Moreover, Pyk2 phosphorylation was also associated with the CD44induced elongated cell spreading. The CD44-induced signaling pathway that leads to Pyk2 phosphorylation and elongated cell spreading involves the activities of SFK, phospholipase C (PLC), and phosphatidylinositode-3-kinase (PI3K), as well as actin polymerization and calcium mobilization. These signaling components identified are also involved in T-cell receptor (TCR)/CD3 signaling, which is initiated during T cell activation; however, the CD44 pathway was distinct. This was supported by the observations that L A T (linker for activation of T cells) phosphorylation and E R K activation were not involved in CD44 signaling, while these events are observed in TCR/CD3 signaling. In the presence of CD45, BW5147 T cells formed F-actin rings and spread round on immobilized CD44 antibody. The formation of F-actin structures in CD45 BW5147 T cells also required Src family kinase activity. Results from confocal +  microscopy studies suggest that CD45 was recruited to CD44 microclusters and this was associated with the prevention of sustained Lck activation. Overall, this work shows that CD44 mediates signals that result in actin reorganization and cell spreading in T cells; however, the outcome of these events is regulated by CD45. This is likely due to the negative regulatory effect of CD45 on SFK during CD44 signaling.  ii  T A B L E OF CONTENTS Abstract  ii  Table of Contents  ' iii  List of Tables  viii  List of Figures  ix  List of Abbreviations  xii  Acknowledgments  xiv  Dedication  xv  C H A P T E R 1: Introduction  1  1.1  Adhesion molecules and the immune system  1  1.1.1  Cellular Migration  3  1.1.2  Antigen Presentation  8  Major classes of adhesion molecules  12  1.2.1  Selectins  12  1.2.2  Integrins  15  1.2.3  Cadherins  16  1.2.4  Immunologlobulin superfamily adhesion molecules  18  1.2  1.3  1.4  1.5  CD44  19  1.3.1  Discovery  19  1.3.2  Structure  20  1.3.3  CD44-hyaluronan interaction  23  1.3.4  Biological functions of CD44  24  1.3.5  CD44 signaling  30  CD45  32  1.4.1  Structure  32  1.4.2  CD45 in T cell development  34  1.4.3  CD45 in T cell activation  34  1.4.4  CD45 in other signaling events  36  Thesis objectives  36  iii  C H A P T E R 2: Materials and methods  37  2.1  37  2.2  Materials 2.1.1  Cell culture  37  2.1.2  Reagents  37  Methods  39  2.2.1  Flow cytometry  39  2.2.2  Cell spreading assay  39  2.2.3  Immunoprecipitation  40  2.2.4  Western blotting  41  2.2.5  Labeling for confocal microscopy  42  2.2.6  Image collection with confocal microscopy  43  2.2.7  Image processing  43  C H A P T E R 3: CD44 proximal signaling associated with actin rearrangement  45  and elongated cell spreading in T cells 3.1  Introduction  45  3.2  Results  45  3.2.1  CD44 mediates cell spreading of T cells  3.2.2  The cytoplasmic domain of CD44 is required for mediating cell spreading  3.2.3  45  49  CD44-induced elongated cell spreading requires extracellular calcium, phospholipase C (PLC), and phosphoinositide-3kinase (PI3K)  3.2.4  49  Cytoskeletal reorganization is required early during elongated cell spreading  3.2.5  54  Induced tyrosine phosphorylation of P L C y l is associated with elongated cell spreading  3.2.6  58  CD44-induced Pyk2 phosphorylation requires extracellular calcium, PI3K, PLC, and actin polymerization  3.2.7  60  Cas/HEF is transiently phosphorylated during elongated cell spreading  62  iv  3.3  3.2.8  CD44 signaling is unique from CD3 signaling  65  3.2.9  CD44 and LFA-1 signaling pathways are similar  67  Discussion  70  3.3.1  Data summary  70  3.3.2  CD44 engagement, cytoskeletal rearrangement, and cell spreading  70  3.3.3  CD44 and lipid rafts  71  3.3.4  CD44 signaling and calcium mobilization  72  3.3.5  P L C y l recruitment, tyrosine phosphorylation, and activation  74  3.3.6  The role of the cytoskeleton in CD44-induced cell spreading, polarity, and signaling in T cells  75  3.3.7  PI3K and cell polarization  76  3.3.8  Pyk2 activation and effector function  77  3.3.9  Cas/HEFl activation and effector function  78  3.3.10 Role of CD45 in CD3, CD44, and LFA-1-mediated cell spreading and random migration  79  C H A P T E R 4: CD45 is a regulator of CD44-induced actin rearrangement  81  through its regulation on Src-family kinases 4.1  Introduction  81  4.2  Results  81  4.2.1  Activated thymocytes from C57/B6 and CD45 knockout mice also displayed different cell spreading morphologies  4.2.2  Receptor tyrosine phosphatase-alpha (PTPa) influences CD44-mediated cell spreading  4.2.3  82  CD44-mediated cell spreading of CD45 and CD45" T cells is +  associated with different actin rearrangement 4.2.4  81  86  SFK activity is required for CD44-mediated actin polymerization in, CD45 or CD45" T cells, but Lck activity is +  sustained in CD45" T cells  88  v  4.2.5  CD44 is recruited into microclusters during CD44-mediated cell spreading in an actin-dependent manner  4.2.6  91  Src-family kinase are differentially recruited into microclusters during CD44-mediated cell spreading in CD45  +  versus CD45" T cells 4.2.7  96  CD45 inhibited the accumulation of tyrosine phosphorylation at CD44 microclusters  96  4.2.8  CD45 co-localizes with CD44 microclusters  99  4.2.9  Inhibition of PI3K leads to F-actin ring formation in CD45" T cells  99  4.2.10 Recruitment of the p85 subunit of PI3K is not inhibited in the presence of CD45  102  4.2.11 Pyk2 recruitment to CD44 microclusters is independent of CD45  105  4.2.12 The chemokine SDF-1 induces segregation of CD45 and CD44 4.3  105  Discussion  112  4.3.1  Data Summary  112  4.3.2  Role of protein tyrosine phosphatase alpha (PTPct) in cell spreading and migration  4.3.3  113  Role of CD45 in CD44 and CD3/TCR signaling through its regulation on SFK  115  4.3.4  CD45 translocation  117  4.3.5  The role of Lck and Fyn in CD44 signaling and T cell activation  119  4.3.6  Lck and Fyn activation  120  4.3.7  CD44 signaling and actin rearrangement  122  4.3.8  CD44 signalosome  123  4.3.9  Association of signal strength and duration with functional outcomes  125  vi  C H A P T E R 5: Summary and Perspectives  126  5.1  CD44 signaling pathway  126  5.2  CD44 as a co-stimulatory molecule  129  5.3  CD44 and lymphocyte migration  131  5.4  CD45 as a regulator of signal strength and duration  133  5.5  Different pools of SFK  134  5.6  Future experiments and conclusion  134  C H A P T E R 6: References  138  vii  List of Tables Table 2.1  List of primary antibodies utilized in immvinoprecipitation or  38  fluorescent labeling Table 4.1  CD44 clustering is an actin-dependent process  Table 4.2  Percentage of cells showing segregation of CD44 and CD45  viii  95 111  List of Figures Figure 1.1  Examples of interactions  2  Figure 1.2  Extravasation  4  Figure 1.3  Immunological synapse  10  Figure 1.4 A , B  Major classes of adhesion molecule  13  Figure 1.4 C, D  Major classes of adhesion molecule  17  Figure 1.5  CD44  21  Figure 1.6  CD45  33  Figure 3.1  CD44 mediates cell spreading on immobillized antibody  47  Figure 3.2  Immobilized transferrin receptor (Tfr) antibody does not  48  induce cell spreading in BW5147 T cells Figure 3.3  Cytoplasmic domain of CD44 is required for cell spreading  50  Figure 3.4  Extracellular calcium is required for CD44-mediated  52  elongated cell spreading Figure 3.5  P L C is required for CD44-induced elongated cell spreading  53  and tyrosine phosphorylation of p i 20/130 Figure 3.6  PI3K is required for CD44-induced elongated cell spreading  55  and tyrosine phosphorylation Figure 3.7  Actin polymerization is required for CD44-mediated  56  elongated cell spreading and tyrosine phosphorylation of pl20/130 Figure 3.8  Dynamics of microtubules is required for CD44-mediated  57  elongated cell spreading, but not for tyrosine phosphorylation ofpl20/130 Figure 3.9  Transient tyrosine phosphorylation of P L C y l is associated  59  with elongated cell spreading of CD45" T cells Figure 3.10  Tyrosine phosphorylation of Y402 was observed with  61  elongated cell spreading of CD45" T cells Figure 3.11  CD44-induced Pyk2 tyrosine phosphorylation requires extracellular C a , PLC, PI3K, and actin polymerization 2+  ix  63  Figure 3.12  Transient tyrosine phosphorylation of Cas is associated with  64  CD44-mediated elongated cell spreading Figure 3.13  CD44 and CD3 signaling pathways are different  66  Figure 3.14  CD45 also inhibits LFA-1-mediated elongated cell spreading  68  Figure 3.15  LFA-1 induces tyrosine phosphorylation of Pyk2 with  69  elongated cell spreading Figure 4.1  Activated thymoctyes from CD45 knockout mice also  83  displayed elongated cell spreading on immobilized CD44 antibody Figure 4.2  CD45" T cells, PTPci, and Y789F-PTPct transfectants  84  expressed comparable levels of CD44 Figure 4.3  Over-expression of CD45-like phosphatase, PTPct in CD45"  85  BW5147 T cells reduced CD44-induced elongated spreading Figure 4.4  Over-expression of PTPct inhibited CD44-induced tyrosine  87  phosphorylation of p i 20/130 and Pyk2 Figure 4.5  CD44-mediated cell spreading of CD45 and CD45" cells is +  89  accompanied by different actin rearrangement Figure 4.6  SFK activity is required for CD44-induced rearrangement in  90  either CD45 or CD45" T cells +  Figure 4.7  CD44 is recruited to microclusters when BW5147 T cells  92  were incubated on immobilized antibody Figure 4.8  Clustering of CD44 is an actin-dependent process  94  Figure 4.9  CD44-induced Lck recruitment into microclusters was  97  independent of CD45 Figure 4.10  Recruitment of Fyn into microclusters was hampered in the  98  presence of CD45 Figure 4.11  Co-localization of tyrosine phosphorylation and CD44 was  100  inhibited by CD45 Figure 4.12  CD45 translocates transiently to the CD44 microclusters  101  Figure 4.13  Inhibition of PI3K in CD45- T cells lead to CD44-induced  103  actin ring formation  x  Figure 4.14  PI3K is recruited to CD44 microclusters in both CD45 and +  104  CD45" T cells during CD44-mediated cell spreading Figure 4.15  Induction of Akt phosphorylation is CD44-independent but  106  SFK-dependent Figure 4.16  Pyk2 is recruited to CD44 microclusters in CD45 and CD45" +  107  T cells Figure 4.17  CD45 and CD44 segregation was observed in BW5147 T  109  cells upon SDF-1 stimulation Figure 4.18  Segregation of CD44 and CD45 was also observed in thymocytes and splenic T cells from Balb/c mice upon SDF-1 stimulation  xi  110  List of Abbreviations Abbreviations aa  Full Names Amino acid  APC  Antigen presenting cells  ATCC  American type culture collection  Ab  Antibody  BSA  Bovine serum albumin  cAMP  Cyclic adenosine monophosphate  CD  Cluster of differentiation  CTL  Cytotoxic T lymphocytes  DC  Dendritic cell  DMEM  Dulbecco's modified Eagle medium  ECM  Extracellular matrix  EGTA  Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid  F-actin  Filamentous actin  f-MLP  Formyl-methionyl-leucyl-phenylalanine  GTP  Guanine nucleotide triphosphate  HA  Hyaluronan or hyaluronic acid  HEV  High endothelial venule  ICAM  Intercellular adhesion molecule  Ig IGF  Immunoglobulin  IL  Interleukin  IP  Immunoprecipitation  IP IS  Inositol-3,4,5-triphosphate  LAT  Linker for activation of T cells  LFA-1  Leukocyte function associated-1  mAb  Monoclonal antibody  MTOC  Microtubule-organizing center  NK  Natural killer  3  Insulin-like growth factor  Immunological synapse  xii  PBMC  Peripheral blood mononuclear cells  PH  Pleckstrin homology  PI3 K  Phosphatidy linositode-3 -kinase  PIP  Phosphatidylinositol 3,4,5-trisphosphate  3  PKC  Protein kinase C  PLCyl  Phospholipase C gamma 1  PKC  Protein kinase C  PMA  Phorbol myristate acetate  pMHC  Peptide-MHC complex  PTEN  Phosphatase and tensin homolog on chromosome ten  PTPa  Protein tyrosine phosphatase alpha  RPMI-1640  Roswell Park Memorial Institute-1640 medium  medium SDF-1  Stomal-derived factor-1  SDS-PAGE  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis  SEB  Staphylococcal enterotoxin B  SFK  Src family kinase  SH  Src-homology  SMAC  Supramolecular adhesion complex  TCR  T-cell receptor  TfR  Transferrin receptor  Thl  T-helper-1  Th2  T-helper-2  VSVG  Vaccinia stomatitis virus glycoprotein  xiii  Acknowledgements I wish to thank my thesis supervisor, Dr. Pauline Johnson, for giving me the opportunity to pursue this work under her guidance. Moreover, I am very grateful for her patience and kindness shown to me.  I also wish to thank the members of my thesis committee: Dr. Michael Gold, Dr. Kelly McNagny, and Dr. Fumio Takei for their suggestions along the pursuit of this thesis work.  The help from the U B C Bioimaging facility was invaluable for the completion of this work. I wish to thank Dr. Elaine Humphrey, Garnet Martens, and the staff at the facility for providing tremendous technical help.  I am also grateful for the past and present members of the Johnson Lab. Particularly, I wish to thank Kelly and Ruihong for helping me to start the project in the lab. I am also thankful for Nina and Jacqueline, whose presence and friendship will always be treasured. Moreover, I thank them for their artistic interpretation of the CD44 and CD45 structures. Furthermore, the help provided by Darlene Birkenhead to keep the lab in a running order is deeply appreciated. In addition, I also thank Shelley Small and Michael Hermawan for their administrative help.  I am also deeply indebted to Joshua Chan, whose encouragement, guidance, and support shall be rewarded by the blessings from above.  xiv  Dedications To  Mom & Dad  and  My Lord and Savior, Jesus Christ, through whom all things were made, without whom nothing was made that has been made.  "It is good for a man to bear the yoke while he is young. Let him sit in silence... For men are not cast off by the Lord forever. Though He brings grief, He will show compassion, so great is His unfailing love." "I say to myself, 'The L O R D is my portion; therefore I will wait for H i m . ' " Lamentations 3:24, 27, 32.  xv  Chapter 1 Introduction 1.1  Adhesion molecules and the immune system The effective functioning of the immune system lies in the recognition of  biological threat and elimination of such by the immune cells. To achieve these ends, the immune cells need to communicate effectively among themselves and with the environment that they are exposed to. The means of communication can be via three different ways. First, interaction occurs between surface molecules of cells, this is known as cell-cell interaction (figure 1.1 A). Second, interaction takes place between components of the extracellular matrix (ECM) and surface molecules of cells; this is often referred as to cell-ECM interaction (figure 1.1B). Third, interaction occurs between soluble molecules and surface receptors of the immune cells (Figure 1.1C). The consequences of these three forms of interactions are usually propagation of cellular signals, leading to behavioral changes of these cells. The importance of these three forms of interactions is exemplified by two essential physiological situations in the immune system: cellular migration and antigen presentation. These are introduced in details in sections 1.1.1 and 1.1.2. In these situations, adhesion molecules are indispensable in mediating the cellular events. Originally identified for their ability to mediate adhesion, as the name suggests, it has become clear, however, that they also transmit signals to alter cellular behavior. These molecules are divided into several main categories, based on structural differences. They are the selectins, the integrins, the cadherins, and the immunologlobin superfamily adhesion molecules. The adhesion molecule of interest of this thesis, CD44, does not belong to any of these classes due to structural differences; however, CD44 does share some of the functional properties of these adhesion molecules. This adhesion molecule will be introduced in detail in section 2.5. One aim of this thesis is to identify the components in CD44 signaling that are associated with changes in T cell morphology, which is essential for the proper functioning of T cells.  1  A  B  C  (A) Cell-cell interaction occurs between a leukocyte and an endothelial cells during extravasation. (B) Cel-ECM interaction occurs when a lymphocyte is migrating in extravascular tissue. (C) Cell-soluble factor interaction occurs when cytokines, e.g. IL-2, interacts with their corresponding receptors. Figure 1.1  Examples of interactions.  2  1.1.1  Cellular migration  Migration of hematopoietic cells in the body is paramount for the normal development and effective functioning of the immune system. For instance, migration of T cell precursors from the bone marrow into the thymus is required for further maturation of the T lymphocytes. Under homeostatic conditions, mature monocytes migrate from bone marrow into the circulation, from where they migrate into various tissues to differentiate into resident macrophages or myeloid dendritic cells (1). The lymphocytes circulate in the body among the bloodstream, the secondary lymphoid organs, and the peripheral organs. Upon detection of a biological threat, immature dendritic cells pick up antigen and migrate to regional draining lymph nodes and lymphocytes are recruited to these specialized organs, where foreign antigen is presented by the mature dendritic cells. The T lymphocytes that bear a reactive TCR that recognizes the antigenic peptide/class II major histocompatibility complex (pMHC) on the mature dendritic cells are activated. After differentiation and proliferation, effector T lymphocytes leave the lymph nodes and enter the blood stream again to travel to the site of inflammation. When leaving the bloodstream, during lymphocyte homing under homeostatic conditions or during specific leukocyte recruitment under inflammatory conditions, the leukocytes go through a process called extravasation. This is a highly orchestrated process, which begins with the initial contact between the leukocytes and the vascular endothelium, resulted in rolling of the leukocytes on the contacting surface (figure 1.2). The rolling process is largely attributed to the interaction between the adhesion molecule selectins (section 1.2.2) and their carbohydrate ligands. The productive consequence of rolling is establishment of firm adhesion between the extravasating leukocytes and the endothelial cells, an interaction mainly mediated by integrins (section 1.2.1). The leukocytes will then undergo cytoskeletal rearrangement that leads to a change of cell shape. When in the circulation, the leukocytes assume a comparatively rigid spherical shape. However, during extravasation, the leukocytes need to undergo a dramatic shape change before they may go between the endothelium and exit the bloodstream (transmigration). A similar process occurs also during inflammation, when leukocytes are recruited to where intrusion of biological threats occurred. The general paradigm, mentioned above, has provided a framework to study the process of how leukocytes  3  The initial tethering and roling contact of the extravasating leukocyte is mediated byselectins and their ligands. Upon activation of integrins, firm adhesion of the leukocyte on the endothelium is established. The leukocyte then undergoes dramatic cytoskeletal changes before and during transmigration through the endothelium. Figure 1.2  Leukocyte Extravasation.  4  exit the bloodstream (2, 3). Other observations have added to the understanding of this process. For instance, CD44, the adhesion molecule of interest in this thesis, may also participate in mediating the rolling of lymphocytes on endothelium or in establishing firm adhesion (discussed below). Moreover, a short-distance migration of monocytes on the endothelium, called locomotion, has been observed between the firm adhesion and the transmigration steps (4). Finally, leukocyte-exiting strategies differ in an organ-specific manner, where leukocyte extravasation from liver and brain vasculature does not follow the general paradigm (reviewed in 5). The migration patterns of leukocytes within the body are regulated by the combination of chemokine receptors and adhesion molecules expressed. For instance, the interaction between secondary lymphoid chemokine (SLC) and its receptors CCR7 helps bring circulating lymphocytes into lymph nodes or the spleen (reviewed in 6) while the integrin a4|37 is usually involves in lymphocyte homing to mucosal tissue (reviewed in 7). After leaving the blood circulation, leukocytes migrate in the extravascular tissue to carry out any necessary function. Under homeostatic conditions, lymphocytes migrate through secondary lymphoid tissues to provide surveillance. During inflammation, leukocytes, such as monocytes or effector T lymphocytes, migrate to sites where the biological threats need to be removed. In both cases, directed cellular migration is required. During this process, the leukocytes assume a polarized cell morphology where the distinction between the leading front and the trailing tail of the cell is clear. The leading edge of a migrating leukocyte is characterized by the presence of actin polymerization and a concentration of chemokine receptors. The migrating cell body contains the nucleus while the trailing edge, known as the uropod, contains the microtubule organizing center (MTOC) and surface molecules, such as CD43 and CD44 (reviewed in 8, 9). Effective cellular migration in the extravascular tissue or extracellular matrix (ECM) requires transient contact between the cell and the components of the matrix. This contact should not be too tight such that the cell may continue to move forward, yet the interaction should not be too loose so that there is enough traction for retracting the trailing tail. The migrating cell is thought to move in cycles of sequential steps, including the extension of the leading edge, formation of firm attachment,  5  retraction of cell body, and detachment at the rear end of the cell (reviewed in 10). The interactions between the migrating cell and the E C M are mediated by adhesion molecules. It is also becoming clear that the adhesion molecules not only provide means for attachment to the E C M , but they also transduce signals to cause appropriate cytoskeletal rearrangement necessary for the process (11,12). A migrating cell may be conceived as a machine integrating input and coordinating output signals to mediate motility. Two major sets of interactions generate the input signals: the interaction between chemotactic cues and their receptors, and the interaction between adhesion molecules and their ligands, mostly E C M components. The output signals consist of the ones that mediate actin reorganization, myosin-dependent contraction, adhesion molecule affinity/avidity modulation, and adhesion molecule turnover and/or shedding. As mentioned above, migrating cells assume a morphological polarization before and during the process. Understanding of these signals has come from studies utilizing two main model systems: Dictyostelium discoideum and neutrophils. During the aggregation phase, Dictyostelium exhibits ameboid motility in response to cyclic adenosine monophosphate (cAMP) (13). Displaying similar migration characteristics, neutrophils move towards sources of formyl-methionyl-leucyl-phenylalanine (f-MLP), which is a chemotactic peptide of bacterial source. In both cases, the chemotactic cue is detected by G-protein coupled serpentine receptors (14). Moreover, the cell polarization caused by chemoattractant stimulation is dependent on polarized accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) (15, 16), which is a product of phosphatidylinositol-3-kinase (PI3K). The polarized accumulation of PIP3 at the leading edge of the migrating Dictyostelium or neutrophil is enhanced by the specific localization of PI3K at the leading edge and the lipid phosphatase P T E N at the back of the cell (17). The enzymatic activity of PTEN is to dephosphorylate the D3 phosphate of PIP3 (18); therefore, its strategic localization away from the front of the stimulated cells enhances the difference of PIP3 levels between the leading edge and the back of the cell. Other regulators of cell polarization include the small Rho GTPases. These are the molecular switches, which are active when bound to GTP. Upon hydrolysis of GTP, the GDP-bound GTPases become inactive (received in 19). There are more than twenty mammalian members of Rho GTPases described thus far and the most studied of them  6  are the Rho, Rac, and Cdc42 (20). These GTPases have been shown to the formation of various actin structures related to cell spreading and migration. For instance, Rac localizes at the leading edge of migrating cells to mediate actin polymerization (21-23). These events are essential for the protrusion of the actin-rich leading front, known as lamellipodia. On the other hand, Rho activity is localized at the trailing edge of a migrating neutrophil, mediating cell body contractility and tail detachment (24). During cell polarization and migration, the tight regulation of actin rearrangement is paramount. The dynamics of actin is regulated by a variety of actin-binding proteins. These include the actin severing and depolymerizing proteins, such as cofilin and gelsolin, whose activity in severing actin filaments has been shown to enhance protrusion of lamellipodia (25-27). This is because lamellipodial protrusion requires initial severing of cortical actin and continual dynamic organization of actin during the advances of the protrusion. There are also actin-binding proteins that promote actin polymerization, such as the Arp 2/3 complex (28, 29) and formin (30, 31), which catalyze branching and unbranched extension of actin filament, respectively. Other than the actin-binding proteins, the organization of the actin cytoskeleton includes many adaptor molecules and kinases, such as Crk (32), Nek (33), Src (34) and Pyk2 (35, 36). Pyk2 is a tyrosine kinase that is related to focal adhesion kinase (FAK) and part of the interest of this thesis is to determine how it is activated upon engagement of the adhesion molecule CD44. This tyrosine kinase is also known as C A D T K (37), CAK(3 (38), F A K 2 (39), and R A F T K (40). The analysis of the Pyk2 knockout mouse revealed a defect in marginal zone B cell development (41). The administration of pertussis toxin to wild-type animals also led to selective loss of marginal zone B cells. Guinamard et al. thus hypothesized that Pyk2 plays a role in the chemotactic response and B cell migration leading to localization of marginal zone B cells. Macrophages from Pyk2 knockout mice also have defects in chemokine-induced polarization and migration. This was observed together with a defect in integrin-mediated signaling (42). In monocytes, Pyk2 is required to mediate spreading and motility (35). Moreover, Pyk2 kinase activity regulates osteoclastic actin organization (36). These observations suggest that Pyk2 plays a key role in chemokine- and integrin-mediated actin organization, relating to cell morphology and migration.  7  Cellular polarization and migration are complex processes that require coordination of signals from chemoattractants and adhesion molecules. The productive consequence is brought about through spatially and temporally controlled rearrangements of cytoskeletal elements. The details of the cellular signaling network, initiated by adhesion molecules, in mediating the cytoskeletal changes is being revealed. CD44, the adhesion molecule of interest of this thesis, has also been shown to play a role in mediating cellular migration (see section 1.3.4); however, the signaling initiated by this molecule is still unclear and one of the aims of this thesis is to identify its signaling components.  1.1.2  A ntigen presentation Another biological phenomenon in the immue system where the adhesion  molecules play an indispensable role is during antigen presentation. To provide surveillance of biological threats, antigen-presenting cells (APC) continuously sample macromolecules through internalization. When a foreign threat is detected, these cells will migrate to nearby secondary lymphoid organs, such as lymph nodes or Peyer's patches, to present the processed antigens to T lymphocytes. During antigen presentation, cell-cell interaction between A P C and T lymphocytes occurs. The activation of T lymphocytes results from the complementary interaction between the T cell receptor (TCR) and pMHC. This interaction may be a prolonged or a series of shortlived contacts (43-48), depending on the combination of A P C type and the microenvironment around the interacting cells. The T cell-APC interaction can be conceived as a sequential process that begins with the migration of a T cell towards an A P C . The initial interaction is mediated through adhesion molecules on both cells in a non-antigen-specific manner. T C R engagement occurs i f there is a complementary interaction between the TCR and pMHC. This engagement is stabilized through the interaction between the co-stimulatory and adhesion molecules on the T cell and their corresponding ligands on the APC. After various periods of engagement, the T cell will break off and migrate away from the A P C (49, 50). These stages of interactions were first observed with T-helper cells interacting  8  with M H C class II-transfected murine fibroblasts pulsed with antigen (51) and later observed in vivo (46, 47). At the initial phase of interaction, the T cell is polarized and it senses the presence of complementary p M H C on the A P C . In general, the polarized T cell migrates at a rate of 2-6 u.m/minute on the A P C surface with a higher sensitivity to p M H C at the leading edge (52). The integrin LFA-1 and chemokine receptors are also localized at the leading edge of crawling T cells. Moreover, the leading edge of the polarized T cell is enriched in F-actin and GM3-containing lipid rafts. The trailing edge of the polarized T cell, the uropod, is enriched in transmembrane molecules, such as ICAM-1, ICAM-3, CD43, and CD44 (reviewed in 53). At present, the specific role of the concentration of these molecules is unclear. However, the presence of a uropod is essential for effective T cell crawling (54). The next phase of T/APC interaction begins in a non-antigen-specific manner. This interaction is likely to be mediated by adhesion molecules or co-stimulatory molecules, such as CD28 or LFA-1 (55). The T cell may migrate away from the A P C after a brief interaction. The length of T/APC interaction depends on whether complementary pMHC is present, the A P C type, and the microenviroment. Should the conditions, such as the presence of complementary pMHC, encourage stable interaction, specific adhesion molecules are localized in a highly organized array on both cells. The apposition of cell membranes of both cells and the resulting cell-cell interaction has been termed immunological synapse (IS, figure 1.3), due to its resemblance to synapses between neuronal cells. Monks et al. first described this form of interaction between T and A P C in terms of surface receptors and signaling molecules (56). The authors demonstrated an antigendependent spatial molecular segregation in terms of TCR, PKC-8, L F A - 1 , and talin. After 30 minutes of interaction between T cell and Ag-pulsed A P C , the integrin LFA-1 and cytoskeletal component talin were enriched in most of the contact surfaces between the interacting cells. However, these molecules were excluded from the central contact region, where the TCR and PKC-0 were localized. The central contact region where the TCR and PKC-6 were located was termed central supramoleuclar activation clusters  9  p-SMAC  d-SMAC  Figure 1.3 Immunological Synapse (IS). The mature immunological synapse consists of a highly ordered array of molecules during antigen presentation. The central supramolecular adhesion complex(c-SMAC) is enriched with PKC, TCR, and CD28. LFA-1 and talin are found at the peripheral SMAC (p-SMAC) and CD45 is enriched at distal SMAC (dSMAC).  10  (c-SMAC) while the region where LFA-1 and talin was observed was named peripheral supramolecular activation clusters or p-SMAC (figure 1.3). Moreover, the authors reported that the Src-family kinases, Lck and Fyn, which are essential for T cell activation, were recruited to the c-SMAC during early time points. The Ag-specific interaction with TCR has been shown to deliver a stop signal to T cells crawling on immobilized ICAM-1 (57). This signal is associated with an increase in intracellular calcium concentration and reorientation of the microtubule-organizing center (MTOC) of the T cell. However, this occurs prior to the formation of the mature synapse (58), which is characterized by the segregation of the c-SMAC and the p-SMAC (56). The induced increase in intracellular calcium signaling is required for the immobilization of the T cell on the A P C (51, 57). During the third phase, segregation of surface receptors is completed. TCR, PKC-6, CD28 (55), and CD2 (59) are enriched at the c-SMAC. The co-stimulatory molecule CD4 and transmembrane phosphatase CD45 (section 1.3) transiently associate with the c-SMAC and later move away, while LFA-1 and talin are localized at the pS M A C . Indeed, the concept of a distal S M A C (d-SMAC) was introduced due to the observation of CD45 localization outside of the p-SMAC contact area (60). Although this phase is thought to be essential for sustained signaling delivered to the T cell, there is evidence that sustained interaction between the T cell and A P C may not be necessary for T cell activation. Faroudi et al. showed that C D 4 T cells were activated by antigen+  pulsed A P C under repeated interruptions through the addition and removal of Src family kinase inhibitor (61). Moreover, using two-photon microscopy and flow cytometry, Miller et al. observed short-lived and serial interactions between C D 4 T cells and +  dendritic cells, resulting in up-regulation of activation marker CD69 (46). The last phase of the T-APC interaction is initiated by the onset of migration of the T cell away from the APC. At present, the exact mechanism that ends the T/APC is unknown. However, several mechanisms have been proposed. One is that internalization and degradation of the TCR may bring about a reduction in signal delivery and end T/APC interaction. Other mechanisms include down a regulation of adhesion molecules, local expression of chemokine and chemokine receptors on T cells, and redistribution of inhibitory molecules (e.g. CD43) (50).  11  The IS described above is considered as a stable or monocentric IS. However, there is increasing evidence that other forms of IS exists (reviewed in 50). This is dependent on the cell types involved, the activation stages of these cells, the organization of adhesion molecules, the duration of signals generated, and the presence or absence of soluble factors secreted. For instance, natural killer (NK) cells interact with target cells and form an IS, yet both stimulatory (62) and inhibitory IS can occur (63). The formation of an IS exemplifies the specific temporal and spatial organization of surface and intracelullar molecules during antigen presentation, an instance of cell-cell interaction in the immune system. During this process, adhesion molecules play a prominent role. First, they mediate the initial interaction between the T cell and the A P C since the numbers of TCR and p M H C are relatively low and p M H C does not have to be present (64, 65). Second, adhesion molecules have been shown to mediate cytoskeletal changes during T/APC interaction (66). Third, signals generated from adhesion molecules also affect the signaling threshold of T cells (67). Cell migration and antigen presentation are examples of the significance of adhesion molecules in the effective functioning of the immune system. The adhesion molecules are classified in four major classes. The following sections provide a general survey of these major classes of adhesion molecules and their role in cellular migration and antigen presentation.  1.2  Major classes of adhesion molecules  1.2.1  Selectins The selectins (figure 1.4A) are C-type lectins that include three members: L -  selectin, E-selectin, and P-selectin. The letters " L " , " E " , and "P" stand for leukocyte, endothelial, and platelet, respectively. L-selectin is generally expressed on myeloid cells and naive T cells, as well as a small subset of effector and central memory T cells. Eselectin is constitutively expressed by skin endothelium. Its expression is also induced in microvascular endothelium at inflamed tissue. Other than being expressed on platelets, P-selectin is also induced in inflamed endothelium of most tissues. The selectins share a common structural organization of protein domains. These include the amino-terminal  12  B P-  LFA-1  human selectins  p subunit  a subunit y  Lectin domain  G  EGF-like domain  0  Short consensus repeat  (J propeller structure  I domain  \ Figure 1.4  l-like domain  Major classes of adhesion molecules.  (A) Selectins. Al selectins contain an N-terminal lectin domain, folowed by an EGF-like domain, various copies of the short consensus repeats, a transmembrane domain, and a short cytoplasmic tail. (B) Integrins. These are heterodimers made up of one a and one p subunit. The p subunit contains the I domain, the conformation of which determines the affinity of the integrin to its counter-receptor.  13  lectin domain, an epidermal growth factor (EGF)-like domain, a series of short consensus repeats, a transmembrane region, and a short cytoplasmic domain (68). These single-pass transmembrane adhesion molecules bind to carbohydrate structures in a calcium-dependent manner. The carbohydrate structures were identified as sialyl Lewis and sialyl Lewis (69-71). These binding determinants of selectins are x  3  presented on specific scaffold proteins. The P-selectin glycoprotein ligand (PSGL)-l, expressed mostly on leukocytes, presents these structures to P-selectin (72, 73). Other than the carbohydrate structures, tyrosine sulfation of PSGL-1 is also essential for its interaction with P-selectin (74-76). The high affinity ligand for E-selectin, known as Eselectin ligand (ESL)-l, has also been identified on myeloid cells and lymphocytes (77, 78). L-selectin was shown to interact with glycosylation-dependent cell adhesion molecule (GlyCAM)-l expressed on endothelial cells of the high endothelial venules (79). Another scaffold protein that presents sialyl Lexis" to L-selectin is CD34, which is expressed by endothelial cells and hematopoietic cells (80). The highly glycosylated mucosal addressin cell adhesion molecule (MadCAM)-l is also a counter-receptor of L selectin (81). The major physiological function of selectins is to mediate the rolling of leukocytes through their interaction with the carbohydrate-presenting ligands (82). This is the first interaction between an extravasating leukocyte and the endothelium. The involvement of selectins in leukocyte recruitment is both cell-type and organ-specific. For instances, L-selectin is essential for in lymphocyte homing to lymph nodes (83) while neither P- nor E-selectin is involved. Both L - and P-selectins are implicated in neutrophil recruitment to inflamed peritoneum (84), while P- and E-selectins mediate recruitment of Thl cells to inflamed skin. Selectins also possess signaling properties that enhance integrin function, which is thought to be necessary for establishing firm adhesion between the extravasating leukocyte and the endothelium (85). Although selectins have not been observed in the immunological synapse, a member in the C-lectin family expressed on denderitic cells (DC), DC-SIGN, interacts with an IgSF adhesion molecule ICAM-3 during T/DC interaction (86).  14  1.2.2 Integrins The integrins (figure 1.4B) are among the most studied adhesion molecules. These transmembrane heterodimers, composed of alpha and beta subunits, are involved in a variety of biological functions. Thus far, 18 alpha and 8 beta chains, making up 24 integrins, have been identified (87). Each integrin subunit contains an ectodomain, a transmembrane region, and a cytoplasmic domain. They are expressed on virtually all cells in the body. The ligands of integrins include other adhesion molecules and components of the E C M , such as fibronectin and collagen. The integrins can be classified by their alpha or beta subunits. For instance, lymphocyte function-associated antigen (LFA)-l and macrophage differentiation antigen, M A C - 1 , are made up of aL|32 and aM(32 subunits, respectively, and they are referred to as |32 integrins. Among the integrins, LFA-1 is the most studied. The functioning of L F A - 1 , as for other integrins, is closely associated with its ability to bind to its ligand. In the case of L F A - 1 , the ligands are intercellular adhesion molecules (ICAM) -1,-2, and -3. The ability of integrins to bind to their appropriate ligands is tightly regulated. The binding ability of LFA-1 is dictated by the affinity and the avidity of the molecule. The affinity of LFA-1 is regulated by conformational changes, particularly a shift at the I, or inserted, domain of the a chain (88, 89). Increased affinity of LFA-1 has been demonstrated with chemokine stimulation (outide-in signaling) (90) and M g  2 +  or M n  2 +  (91, 92). The avidity  of L F A - 1 , on the other hand, is enhanced by clustering of this adhesion molecule. This is the result of lateral movement of LFA-1 after the release from actin cytoskeleton (93). At present, it is still contraversial as to whether avidity or affinity modulation is the dominant contributor to the enhanced adhesiveness of LFA-1 to its ligand in physiological situations (reviewed in 94, 95). The physiological functions of integrins are widespread, ranging from a role in embryonic development to specific immune functions. During certain types of lymphocyte extravasation, the integrins very late antigen (VLA)-4 and, to a lesser extent, LFA-1 mediate rolling (96-99). Moreover, these integrins also mediate the firm adhesion of extravasating leukocytes to endothelial cells (100, 101). This likely results from activation of integrins through chemokine presentation on the endothelial cell surface (102, 103). Moreover, LFA-1 is also responsible for mediating transmigration of  15  lymphocytes through endothelial layers via its interaction with a recently identified ligand, JAM-1, at the endothelial junction (104). After leaving the bloodstream, the leukocytes will need to migrate in an extravascular environment. The migratory function of the leukocytes may be regulated by the integrins that interact with E C M components, such p i integrins (105). During T/APC interaction, LFA-1 also plays various roles. First, it contributes to the non-antigen-dependent scanning of A P C . Second, LFA-1 is found at the immunological synapse (56). Third, it lowers the signaling threshold for T cell activation (67). Fourth, it contributes to the signals that mediate cytoskeletal rearrangement (106). Indeed, the signaling function of LFA-1 is possibly mediating the cytoskeletal rearrangement of lymphocytes during extravasation (107-109).  1.2.3  Cadherins The cadherins (figure 1.4C) belong to a large family of adhesion molecules that  are categorized into different subclasses (reviewed in 110). The four major categories include classic cadherins, Fat-like cadherins, seven-transmembrane cadherins and cadherins that are related to Drosophila Cadl02F. A l l cadherins are characterized by the presence of cadherin domains (reviewed in 111). These domains are about 110 amino acids in size and are responsible for mediating calcium-dependent homophilic interactions between cadherin molecules. These interactions may occur in both cisinteracting and trans-interacting manners. When interacting in cis, the cadherins form homodimers on the plasma membrane of the same cell. On the other hand, when interacting in trans, the cadherins form adhesion complexes with each other on the plasma membrane on opposing cells. The function of cadherins has largely been implicated in embryogenesis, particularly in development and maintenance of organ architecture (112). At present, little about the role of cadherins is known in mediating leukocyte migration or in antigen presentation, even though a report showed that Ecadherin can interact with a4p7 integrin to mediate adhesion between T cells and epithelial cells (113).  16  D  c  ICAM-1  E-cadherin dimer  Q  Ig-like domain  cadherin domain a-cantenin (5-cantenin  Rgure 1.4  Major classes of adhesion molecules.  (C) Cadherins. The classical cadherins contain various repeats of the cadherin domain at the extracelular region. The cytoplasmic domain is associated with a-catenin, to which p-catenin interacts. (D) IgSF adhesion molecule. All IgSF adhesion molecules contain various copies of the immunoglobulin-like domain.  17  1.2.4  Immunoglobulin superfamily adhesion molecules The members of the immunoglobulin superfamily (IgSF) adhesion molecules  (figure 1.4D) are characterized by the presence of Ig-like domains in their extracellular region. These domains are about 100 amino acids in size containing a characteristic fold that includes two (3 sheets with anti-parallel stands. The domains are found also in the (32 microglobulin of M H C and Thy-1 (reviewed in 114). The Ig adhesion molecules also contain a single helical transmembrane domain and a cytoplasmic tail. The IgSF adhesion molecules are expressed in a variety of cell types, including endothelial cells, A P C , and lymphocytes. The counter-receptors of L F A - 1 , the ICAMs and JAM-1, are members of the IgSF adhesion molecules. So is the counter receptor of V L A - 4 , V C A M 1. As ligands for these integrins, their roles in cellular migration and antigen presentation are important. ICAM-1 and V C A M - 1 expressed on endothelial cells mediate the rolling and firm adhesion of leukocytes, in conjunction with LFA-1 and V L A - 4 , during leukocyte extravasation (96-98, 102). Moreover, ICAM-2 and ICAM-3 may interact with DC-SIGN to mediate T cell scanning of A P C (86). Furthermore, there is increasing evidence that ICAM-1 is capable of mediating signaling that brings about recruitment of M H C molecules; thereby, enhancing the efficiency of antigen presentation (reviewed in 115). The co-stimulatory molecules CD28 and CTLA-4 are also members of the IgSF that possess important signaling functions, although they are not considered efficient adhesion molecules (116). Although these major classes of adhesion molecules exhibit structural differences that ensure clear distinction, there are similarities that deserve attention. Other than their ability to mediate adhesion processes, either in the context of cell-cell or cell-ECM interactions, there is accumulating evidence that these receptors also mediate signaling functions that alter cell behavior (12, 66, 85, 115, 117). Moreover, the cytoplasmic domains of most of these adhesion molecules associate with scaffolding proteins that interact with the cytoskeleton. Although little is known about the cytoskeletal association of selectins, it has previously been reported that L-selectin interacts with actin cytoskeleton through a-actinin (118). The integrins are known to interact with the cytoskeleton through talin, vinculin, a-actinin, and tensin (reviewed in 119). The cytoplasmic domains of cadherins interact with |3-catenin, to which the ct-catenin binds,  18  providing a link to the actin cytoskeleton (120). Members of the IgSF adhesion molecules interact with the E R M family of adaptors, which also provide a link to the actin cytoskeleton (121). The interactions between these adhesion molecules and the cytoskeletal elements may mediate the formation of scaffolds that promote signaling functions (122). CD44, the adhesion molecule of interest in this thesis, does not belong to any of the adhesion molecule classes presented above. However, CD44 also shares the similarities, mentioned above, with other adhesion molecules. A more detail description of this adhesion molecule is provided in the following section.  1.3  CD44  1.3.1  Discovery CD44 is a widely expressed adhesion molecule; its expression has been observed  on most nucleated cells. The identification of CD44 originates from studies that utilized monoclonal antibodies that were raised against different cell types. CD44 was first described as a polymorphic molecule that is about 80 kDa in size. It is expressed by macrophages, granulocytes, T cell, cortical thymocytes, and certain brain cells (123-127). Another isoform of this transmembrane glycoprotein, of 95 kDa in size, was also found in early T-cell precursors (128). In fibroblasts, CD44 was described as a cytoskeletonassociated protein of 80-100 kDa in size (129) that binds to types I and VI collagen and to fibronectin(130, 131) Jalkanen et al. developed a series of antibodies against surface molecules on lymphocytes that interact with H E V . The first reported anti-human antibody by this group, Hermes-1, precipitated a 90-kDa glycoprotein. This antibody blocks the adhesion between lymphocytes and H E V in frozen sections of lymph nodes (132). Hermes-1 was later utilized to immunoprecipitate a potential adhesion molecule that interacts with mucosal H E V . This was done by immunization of Balb/c mice with a human B lymphoma cell line that preferentially binds to mucosal H E V (133). These antibodies were later found to react with CD44. In 1989, Gallatin and co-workers also described a mouse monoclonal Ab, Hutch1, that was generated by immunizing Balb/c mice with whole macaque peripheral blood  19  mononuclear cells (PBMC). This antibody was found to precipitate a 90-kDa glycoprotein from macaque P B M C , human P B M C and a fibrosacoma cell line (134). This 90-kDa glycoprotein was found to share epitopes with Hermes-1. Originally given the names phagocytic glycoprotein-1 (Pgp-1), extracellular matrix receptor III (ECM-III), Ly-24, and In(Lu)-related p80, the identity of CD44 was revealed by comparing the reactivity of the monoclonal antibodies (134-137) and cloning of the antigen (138-141).  1.3.2  Structure CD44 is a single pass transmembrane protein that consists of an extracellular, a  transmembrane, and a cytoplasmic domain (figure 1.5). The ectodomain of human CD44 contains 248 amino acid residues (141). One peculiar feature of this ectodomain is that sequences encoded by the variant exons are inserted at the membrane-proximal region of this molecule by alternative splicing. In the CD44 gene, there are 10 variant exons and any combination of these variant exons can be inserted in the stem region of CD44 (reviewed in 142). Another characteristic feature of CD44 is post-translational modification of this ectodomain. The amino acid sequence of CD44 predicts a molecule of only 37 kDa in size; however, CD44 runs as an 80-90-kDa protein on SDS-PAGE gels, suggesting extensive post-translational modification. Both O-linked and N-linked glycosylation, as well as sulfation, have been shown to modify the ectodomain of CD44 and its ligand binding ability (143-146). Therefore, the combination of alternative splicing and posttranslational modification generates a highly heterogeneous group of CD44 isforms. The physiological role of generating such diverse family of CD44 proteins is still unclear. The CD44 isoform that contains no variant exon insertion is referred to as the standard (CD44s) or hematopoietic (CD44H) isoform. The transmembrane domain of CD44 consists of a region of 23 hydrophobic residues. In this region is a cysteine residue that has been proposed to enable dimerization of CD44 upon stimulation (147). The transmembrane region of CD44 may be responsible for targeting CD44 to a specialized membrane compartment, known as the lipid rafts (148, 149). These are detergent-resistant domains of the plasma membrane  20  transmembrane region  [—cytoplasmic domain  SS  Chrondroitin sulfate  disulfide bridge  O-glycosylation site N-glycosylation site  Figure 1.5  CD44  CD44 is a type 1 transmembrane glycoprotein. The chrondriotin sulfate at the stem region may influence HAbinding abilityof CD44. The site to which variant exons are insert is also located at the membrane proximal region of the ectodomain. The cytoplasmic tail has been shown to interact with ankyrin and the ERM proteins.  21  enriched in cholesterol and sphingolipids. It has been proposed that these compartments are essential for sorting signaling molecules and providing a platform for signaling functions (150). The cytoplasmic domain of CD44 is a sequence of 72 amino acids, through which this adhesion molecule interacts with the actin binding proteins, E R M (151, 152) and ankyrin (153). The E R M proteins belong to a family of three structurally related proteins, ezrin, radixin, and moesin. These actin-binding proteins are responsible for providing linkages between cell surface molecules and filamentous actin (F-actin) (121). Other cell surface molecules that interact with F-actin through the E R M proteins include ICAM-3 (154) and CD43 (155). The ERM-interacting region of the cytoplasmic domain of CD44 can also interact with another ERM-related protein, merlin (156). However, merlin does not provide a linkage between its interacting molecules and F-actin (157). Legg et al. (158) showed that CD44 interacts with ezrin in a protein kinase C (PKC)dependent mechanism. In resting cells, serine 325 at the cytoplasmic domain of CD44 is constitutively phosphorylated and this is correlated with the association of ezrin. Upon stimulation with phorbol ester, a P K C activator, deposphorylation of serine 325 was concomitant with phosphorylation of serine 291. The dephosphorylation of serine 325 and phosphorylation of serine 291 was coordinated in such a manner that the overall phosphorylation level of CD44 was maintained. This shift of serine phosphorylation is associated with the decrease in ezrin binding to CD44. The negative regulatory role of phosphorylated S291 on ezrin-CD44 interaction was supported by the observation that S291A CD44 mutant showed a decrease in association between ezrin and CD44. As mentioned above, the cytoplasmic domain of CD44 interacts with the cytoskeletal linker protein ankyrin (153). Moreover, this association is promoted by P K C activation (159), acylation of CD44 (160), and GTP-binding (161). The ankyrin binding site in the cytoplasmic domain of mouse CD44 has been mapped to the sequence containing amino residues 305 to 355. In this sequence, two sub-regions contributing to ankyrin association were identified: region I (a.a. 305-320) and region II (aa 320-355), where region II is the high-affinity binding site for ankyrin (162). The results from Lokeshwar et al. also suggest that the association between ankyrin and the cytoplasmic domain of CD44 is essential for hyaluronan (HA) binding to CD44 in COS cells (162).  22  1.3.3  CD44-HA interaction The sequence of CD44 revealed a homology between the N-terminus of CD44  and cartilage link protein and proteoglycan core proteins (138, 140, 141, 163). Since these proteins bind the glycosaminoglycan hyaluronan (HA), it was postulated that CD44 might be a HA-binding protein. Later, the HA-binding ability of CD44 was observed by various groups (164-166). H A is a polymer containing repeating units of a disaccharide of glucuronic acid and N-acetylglucosamine (167). It is a widely expressed component of the E C M . Other than H A , CD44 also binds other ligands, including collagen (130, 131), fibronectin (168), osteopontin (169), and serglycin (170). The interaction between CD44 and H A is the most studied. The interaction was shown to be a highly controlled process - the expression of CD44 alone is not sufficient for H A binding. There are three binding variantes of CD44: 1. CD44 that binds H A ; 2. CD44 that does not bind HA, yet it can be induced to do so; and 3. CD44 cannot be induced to bind H A (144). Efforts have been invested to study how the CD44-HA interaction is regulated. The post-translational modification of the CD44 ectodomain, in particular glycosylation plays a role in its regulation (144, 171-173). Other than glycosylation, CD44 may also be modified by sulfation, which enhances CD44-HA interaction (145, 174, 175). Moreover, insertion of variant domains into CD44 also affects this interaction (176, 177). Clustering of CD44 also contributes to its ability to bind H A . This was suggested by enhanced H A binding upon CD44 isoform oligomerization (178). Moreover, forced dimerization of CD44 by substituting the transmembrane domain with CD3 t, chain induces H A binding (179). In addition, clustering of CD44 with the mAb, IRAW14, enhances H A binding (180). Furthermore, H A binding induction correlates with CD44 dimer formation upon P M A stimulation of CD44-transfected Jurkat cells (147). The cytoskeleton also seems to play a role also in CD44-mediated H A binding, since the induced CD44/HA interaction is significantly reduced when cells are treated with F-actin disrupting agent cytochalasin D (181). Moreover, the interaction between ankyrin and the cytoplasmic domain of CD44 was shown to be essential for H A binding (162). In  23  conclusion, CD44/HA interaction is regulated by multiple mechanisms. The mechanism that affects CD44/HA interaction depends on the cell type and the specific stimuli applied to the cells.  1.3.4  Biological functions of CD 44  Leukocyte migration As a widely expressed and heterogeneous group of cell surface molecules, CD44 has been implicated in a wide variety of biological functions. One of the first described functions of CD44 was its role in mediating lymphocyte trafficking. This was first suggested by the blocking of lymphocyte interaction with frozen H E V section with the Hermes class Ab (132, 133). It was later shown that lymphocytes can roll on endothelial cells in a CD44/HA-dependent manner (182). In the same study, this observation was also replicated in a parallel flow chamber with immobilized H A . DeGrendele et al. subsequently showed that TCR cross-linking or antigen-specific activation of T cells lead to induced CD44/HA interactions and T cell rolling on immobilized H A (183). A CD44/HA interaction, resulting in rolling of T cells under flow conditions, was also observed by Gal et al. (184). They also showed that the cytoplasmic domain of CD44 and its interaction with the cytoskeleton were not required to support rolling. Several experimental observations have suggested that, like integrins, CD44 may be able to mediate firm adhesion through high affinity interaction with its substrate H A . Gal et al. showed that a deletion mutation at the membrane proximal region of the CD44 ectodomain resulted in firm adhesion of murine lymphocytes, even under flow conditions (184). Recently, Ruffell and Johnson showed that chondroitin sulfate addition to CD44 at serine 180, which is in the membrane-proximal region that Gal et al. deleted, discourages CD44/HA interaction (185). When a S180A mutation was introduced into CD44, preventing addition of chondroitin sulfate, a significant increase in the affinity of CD44 for H A resulted. This result suggests that chondroitin sulfate addition to CD44 may be a means of controlling CD44/HA interaction and the resultant high-affinity interaction, by removal of chondroitin sulfate, leads to firm adhesion. It has been shown in an in vivo acute inflammation model that CD44/HA interaction is required for effective adherence of neutrophils to vascular endothelium and their subsequent emigration out of  24  the blood vessel (186). Furthermore, this interaction was shown to be dependent on the expression of CD44 on both the neutrophils and the endothelial cells. Recently, CD44-dependent rolling on immobilized H A has been shown to take part in mediating firm adhesion of human and mouse T cells through enhanced V L A 4 / V C A M - l interaction (187). This phenomenon was also observed with T cells rolling on murine endothelial cells. Later work by the same group showed that this was a result of induced bimolecular complex formed by CD44 and V L A - 4 on the lymphocytes (188). The authors reported that the cytoplasmic domain of CD44 was indispensable for the formation of this complex. Moreover, the T cells expressing the cytoplasmic deletion of CD44 failed to migrate to the inflamed peritoneum in a mouse model (188). Engagement of CD44, resulting in cell spreading on a substratum, has also been observed in B cells and T cells. Partida-Sanchez et al. reported that incubation of activated murine B cells on immobilized CD44 antibody induced actin-dependent dendrite formation and cell spreading (189). However, this observation was not replicated in resting B cells, suggesting the capability of CD44 in mediating actin rearrangement is associated with the activation stage of the cells (190). Pre-treatment of human peripheral blood T cells with IL-2 and subsequent exposure to pro-inflammatory cytokines or chemokines also resulted in adhesion and polarized cell spreading of these cells on immobilized H A (191). Kim et al. have also reported that incubation of T cells on immobilized CD44 A b resulted in actin-dependent spreading and this spreading requires the cytoplasmic domain of CD44. Together, these observations suggest that CD44 is capable of mediating actin rearrangement in B and T cells, resulting in cell spreading on an underlying substratum. During extravasation, one necessary step is transmigration of leukocytes through a layer of endothelial cells. Using blocking antibody, Katakai and co-workers demonstrated that Thl-polarized T cells can transmigrate through a layer of murine endothelial cells in an L F A - 1 - and CD44-dependent manner (192). After extravasation of leukocytes, they need to move to the specific site of inflammation to exert their effector functions. Recently, Fanning et al. (193) observed that activated human T cells displayed a polarized cell spreading morphology when incubated in three-dimensional matrix containing high molecular weight H A . Moreover,  25  migration of these cells within the matrix was observed. This observation implies that CD44 can mediate lymphocyte migration in extravascular tissue. Several mouse models support the possibility that CD44 mediates T lymphocyte trafficking during inflammatory conditions. When a superantigen, Staphylococcal enterotoxin B (SEB), was injected into the peritoneum of Balb/c mice, V 8 T cells were +  p  preferentially stimulated. The proportion of these cells in the peripheral blood was decreased by 4 hours after intraperitoneal administration of SEB. A gradual accumulation of V 8 T lymphocytes in the peritoneum exudate and in the peripheral +  p  blood at the 8-hour and subsequent time points was observed. The migration of V 8 T +  p  lymphocytes was accompanied by an increase in H A binding ability of these cells. Intravenous administration of anti-CD44 antibody effectively blocked the migration of V 8 T cells into the inflamed peritoneum (194). These observations suggest that +  p  recruitment of antigen-specific T lymphocytes to the inflamed site may depend on CD44H A interaction. Other in vivo studies have also suggested a role for CD44 in recruiting lymphocytes to inflammatory sites. Brocke et al. showed that administration of antiCD44 or anti-ct4 antibodies alleviated the influx of encephalogenic T lymphocytes and clinical presentation of myelin-induced experimental autoimmune encephalomyelitis (195) . Moreover, administration of CD44 antibody has also been shown to prevent leukocyte recruitment to the synovial cavity of D B A mice with collagen-induced arthritis (196) . Furthermore, CD44-null D B A mice were also shown to have reduced symptoms of collagen-induced arthritis, even though a normal response to the immunization was observed (197). In addition, peripheral lymphocytes with low CD44 surface expression, resulted from shedding induced by CD44 antibody, show a significantly reduction in recruitment to cutaneous sites of delayed-type hypersensitivity. However, these lymphocytes exhibit normal homing to peripheral and mesentery lymph node (198). Together, these observations strongly suggest that CD44 mediates leukocyte recruitment, particularly during inflammation. These in vitro studies suggest that the in vivo effects may be due to the ability of CD44 to mediate rolling, firm adhesion, transmigration, and extravascular migration of leukocytes.  26  Co-stimulation of T cells  The two-signal model predicts that effective T lymphocyte activation, leading to proliferation and differentiation, requires a signal from the T-cell receptor (TCR) and a signal from a co-stimulatory molecule. The failure to receive a co-stimulatory signal can result in T cell anergy. At present, the most studied co-stimulatory molecule is CD28 (55, 199). However, other candidate co-stimulatory molecules, such as CD2 (200), CD5 (201), OX-40 (202), and members of the TNFct receptor family (203) have also been proposed. CD44 has also been proposed as a co-stimulatory molecule. This was due to the observation that a CD44 monoclonal antibody can induce a strong mitogenic signal in conjuction with CD3 or CD2 cross-linking (204). Moreover, co-ligation of CD44 and CD3 with monoclonal antibodies leads to enhanced production of IL-2 (205). Furthermore, co-culture of T and B cells with antigenic peptide stimulation showed reduced IL-2 production in the presence of CD44-receptor globulin protein (206). Since the CD44-receptor globulin protein contains the extracellular domain of CD44 (207), the observation suggests the need for CD44-ligand interaction during antigen presentation. Recently, Do et al. reported that CD44 was required for T cells to effectively form conjugates with antigen-pulsed DC. In contrast, CD44-deficient T cells did not form conjugates with DCs. Moreover, the CD44-deficient T cells showed a significantly reduced proliferative response when co-cultured with antigen-pulsed DC (208). Therefore, these results suggest a role for CD44 in T/APC interaction and co-stimulation. Hematopoiesis  CD44 has also been implicated in the process of hematopoiesis. This may be attributed to the ability of CD44 to bind HA, to initiate intracellular signaling, and to mediate cytoskeletal rearrangement and cell migration. Early hematopoietic cells express high levels of CD44 (128, 209, 210). Moreover, human hematopoietic cells are capable of binding HA (211,212). The bone marrow ECM is enriched with HA and the homing of human CD34 progenitor cells to +  the bone marrow of NOD/SCID mice depends on CD44/HA interaction (213). In a long-term bone marrow culture experiment, the presence of CD44 blocking antibody inhibited the production of lymphoid and myeloid cells from progenitors (214).  27  This suggests that blockage of hematopoietic precursor and stromal cell interaction is disruptive to hematopoiesis. This is also supported by the observation that blocking the stromal/progenitor interactions, by using integrin antibodies, also disrupted development development of hematopoietic cells (reviewed in 215). Moreover, it has been suggested that the adhesive interaction between hematopoietic progenitors and stromal cells results in subsequent cytoskeletal rearrangement and signaling that is necessary for blood cell development (216). Thus, the role of CD44 in hematopoiesis may also be to mediate cytoskeletal rearrangement. This is suggested by the observation that SDF-1 stimulated CD34 progenitor cells are able to spread on immobilized H A (213). +  Target cell killing by cytotoxic T lymphocytes and natural killer cells CD44 engagement can trigger the lytic activity of cytotoxic T lymphocytes (CTL) (217, 218) and natural killer (NK) cells (219). Matsumoto and co-workers reported that the IL-2-activated killing of various tumor cell lines by N K cells is defective in CD44 knockout mice (220). This was due to the inability of CD44 " N K cells to form _/  conjugates with target cells. Moreover, H A was identified as the principal CD44 ligand involved in CD44-dependent N K killing. Tan et al. showed that a CD44 antibody, S5, was able to enhance N K killing of target cells (221). The same group later showed that a CD44 antibody enhances conjugate formation between canine N K and target cells by promoting conjugate formation and T N F a secretion (222). Galandrini et al. showed that the CD44-dependent increase in N K killing required an increase in intracellular calcium and tyrosine kinase activity, leading to T N F a secretion (223). Other than mediating adhesion of N K cells to target cells, CD44 engagement also leads to cellular signaling promotes target cell killing. Moreover, intact actin was required for CD44-mediated lysis of target cells (224). Together, these observations suggest that the contribution by CD44 to N K killing is attributed to its ability to bind H A and its ability to signal, leading to cytokine secretion.  Cancer metastasis and adherent cell migration In 1991, Gunthert et al. reported that two metastatic rat pancreatic carcinomas expressed a high molecular weight isoform of CD44. When this CD44 isoform was  28  overexpressed in a non-metastazing tumor cell line, metastatic behavior of this cell line was observed (225). Much interest in studying the relationship between CD44 and cancer metastasis and progression was stimulated. The first demonstration that CD44 does mediate cell motility was provided by Thomas et al. (226). Stable transfection of CD44H into a human melanoma cell line conferred motile behavior on HA-coated surface to this cell line. Moreover, the ability of a CD44 isoform in mediating rolling of a lymphoma cell line is associated with its ability to accumulate in lymph nodes (227). The interaction between CD44 and H A not only confers migratory ability to cancer cells (228), but also to fibroblasts (229), endothelial cells (225), osteoclasts (230), and dendritic cells (231).  Angiogenesis  H A , the principal ligand of CD44, has been shown to stimulate endothelial cell proliferation and migration (232), which are essential processes in angiogenesis. However, the action of H A was shown to be restricted to H A of specific sizes. In particular, H A molecules of 3 to 16 disaccharide units (oligo-HA) were internalized and stimulated angiogenic responses of endothelial cells. In response to oligo-HA, bovine microvascular endothelial cells invade a three-dimensional matrix and form capillary structures, which are processes that are indicative of an angiogenic response (233). Similar observations with calf pulmonary artery endothelial cell and human microvascular endothelial cells (HMEC-1) were also made (234). The induced capillary formation of HMEC-1 in response to H A was blocked by a CD44 antibody, suggesting that CD44/HA interactions mediate this process (234). Singleton and Bourguignon showed that an isoform of CD44 containing variant exon vlO (CD44vl0), expressed by bovine aortic endothelial cell, interacts with H A . This interaction resulted in calcium mobilization and migration of these cells (235).  Knockout mouse phenotype  Two independent CD44 knockout mouse lines have been generated (236, 237). In both cases, it was surprising to find that the mice survived embryonic development without gross anomaly, even though impaired migration of lymphocytes into peripheral  29  lymph nodes and thymus was observed by Protin and co-workers. Overall, the mice were healthy and fertile. These results were counter-intuitive since CD44 has been implicated in such a wide range of biological functions, particularly when the role of cellular migration has been suggested in a variety of cell types. Moreover, CD44 expression pattern at days 9.5 to 12.5 of mouse embryo was found to coincide with where H A was found to mediate developmental events (238). Moreover, gene-targeted disruption of hyaluronan synthase-2 proved lethal at the embryonic stage (239). These could be explained, however, by several possibilities. First, the role of CD44 may be mainly to mediate inflammatory events, such as leukocyte recruitment, lymphocyte activation, or wound healing. Therefore, the role in embryonic development may not be the major function of this adhesion molecule. Second, there might be H A binding molecules that compensate for CD44 during development. The knockout mice generated were C57/B6, which Lynch et al. reported as low CD44 expressors (240). Since this genetic background was "purified" through generations of inbreeding, a possibility is that this strain with low dependence on CD44 during embryonic development was selected. In other words, compensation mechanisms by other adhesion molecules were selected for during the inbreeding process. The biological functions of CD44 in various cell types described above are attributed mostly to the binding of CD44 to H A . Moreover, in most of these processes, CD44 signaling and CD44-mediated cytoskeletal rearrangement seem to play a fundamental role. However, it is only recently that the role of CD44 as a signaling molecule was appreciated.  1.3.5  CD 44 signaling The signaling ability of CD44 has been demonstrated in a variety of cell types.  Local application of soluble H A has been shown to induce localized lamellipodial outgrowth in Eph4 murine epithelial cells (241). This formation of this CD44-induced localized cytoplasmic extension was inhibited by a CD44 blocking antibody and microinjection of a dominant-negative form of Rac. This was not inhibited, however, by a dominant-negative Cdc42. This observation suggests that the CD44/HA interaction activates the small GTPase Rac, resulting in local actin rearrangement. Moreover,  30  several studies with cancer cell lines suggest that CD44 is capable of activating Rac through its association with guanine nucleotide exchange factors, such as Tiaml (242244). Interaction between oligomer H A and CD44 on endothelial cells has been shown to induce migration, as well as angiogenic response, in bovine vascular endothelial cells and human microvascular endothelial cell line HMEC-1 (232, 245). This involved the stimulation of P K C , Raf-1, M E K - 1 , and ERK-1 (246). Moreover, a CD44 isoform expressed by bovine aortic endothelial cells was shown to mediate calcium signaling that is associated with cellular migration (235). The co-stimulatory property of CD44 suggests that cellular signals were initiated by engagement of this adhesion molecule (204, 205). Bourguignon et al. showed that CD44/HA interaction on BW5147 murine T cells induces the capping of CD44, in conjunction with an increase of intracellular calcium (247). Taher et al. showed that cross-linking of CD44 with antibodies generate tyrosine phosphorylation signals (248). This signal was dependent on Src-family kinases (SFK), Lck, leading to phosphorylation of ZAP-70. Later, it was shown that a population of CD44 is partitioned in lipid rafts and this population of CD44 is associated with the SFKs Lck and Fyn (249). CD44 is also capable of mediating cell signals in T cells that lead to cell spreading. Foger et al. (250) reported that incubation of a murine T helper cell line on immobilized CD44 antibody results in actin- and microtubule-dependent cell spreading. This CD44-induced cell spreading is accompanied by the recruitment of Rac with CD44 and Fyn into lipid rafts. The activities of both Rac and Src-family kinases are required for this cell spreading. However, the cell spreading does not require activities of PI3K. The CD44-mediated elongated cell spreading reported by L i et al. is also dependent on Src-family kinases (251). However, this cell spreading observed was negatively regulated by CD45. Associated with this cell spreading was the induction of tyrosine phosphorylation of proteins that were 120-130 kDa in molecular weight. Three signaling molecules around that range of molecular weight were identified. They were Pyk2, F A K , and an isoform of Cas. Recently, Fanning et al. demonstrated that activated human peripheral T lymphocytes were able to migrate on immobilized CD44 antibody or in three-dimensional matrix incorporated with H A (193). Moreover, they showed that this  31  CD44-dependent migration required P K C activity and that CD44-induced cell polarization was associated with translocation of PKCfS and PKCS to the M T O C , which is similar to what occurs LFA-1-mediated migration on immobilized ICAM-1 (252). Furthermore, Katakai and co-workers have shown that stimulation of T h l cells with CD44 and a capturing secondary Ab induces elongated cell spreading. By using specific pharmacological inhibitors, the authors showed that this cell spreading is dependent on SFK, PI3K, and PLC (192). These reports have clearly demonstrated that CD44 is capable of mediating cellular signals, particularly in relation to cytoskeletal changes and cell migration. However, the CD44 signals leading to such cytoskeletal rearrangement are not well understood, especially in hematopoietic cells. Therefore, one of the aims of this thesis was to determine the signaling components involved in CD44 signaling pathways in that leads to actin rearrangement and elongated cell spreading in T cells. Moreover, as mentioned above, since the transmembrane phosphosphaste CD45 was shown to inhibit CD44-mediated elongated cell spreading (251), it is also the aim of this thesis to determine how CD45 regulates such signaling.  1.4  CD45  1.4.1  Structure CD45 was first described as an abundant protein that made up about 10% of  surface proteins on lymphocytes (253). The sequence homology of CD45 with PTP1B first suggested that it was a protein tyrosine phosphatase (254) and experimental evidence supporting this was first provided by Tonks et al. (255). CD45 is expressed by all nucleated hematopoietic cells and its role in T cell development and activation has been well established (reviewed in 256, 257). CD45 is a type I transmembrane glycoprotein (figure 1.6). It possesses an extracellular domain of various sizes resulting from alternative splicing, giving it a size from 180 to 220 kDa when resolved on SDS-PAGE. The three exons that can be variably added to the extracellular domain of CD45 are termed as A , B , and C. When all three variant exons are present, CD45 is referred to as CD45 R A B C . When none of the varaint exon is present, CD45 is referred to as CD45RO. The expression of CD45 variants is  32  Alternatively Spliced Exons  Cysteine-rich Domain  Fnlll-like Repeats  Transmembrane  Protein Tyrosine Phosphatase Domains  Figure 1.6  CD45  is a prototypic protein tyrosine phosphatase, the activity of which lies in cytoplasmic domain D1. The ectodomain of CD45 is heavily glycosylated. Various isoforms of CD45 exist as a result of alternative splicing. CD45  33  related to the differentiation stage of certain cell types (reviewed in 258). For example, memory T cells usually express CD45RO while naive T cells express the CD45 R B isoform. The extracellular domain of CD45 is extensively modified by N - and O-linked glycosylation (259, 260). The ectodomain modification and the change in isoform expression suggest potential ligand of CD45, possibly associated with cell stage-specific functions. However, no specific ligand has been identified thus far. The cytoplasmic domain of CD45 contains two phosphatase-like domains. It has been found that the membrane-proximal domain D l is where the phosphatase activity of CD45 resides (261-263). At present, it is still uncertain what the main function of D2 is; however, it has been suggested that it may be involved in regulation of CD45 activity (264) or modulate substrate binding by CD45 (265).  1.4.2  CD45 in T cell development The importance of CD45 in T cell development was demonstrated by targeted  disruption of the CD45 gene. A significant defect in T cell development was observed in CD45" mice constructed by different groups (266, 267). Moreover, a CD45 gene A  mutation, resulting in lack of CD45 expression, was identified in a patient. Similar to the CD45-defective mice, this patient also showed low peripheral blood T cell count (268). In the knockout mice, transitions from CD4"CD8" to C D 4 C D 8 and C D 4 C D 8 +  +  +  +  to single positive (CD4 or CD8 ) T cells are impaired. However, re-expression of Lck +  +  Y505F restored thymocyte development (269). This further supports the role of CD45 in dephosphorylating the inhibitory tyrosine phosphorylation site at the C-terminus of Lck.  1.4.3  Roles of CD45 in T cell activation The role of CD45 in T cell activation was first provided by Pingel and Thomas.  The researchers showed that the expression of CD45 was required for T cell proliferation in response to antigen (270). Others have also observed the requirement of CD45 in effective T cell activation (271-274). In particular, these studies showed that the phosphatase activity of CD45 is indispensable for TCR signaling. CD45 is thought to regulate T C R signaling through its regulation of Lck and Fyn, since these SFK are the major substrates of CD45 in T cells (reviewed in 275). The  34  positive regulatory function of CD45 is supported by several lines of evidence. First, Lck is indispensable for T cell activation (276, 277). Second, CD45 can dephosphorylate Lck efficiently and the consequence of such dephosphorylation is enhanced kinase activity (278, 279). Third, crystal structures of SFK members, Hck, Src and active Lck (280282), have suggested an intramolecular model of kinase activity regulation. In this model, the C-terminal tyrosine phosphorylation is inhibitory to the kinase function. This is due to an intramolecular interaction between the phosphotyrosine and the SH2 domain. This forms a closed conformation, inhibiting accessibility of the kinase domain. Moreover, this interaction is stabilized by another intramolecular interaction bewteen the SH3 and the linker region between the SH2 and the kinase domains. Fourth, in CD45defective cell lines, the C-terminal tyrosine was preferentially phosphorylated (283, 284). Moreover, the C-terminal Src kinase (Csk) that preferentially phosphorylates the Cterminal tyrosine residue of Lck has also been identified (285), suggesting the opposing functions of Csk and CD45 in regulating Lck activity. In view of the above observations, a model of CD45 function, in positively regulating TCR signaling, has been widely accepted. However, recent observations have suggested that CD45 may also negatively regulate Lck. First, the hyperphosphorylated Lck from thymocytes of CD45" mouse was found to possess higher kinase activity A  (286). Second, CD45 was shown to be able to dephosphorylate Y394, which is required for full kinase activity (284, 287). Third, lipid raft-targeting of a CD45 chimera was shown to down-regulate TCR signaling (288). Since Lck is recruited to lipid rafts to initiate TCR signaling (289), the forced association between lipid rafts and CD45 that resulted in inhibition to TCR signaling suggests that CD45 can down regulate Lck activity. It has been suggested that this dual role of CD45 in T cell activation may be explained by segregation of CD45 and TCR/pMHC. This is supported by the initial recruitment of CD45 to the c S M A C and exclusion of CD45 at later time points (60). Moreover, it has been observed that a small population of CD45 resides in the lipid rafts in resting T cells, yet upon TCR stimulation, CD45 was excluded from the lipid rafts (290).  35  1.4.4  CD45 in other signaling events Other than its regulatory role in T cell activation, CD45 has also been shown to  play a positive role in SDF-1 signaling. T cell migration in response to SDF-1 stimulation was significantly reduced in the absence of CD45. Moreover, SDF-1 stimulation was shown to activate Lck, but this does not occur in CD45" T cells (291). CD45 has also been shown to down-regulate integrin-mediated adhesion and signaling. T cells that are CD45-deficient show enhanced adhesive function through a5|31 integrin, yet the adhesive function of a4(31 integrin is not affected (292). Moreover, re-transfection with a CD45 construct containing the transmembrane and cytoplasmic domains, with intact phosphatase activity, was able to restore normal ct5|31 integrin adhesive function. Furthermore, deregulation of (32 integrin-mediated adhesion was also observed in CD45" macrophages derived from knockout mice (293). The CD45 macrophages showed initial enhanced adhesion; however, these macrophages were unable to maintain (32-integrin-dependent adhesion. The authors suggested that the phenomenon was likely due to deregulation of the SFKs Hck and Lyn in the CD45" " 7  macrophages.  1.5  Thesis Objectives  The observations reported above suggest that the role of CD45 is mainly exerted on its regulation of SFKs. Since SFKs are involved in a variety of signaling functions, the biological significance of CD45 is likely not limited to its role in TCR signaling. Consistent with the negative regulatory role of CD45 on integrin function, observations made by L i et al. have also suggested that CD45 negatively regulates CD44 signaling (251). As described above, CD44 is family of adhesion molecules that has been shown to play a role in cellular migration. Moreover, the ability of CD44 in inducing cellular signals, leading to cytoskeletal rearrangement, has been shown. However, since the specific signals that CD44 induces were unclear, the aim of this study was to identify the signaling components in this pathway. Furthermore, although the role of CD45 in T cell activation and development is well established, its role in adhesion-related events is not. Another aim of this thesis was, thus, to determine how CD45 regulates CD44 signaling.  36  Chapter 2  Materials and Methods  2.1  Materials  2.1.1  Cell culture CD45 and CD45" BW5147 T cells were obtained from American Type Cell +  Culture (ATCC, Manassas, V A ) and were transfected with CD3£ and 5 chains for T cell receptor (TCR) expression (294). These cells were kept in Dulbecco's modified Eagle medium (DMEM)/10% heat-inactivated horse serum/2 m M L-glutamine/2 m M sodium pyruvate at 37°C with 5% C 0 . L-histodinol (Sigma-Aldrich, St. Louis, MO) at 0.3 m M 2  was utilized to maintain plasmid expression. Murine A K R T cells were from A T C C and were transfected with full-length CD44 (CD44wt) (179) or CD44 containing only 2 amino acids of the cytoplasmic domain (CD44Acy). These cells were kept in D M E M with 10% heat-inactivated horse serum, 2 m M L-glutamine, and 2 m M sodium pyruvate at 37°C with 5% CO2. Cells were kept in the presence of 1.5 mg/mL Geneticin 418 (50-75% active) (Invitrogen, Burlington, ON) to maintain CD44 expression. Thymocytes were harvested from CD45 knockout (266) and C57/B6 mice (The Jackson Laboratory, Bar Harbor, ME) were kept in RPMI/10% heat-inactivated fetal bovine serum (FBS)/2 m M L-glutamine/2 m M sodium pyruvate at 37°C with 5% CO2. The thymocytes were activated with 12.5 ng/mL P M A (Sigma-Aldrich, St. Louis, MO) and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence of 20 units/mL recombinant IL-2 (R&D Systems, Minneapolis, MN). On the 4 day, the medium containing P M A th  and ionomycin were removed and replaced with fresh medium containing IL-2. Activated thymocytes were used for cell spreading assays between days 5 and 10.  2.1.2 Reagents A l l the antibodies utilized for immunoprecipitation, Western blotting, and fluorescent labeling are listed in table 2.1. The pharmacological reagents PP2, LY294002, U73122, U73343, and Latrunculin A were from Calbiochem (La Jolla, CA). These agents were reconstituted with dimethyl sulfoxide (DMSO, Sigma-Aldrich) according to the suggestion of the  37  manufacturer. After reconstitution, the pharmacological agents were kept in small aliquots at -20°C.  Table 2.1  List of primary antibodies utilized in immunoprecipitation or fluorescent labeling  Antibodies  Antigen Recognized  Manufacturer/Reference  4G10  Phosphotyrosine residues  Upstate Biotechnology  54-3B  Lck  (295)  Anti-Fyn (sc-16)  Fyn  Santa Cruz Biotechnology  Anti-PLCyl (sc-1249)  PLCyl  Santa Cruz Biotechnology  Anti-Pyk2(sc-1515)  Pyk2  Santa Cruz Biotechnology  Anti-Cas  Cas  B D Transduction  13/2  CD45  (296)  J1WBB  CD44  (251)  KM201  CD44  (214)  KM81  CD44  (166)  Phospho-p44/42 M A P  Phosphorylated Thr 202 and  Cell Signaling Techology  kinase  Tyr 204  pS473-Akt  Phosphorylated S374-Akt  Cell Signaling Technology  pY394-Lck  Phosphorylated Y394-Lck  (372)  pY402-Pyk2  Phosphorylated Y402-Pyk2  Biosource Inc.  TIB213  LFA-1  ATCC  R17  Transferrin receptor  (297)  38  2.2  Methods  2.2.1  Flow cytometry Flow cytommetry was used to assess levels of expression of cell surface  molecules. These include CD44, L F A - 1 , and VSVG-tagged PTPa. Cells were labeled by incubation with primary antibody in 96-well plate for 20 minutes on ice. The cells were then washed once with PBS/2 % horse serum and incubated with FITC-conjugated secondary antibody. After the incubation with secondary antibody on ice for 20 minutes, the cells were washed twice with PBS/2 % horse serum and transferred to flow cytommetry tubes. Non-viable cells were labeled with either propidium iodide or 7-AAD and excluded from analysis. Generally, 2 x 10 cells were labeled for each sample and 5  5000 counts were collected. To avoid measurement of auto-fluorescence, cells without incubation of antibody were included as a control. To ensure specificity of fluorescence signals, cells incubated with only FITC-conjugated or Alexa488-conjugated secondary Ab were also included as controls.  2.2.2  Cell spreading assay Monoclonal antibodies (mAb) were immobilized on 96-well plates (tissue  culture-treated plates, Falcon or Nunc) at 40 ixg/ml in PBS overnight at 4°C. The wells were blocked with 2% B S A (Sigma-Aldrich) in PBS at 37°C for 2 hours, and were washed three times with PBS before the incubation of cells. Cells were washed once with pre-warmed D M E M alone and resuspended at 10 cells/mL in pre-warmed cell 6  spreading medium (DMEM/0.1% heat-inactivated fetal bovine serum/2 m M L glutamine/2 m M sodium pyruvate). Fifty microliters of cell suspension was applied to each well with either immobilized mAb or B S A alone as a control. Cells were incubated at 37°C with 5% CO2 for various periods before fixation by the addition of 17 uL of 16% paraformaldehyde. Digital images of cells were taken with Nikon coolpix 950 mounted on Nikon inverted microscope TS 100 with a 20x objective. Measurement of cell length was carried out and calibrated with a microscope stage ruler. For each sample, three independent experiments were done and more than 100 cells/sample were measured from randomly selected fields. Significant difference of cell length was determined using  39  Student's 2-tail unpaired t-test. Graph plotting and statistical analysis were carried out with Microsoft Excel. For determination of the effect of the pharmacological or chemical agents on cell spreading, cells were incubated in cell spreading medium with the agent at the desired concentration for 30 minutes at 37°C with 5% CO2. Control pretreatment with cell spreading medium containing D M S O was included when it was utilized for reconstitution of the pharmacological agent. After incubation, the cells were applied to wells containing immobilized Ab or B S A . The experiment was then carried out as described above. To detect the tyrosine phosphorylation signal from cells incubated on immobilized Ab or BSA, 30 uL of 3x reducing gel loading buffer (0.375M Tris-HCl, pH 6.8; 30% glycerol; 6% SDS; 15% |3-merceptoefhanol; 0.03% bromophenol blue) was added to each well. Cell lysate was collected and boiled for 5 minutes before resolving with polyacrylamide gel. The proteins were then transferred to PVDF (Immobilon P, Millipore) membrane for Western blotting.  2.2.3  Immunoprecipitation CD45 and CD45" T cells were washed once with pre-warmed D M E M and +  suspended in cell spreading medium at 5 x 10 cells/mL. One milliliter of cell suspension 6  was applied to each well of a 6-well plate (Nunc) pre-coated with KM81 and blocked with 2% BSA/PBS. For pre-treatment with pharmacological agents, cells were incubated for 30 minutes at 37°C with 5% CO2 before applying onto wells with immobilized mAb. To harvest cells at due time, 250 ul of ice-cold 5x lysis buffer (5% Triton X-100; 50 m M Tris-HCl, pH7.2; 700 m M KC1; 10 m M E D T A , pH8.0; 2.5 m M sodium orthovanadate; 1 m M sodium molybdate; 5 mg/mL aprotinin; 5 mg/mL leupeptin; 5 mg/mL pepstatin; and 1 m M phenylmethyl sulfonyl fluoride) was added to each well. For 0-minute time point, cells were kept in suspension before lysis. Cell lysate samples were then transferred to Eppendorf tubes and allowed to sit on ice for 5 minutes. The Triton-insoluble fraction was cleared by centrifugation at 16.1 x 10 g at 4°C for 10 minutes. Cell lysate was 3  transferred to fresh Eppendorf tubes with the addition of 1 u,g of immunoprecipitating antibody to each sample. The samples were incubated at 4°C for 1 hr with rotation.  40  Protein A (Repligen, Waltham, M A ) or G beads (Amersham Pharmacia) were washed with ice-cold l x lysis buffer before application to each IP sample. The usage of either protein A or protein G beads was determined by the host species of the IP Ab; the beads/Ab combination that gives better affinity was utilized for IP. After 1 hour of rotation at 4°C, 20 uL of protein A or G beads 50% slurry were added to each IP sample. The tubes were incubated further at 4°C for 1 hour. After the incubation, protein beads were collected by brief centrifugation. The cell lysate was removed with suction. Beads from each IP sample were washed three times with ice-cold l x lysis buffer before the addition of 20 uL of 3x reducing SDS-PAGE loading buffer to harvest precipitated proteins. The samples were then boiled for 5 minutes before loading onto 7.5-12.5% polyacrylamide gel for resolution of samples. Protein bands were transferred to PVDF membrane at a constant voltage of 100V for 60 minutes. Membrane was air dried before probing with antibodies. Immunoprecipitation (IP) controls include cell lysate only, cell lysate with protein A or G beads only, and antibody with no cell lysate.  2.2.4  Western blotting For detection of tyrosine phosphorylation signal, the blot was incubation with  4G10 at 1/5000 in 0.5% w/v BSA/TBST for 1.5 hours with gentle shaking at room temperature. After brief washes with TBST (20 m M Tris-HCl, pH 7.5, 150 m M NaCl, 0.1% v/v Tween 20), the blot was incubated with horseradish phosphatase (HRP)conjugated anti-mouse IgG (Southern Biotechnology) for 1 hour. The blot was then washed at least three times with TBST. A final wash of at least 30 minutes with TBST was carried out before visualization of bands with E C L or E C L plus (Amersham Biosciences, Uppsala, Sweden). To detect signals for Pyk2, pY402-Pyk2, Cas, P L C y l , pS473-Akt, or Akt, the antibody was diluted in 5% w/v skim milk powder/TBST. The PVDF membrane was incubated with primary Ab at room temperature for 1 hour. After a few brief washes with TBST, the membrane was incubated in 1/5000 HRP-conjugated anti-goat IgG or HRP-conjugated anti-rabbit IgG, depending on the host species of the primary antibody, for 1 hour. After washes with TBST, the protein bands were visualized chemi luminescence.  41  When reprobing and stripping was required, the membrane was first incubated in methanol and washed briefly with TBS before incubation in stripping buffer (50 m M glycine, pH 2.5, 150 m M NaCl, 0.1% v/v Nonidet P-40) for 30 minutes at RT. Membrane was washed several times with TBST before reprobing.  2.2.5  Labeling for Confocal Microscopy Chamber slides (Lab-Tek®, Naperville, IL) were coated overnight with KM81 at  40 mg/ml in PBS at 4°C. The wells of the chamber slides were blocked with 2% BSA/PBS for 2 hours at 37°C and were washed three times with PBS prior to incubation with cells. CD45 or CD45" T cells were washed once with pre-warmed D M E and +  resuspended in cell spreading medium at 10 cells/ml. For pretreatment with 6  pharmacological agents, cells were resuspended in the same cell density with the desirable concentrations of the pharmacological agents. The optimal concentrations for the pharmacological agents were determined by titration. For the experiments carried out in this study, the concentrations utilized were 20 u M PP2, 20 u M LY294002, 0.5 u M U73343, and 0.5 u M U73122. Cells were then incubated at 37°C with 5% C 0 for 30 2  minutes prior to application to wells with immobilized antibody or BSA. One hundred and fifty microliters of cells were added to each well. At various time points, cells were fixed with 4% paraformaldehyde at room temperature (RT) for 20 minutes. The cells were washed three times with PBS before they were permeabilized with 0.1% Triton X 100 in PBS for 10 minutes at RT. To avoid non-specific binding of labeling antibodies, cells were incubated with 1% B S A at RT for 30 minutes. Dilutions of primary antibodies were made in 1% B S A and the dilutions utilized were as follows: 1/50 4G10, 1/50 Alexa Fluor 488-conjugated 13/2, 1/10 anti-pY394-Lck, 1/100 54-3B, 1/100 J1WBB, 1/100 anti-Fyn antibody. Labeling with primary antibodies was carried out at RT for 1 hour. For F-actin labeling, 1/40 dilution of Alexa488-conjugated phalloidin was made in PBS and overnight labeling was carried out at 4°C. After washing the cells three times with PBS, corresponding Alexa Fluor-conjugated antibodies were applied at 1/100 dilution in 1%BSA/PBS. After another hour of incubation, cells were washed at least three times with PBS. The slides were then air dried and mounted with 90% glycerol/2.5% 1,4Diazabicyclo-(2,2,2)-octan (DABCO, Sigma) and coverslip (no.1 thickness, 0.13 to 0.17  42  mm, V W R International). Alexa Fluor-conjugated secondary antibody alone control was included in each experiment to ascertain the absence of non-specific reactivity between the secondary antibody and any cellular component. For double labeling, the staining was carried out in a sequential manner with four antibody labeling steps. The sequence of staining with double labeling of phosphotyrosine and CD44 was 4G10, Alexa488conjugated anti-mouse IgG, J l W B B , and Alexa568-conjugated anti-rabbit IgG. Control labeling without J l W B B was also carried out to ensure no cross-reactivity occurred between Alexa568-conjugated anti-rabbit IgG and 4G10 or Alexa488-conjugated antimouse IgG. Same controls were included in CD45 and CD44 double labeling.  2.2.6  Image collection with confocal microscopy Images of labeled cells were captured with Bio-Rad Radiance 2000 with krypton  lasers (Bio-Rad, Hercules, CA) on Nikon Eclipse TE300 at RT, using a 60x oil immersion objective. To ensure specific fluorescent signals, fluorescence intensities between the experimental proper was always compared with secondary antibody alone control (no primary antibody). Optimal iris sizes were utilized for image collection for all experiments. For this specific setup, the iris sizes for 488 nm and 568 nm signals were 1.6 and 1.8 mm, respectively. The images were collected with scanning speed of 166 lines per second and Kalman collection filter (2x) with step size of 0.3 u.m. Image size was 512 by 512 pixels covering the dimension of 162 by 162 u.m. For double labeling experiments, images were collected with the same settings in a sequential manner. For any given experiment, the same laser power and gain controls settings were utilized to ensure consistent signal intensities. Cells from random fields were collected and analyzed from at least three independent experiments.  2.2.7  Image Processing Light microscopy images taken with Nikon coolpix 950 were opened in Adobe  Photoshop 4.0 L E or CS. Cell images were made into grayscale pictures with the adjustment of image size to 4" x 3" and with the resolution of 300 pixels per inch. Image/adjustments/levels command of Photoshop was utilized to enhance visibility of cell border for cell length measurement. Images presented in figures 1 and 2 were 1" by  43  1" images cropped as representative images and saved with resolution of 600 pixels per inch. Fluorescence images collected using confocal microscopy were first processed in either N I H image or ImageJ (NIH, Bethesda, MD). After a stack of confocal images was opened in NIH image or ImageJ, one of the images close to the interface between the cells and the slide was selected. A duplicate image was then made and re-opened in Photoshop. The image/adjustments/levels command was utilized to adjust image contrast and all adjustments were applied to the whole image. Representative image was cropped as 1" x 1" picture and saved with the resolution of 600 pixels per inch. For images required for direct comparison (tyrosine phosphorylation levels in figures 4.6B and 4.11), enhancement of visibility by adjusting brightness/contrast was applied to all images at the same time. The side view cell image in figure 4.5B was generated by three-dimensional projection. It was produced with the Image/Stacks/3D projection function of ImageJ with the following parameters: mean value projection, rotation around x-axis, slice spacing of 0.3 micron, initial angle of 0°, total rotation of 180°, rotation angle increment of 10, lower transparency bound as 1, upper transparency bound as 255, opacity of 0%, surface depthcueing as 100%, and interior depth cueing of 50. Re-coloring of images for merging was done in ImageJ by converting the image to R G B type image and by using R G B recolor plug-in. Red or green re-coloring was done by adjusting red or green scaling factor as 1 while the constants remained as 0. Merging of color image was done by using Image/Color/RGB merge command in ImageJ.  44  Chapter 3  CD44 proximal signaling associated with actin rearrangement and elongated cell spreading in T cells  3.1  Introduction CD44 is a widely expressed adhesion molecule, involved in mediating cellular  migration, particularly in cancer metastasis. In T cells, the role of CD44 in T cell migration to sites of inflammation has also been demonstrated in several mouse models (194, 195, 197). Another function of CD44 that has also been implicated in T cell biology is co-stimulation (204, 205, 298). In these two processes, a common requirement is the efficient re-organization of the actin cytoskeleton (8). Previously, our laboratory observed that CD45" BW5147 and S A K R murine T cells displayed an elongated cell spreading morphology when plated on immobilized CD44 antibody (251). Since cell spreading on a substratum requires actin rearrangement, this observation suggests that CD44 is capable of mediating this cytoskeletal reorganization. Moreover, these observations suggest that CD45 plays a role in regulating CD44-mediated actin rearrangement and cell spreading. When this study was initiated, the signaling pathway that CD44 utilizes to regulate actin rearrangement was unclear. Therefore, one aim of this study was to identify the signaling pathway that is associated with CD44-mediated actin rearrangement and cell spreading.  3.2  Results  3.2.1  CD44 mediates cell spreading of T cells To determine i f CD44 is capable of mediating cell spreading in CD45 BW5147 +  T cells, these cells were incubated on immobilized CD44 antibody (KM201 or KM81) for 2 hours. These cells were then fixed with 4% paraformaldehyde and photographed. Since spreading cells should display changes in cell length (the longest measurement), this measurement was used, in a quantitative manner, as a means to determine if cell spreading occurs. CD45" T cells were included as positive cell spreading control. These cells were also incubated on immobilized B S A as a negative cell spreading control. In general, elongated cell spreading of CD45" cells was observed within 30 minutes of incubation on immobilized CD44 antibody. By 2 hours, the development of  45  spread morphology was completed. Neither CD45 nor CD45" BW5147 T cells were +  adherent to immobilized B S A (fig. 3.1 A); these cells, therefore, did not display cell spreading and they showed comparable cell length (fig. 3. IB). The lengths of these cells were smaller, when compared to the length of spreading cells incubated on immobilized KM81 (fig. 3. IB). When plated on immobilized anti-CD44 (KM81), CD45 T cells +  displayed a roughly round (non-directionally biased) cell spreading, while the CD45" T cells displayed an elongated cell spreading, as previously observed (251). When the lengths of the cells were measured and compared, there was a significant difference between the CD45 T cells incubated on B S A and those incubated on K M 8 1 . A n 11% +  (p<0.01, n>250 from 3 independent experiments) increase in cell length was observed in CD45 T cells due to cell spreading on immobilized CD44 antibody. In CD45" T cells, a +  dramatic difference of cell length was observed between cells incubated on B S A and those incubated on KM81 (69% increase, p<0.01, n>250 from 3 independent experiments). The difference in cell spreading lengths or morphologies was not due to a difference in CD44 expression levels in CD45 versus CD45" T cells (fig. 3.1C). +  To determine if cell spreading is induced by incubation of cells on immobilized antibody directed against other cell surface molecules, CD45 and CD45" BW5147 T +  cells were incubated on immobilized transferrin receptor (TfR) antibody as a comparison to CD44-mediated cell spreading. Figure 3.2A shows representative figures of at least three independent experiments. Incubation of BW5147 T cells on immobilized CD44 antibody resulted in cell spreading, as reported above. However, incubation of BW5147 T cells on anti-TfR did not generally result in cell spreading. Complete absence of cell spreading was observed in CD45 T cells, while only 14% of CD45" T cells displayed +  spreading (n=348 from 3 independent experiments). Both CD45 and CD45" BW5147 T +  cells express comparable levels of transferrin receptor, as shown by results of flow cytometry in figure 3.2B. These results show that CD44 is capable of mediating cell spreading, in both CD45 and CD45" T cells, but with different cell morphologies. On +  the other hand, TfR is ineffective in mediating cell spreading.  46  A  Light microscopy photographs  B  Histogram of mean cell length with standard deviation p<0.01  CD45"  1  I  E  30  •&S 20 c «  BSA  p<0.01 I 1  -J—, J_  O  CD45 Substratum  + + BSA CD44 BSA CD44 mAb mAb  KM81 C  CD44 expression levels measured by flow cytometry CD44  c 3  o o  a> O  I  2° Ab control  +  Cells only  V  I 0  CD45  CD44  2° Ab control  10  1  10 10 2  CD45"  Cells only  3  Fluorescence Intensity  Figure 3.1 CD44 mediates cell spreading on immobilized antibody. (A) C D 4 5 and CD45" BW5147T cells incubated on immobilized CD44 Ab for 2 hours were fixed in 4% paraformaldehyde and photographed. Neither did C D 4 5 nor CD45" T cells spread on immobilized BSA. However, C D 4 5 cells displayed a round spreading on immobilized CD44 antibody while CD45" T cells displayed elongated cell spreading. The scale bar denotes the length of 10 Lim. (B) The histogram shows the mean cell length with the standard deviation. Significant difference (p<0.01) between the mean length of cells incubated on BSA and on CD44 mAb was observed. (C) CD44 expression levels on C D 4 5 and CD45" BW5147T cells. +  +  +  47  +  A  Light microscopy photographs CD45  CD45"  +  KM81  R17 B  TfR expression levels byflowcytometry TfR 2° Ab control  CD45'  1  Cells only  c 3  o O  "5  TfR  O  2° Ab control  0  10  1  10  2  10  CD45"  Cells only  3  Fluorescence Intensity  Fig. 3.2 Immobilized transferrin receptor (TfR) antibody does not induce cell spreading in BW5147 T cells. (A) After 2 hours of incubation, C D 4 5 T cells and CD45" T cells displayed round and elongated cells spreading on immobilized KM81. However, immobilized transferrin receptor antibody (R17) did not induce similar cell spreading in these cells. The scale bar denotes the length of 10 um. (B) C D 4 5 and CD45" BW5147 T cells express comparable levels of transferrin receptor. +  +  48  3.2.2  The cytoplasmic domain of CD44 is requiredfor mediating cell spreading CD44 has been found to reside in lipid rafts (299). These are sphingolipid-rich  compartments of the cell membrane that are thought to be important for organization of signaling molecules (300). When the BW5147 T cells are incubated on immobilized K M 8 1 , CD44 cross-linking may occur and lead to aggregation of lipid rafts. In order to determine i f CD44-mediated actin rearrangement and cell spreading was due to the aggregation of lipid rafts, A K R murine T cells transfected with either full-length CD44 (AKRwt44) or a cytoplasmic truncation mutant (AKR44Acyto) were utilized. The distribution of wild-type CD44 and cytoplasmic truncation mutant in the lipid rafts was the same (Maeshima and Johnson, unpublished results). The transfected A K R cells were incubated on immobilized KM81 for 2 hours before fixation. The cells were then photographed. Measurement of cell length was taken to determine if cell spreading was influenced by truncation of the cytoplasmic domain of CD44. Figure 3.3A shows that AKRwt44 or AKR44Acyto cells do not spread on immobilized BSA. AKRwt44 showed spreading similar to that of CD45 BW5147 T +  cells, i.e. round, non-directionally biased spreading, whereas AKR44Acyto cells displayed minimal cell spreading. The cell length of the A K R cells is shown in Figure 3.3B. A significant decrease of cell length was observed with AKR44Acyto cells, when compared to the spreading cell length of AKRwt44 cells (pO.Ol, n>100 from 3 independent experiments). These data show that the cytoplasmic domain of CD44 is required for mediating cell spreading, negating the possibility that CD44-induced lipid raft aggregation is sufficient to cause actin rearrangement and cell spreading.  3.2.3  CD44-induced elongated cell spreading requires extracellular calcium, phospholipase C (PLC), andphosphoinositide-3'-kinase (PI3K) The difference between CD45 and CD45" BW5147 T cell spread morphologies +  suggests that CD45 negatively regulates a CD44-mediated signal that leads to elongated T cell spreading. This elongated cell spreading morphology is an asymmetric shape that suggests a directional bias, resembling the morphology of T cells during directional migration. Therefore, I was interested in identifying the CD44 signaling pathway that leads to actin rearrangement and this directionally biased cell spreading morphology.  49  A  Light microscopy photographs AKRwt44  AKRAcyto  BSA r  KM81 B  Histogram of mean cell length with standard deviation  p<0.01 | 20 c  I  p<0.01 II  1  I o  AKRwt44 AKRAcyto BSA KM81 BSA KM81  Figure 3.3 Cytoplasmic domain of CD44 is required for cell spreading. (A) AKR T cells transfected with full-length CD44 (AKRwt44) displayed cell spreading on immobilized KM81 after two hours of incubation. AKR T cells transfected with a cytoplasmic truncation mutant of CD44 (AKRAcyto) showed significantly reduced cell spreading. The scale bar in the figure depicts the length of 10 (im. (B) The histogram shows the mean and standard deviation of cell length measurements. After 2 hours of incubation, the cells were fixed and the measurements were taken. A significant increase of cell length (p<0.01) was observed with AKRwt44 cells, when compared to length of the cells incubated on BSA. The increase of cell spreading length was significantly decreased (p<0.01) when cytoplasmic truncation mutation was introduced into CD44.  50  Previous observations from our laboratory showed that this directionally biased cell spreading morphology was accompanied by tyrosine phosphorylation of signaling proteins with molecular weight of 120-130 kDa (pi20/130) (251). Moreover, one of these phosphorylated signaling proteins was the calcium-dependent tyrosine rich kinase Pyk2. To determine i f CD44-mediated elongated cell spreading is dependent on calcium mobilization, CD45" BW5147 T cells were incubated on immobilized KM81 in the presence of extracellular ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), a known chelator of calcium ion. Cells were either fixed and photographed or lysed for probing of tyrosine phosphorylation signals with immundblots. Figure 3.4A shows the expected elongated, or directionally biased, spreading of CD45" T cells after incubation on immobilized KM81 in the absence of E G T A . In the presence of 5 or 10 m M E G T A , reduced elongated cell spreading was observed. Moreover, this was accompanied by loss of CD44-induced tyrosine phosphorylation of pi20/130 (Fig. 3.4B). Since the E G T A is not cell-permeable, the results indicate that calcium entry is required for CD44-induced signaling to cause elongated cell spreading and tyrosine phosphorylation of p i 20/130. TCR-induced calcium mobilization in T cells is a PLC-dependent process (301). To determine if this is also the case in CD44 signaling, leading to elongated cell spreading, CD45" BW5147 T cells were pretreated with a specific P L C inhibitor, 0.5 u M U73122, for 30 minutes before incubation of the cells on immobilized K M 8 1 . A negative pre-treatment control with a chemical analog, U73343, was also included. The P L C inhibitor abolished elongated cell spreading (Fig. 3.5A) and tyrosine phosphorylation of pi20/130 (Fig. 3.5B), while the inactive structural analog did not affect the CD44induced elongated cell spreading or the tyrosine phosphorylation. This suggests that CD44 signaling was likely to cause calcium mobilization through activation of P L C , leading to elongated cell spreading. Previous studies have shown that PI3K plays an indispensable role in cellular polarization and migration in Dictyostelium (302) and neutrophils (15, 303, 304). These migrating cells display an elongated cell shape where the longitudinal axis of the cell bodies is aligned with the direction of migration. To determine if the elongated cell  51  A  Light microscopy photographs  No EGTA B  5 mM EGTA  10 mM EGTA  Phosphotyrosine blot of whole-cell lysate CD45" -4- p120/130  Figure 3.4 Extracellular calcium is required for CD44-mediated elongated cell spreading and tyrosine phosphorylation of p120/130. (A) The CD44-induced elongated cell spreading of CD45" T cells on CD44 Ab was inhibited by EGTA. The cells were fixed after 2 hours of incubation on immobilized CD44 antibody and photographed. The scale bar denotes the length of 10 um. (B) Whole-cell lysate samples were harvested after 2 hours of incubation on immobilized CD44 antibody. The CD44-induced tyrosine phosphorylation is indicated by the arrowhead.  52  A  Light microscopy photographs  Medium B  0.5J_IM U 7 3 3 4 3  0.5 iM U 7 3 1 2 2 t  Phosphotyrosine blot of whole-cell lysate mw (kDa) 175—I ~4 p120/130 83-  62-  47.5medium Time (min.)  30  0.5 uM 0.5 uM U73343 U73122 30 30  Figure 3.5 PLC activity is required for CD44-induced elongated cell spreading and tyroine phosphorylation of p120/130. (A) When CD45" BW5147 T cells were pre-treated with a specific inhibitor U73122, elongated cell spreading on immobilized CD44 Ab was inhibited. No inhibition was observed with the negative control U73343. The cells shown have been incubated on immobilized CD44 antibody for 2 hours. The scale bar denotes the length of 10 um. (B) Whole-cell lysate samples were harvested after the cells had been incubated on immobilized CD44. Western blot of phosphotyrosine was shown above. The arrowhead indicates the induced phosphorylated p120/130, which was abolished by the pre-treatment of cells with 0.5 uM U73122.  53  spreading induced by CD44 involves also PI3K activity, CD45" T cells were pre-treated with a specific inhibitor, LY294002, before incubation of these cells on immobilized KM81. Pre-treatment of CD45" BW5147 cells with 20 u M LY294002 inhibited the elongated cell spreading induced by anti-CD44 (Fig. 3.6A), and this was accompanied by the abolishment of the tyrosine phosphorylation of pl20/130 (fig. 3.6B).  3.2.4  Cytoskeletal reorganization is required early during elongated cell spreading A n intact actin cytoskeleton is required for integrin signaling (305), although the  exact mechanism underlying this requirement is unclear. Moreover, the integrity of the actin cytoskeleton has been shown to affect CD3 signaling in T cells (306-308). To determine if an intact actin cytoskeleton is required for CD44-mediated signaling, CD45" BW5147 T cells were pre-treated with Latrunculin A , an inhibitor of actin polymerization (309, 310), before incubation of these cells on immobilized KM81. Figure 3.7A shows that inhibition of actin polymerization abolished CD44-induced elongated cell spreading. Moreover, actin polymerization was required to induce tyrosine phosphorylation of p i 20/130 in CD44 signaling, the mechanism of which will be further investigated in section 4.2.5. The cytoskeleton of lymphocytes is also composed of the microtubule network. To assess i f this component of the lymphocyte cytoskeleton is required for CD44mediated signaling and elongated cell spreading, CD45" T cells were treated with Taxol before incubating these cells on immobilized KM81. Taxol is an inhibitor that prevents depolymerization of microtubules into its subunits (311, 312). Therefore, the action of this inhibitor lies in blocking the dynamics of the microtubule network. The results show that breakdown of microtubules is required for CD44-mediated elongated cell spreading (fig. 3.8A), however, the signaling of CD44, leading to tyrosine phosphorylation of pi20/130, does not require microtubule disassembly (fig. 3.8B).  54  A  Light microscopy photographs DMSO control  LY294002  BSA  KM81  B  Phosphotyrosine blot of whole-cell lysate  175-  p120/130 83-  62-  47.5Time (min.)  0  Med 30  D LatA LY 30 30 30  Figure 3.6 PI3K is required for CD44-induced elongated cell spreading and p120/130 tyrosine phosphorylation. (A) The light microscopy photographs show CD45" BW5147 T cells incubated on BSA or KM81 for 2 hours. Pre-treatment with DMSO, the solvent carrier of LY294002, did not affect cell morphology, whether the cells were incubated on immobilized BSA or KM81. However, treatment of cells with 20 u.M LY294002 inhibited the elongated cell spreading of CD45" T cells on KM81. The scale bar denotes the length of 10 urn. (B) In B, "Med" denotes samples from cells incubated in cell spreading medium; "D" was the DMSO control; "Lat A" was samples with 1 u.M Latruculin A pre-treatment (section 4.2.4), and "LY" was samples with cells pre-treated with 20 u,M LY294002. The amount of time that the cells were incubated on immobilized KM81 was also indicated in minutes. The arrowhead indicates the phosphorylated p120/130. 55  A  Light microscopy photographs  v JM H S B M U H H I B I H P S H R H H H H ^ I H H H I ^ H B H H H medium  B  DMSO control  1 uM Lat A  2 uM Lat A  4 uM Lat A  Phosphotyrosine blot of whole-cell lysate mw(kDa) 175 ^  p120/130  47.5  32.5  B  M  D  1  2  4  Lat A (uM)  Figure 3.7 Actin polymerization is required for CD44-mediated elongated cell spreading and tyrosine phosphorylation of p120/130. (A) Pre-treatment of CD45" BW5147 T cells with Latrunculin A, and inhibitor actin polymerization, abolished CD44-mediated elongated cell spreading. Cells were fixed after two hours of incubation and photographed. The scale bar represents the length of 10 Lim. (B) Whole-cell lysate samples were obtained after cells were incubated on immobilized CD44 antibody for 30 minutes. Western blot of whole-cell lysate shows that CD44-induced tyrosine phosphorylation of p120/130 (indicated by the arrow) was inhibited also by Latrunculin A. "B" was sample from cells incubated on BSA. "M" was sample from cells incubated on KM81 with normal cell spreading medium. "D" was the DMSO control.  56  A  Light microscopy photographs  DMSO control  Medium  B  5 LIM Taxol  1 uM Taxol  Phosphotyrosine blot of whole-cell lysate  CD45"  CD45  ~< p120/130  B M  KM81 D 1  5  M  (iM Taxol  KM81 D 1  5  uM Taxol  Figure 3.8 Microtubules depolymerization is required for CD44-mediated elongated cell spreading, but not for tyrosine phosphorylation of p120/130. (A) After CD45" T cells were pre-treated with Taxol, an inhibitor of microtubule depolymerization, these cells no longer spread as elongated. Cells were fixed after 2 hour of incubation on immobilized CD44 antibody and photographed. The scale bar denotes the length of 10 um. (B) When CD45" T cells were incubated on BSA (samples marked as "B" in the figure), no induction of tyrosine phosphorylation was observed. Tyrosine phosphorylation of p120/130 was induced when cells were incubated on KM81 with cell spreading medium (M) or with the DMSO control (D). However, no inhibition of tyrosine phosphorylation was observed with Taxol pre-treatment.  57  3.2.5  Induced tyrosine phosphorylation of PLCyl is associated with elongated cell spreading As suggested by the results in section 3.2.3, the activity of PLC was required for  CD44-induced elongated cell spreading. In T cells, the major PLC isoform that is responsible for mediating TCR-induced calcium mobilization is P L C y l (301). To determine i f P L C y l was also activated during CD44 signaling, leading to elongated cell spreading, immunoprecipitation of P L C y l was performed. The activity of P L C y l correlates with its tyrosine phosphorylation (313-315). Therefore, induced tyrosine phosphorylation signal from Western blot with the immunoprecipitated P L C y l was used as the readout for activation. Results presented in figure 3.9 indicate that induced tyrosine phosphorylation of P L C y l in CD45" T cells was early and transient. Induced tyrosine phosphorylation was observable at 5 minutes after adding the cells to immobilized KM81. By 120 minutes, P L C y l was dephosphorylated. On the other hand, induced tyrosine phosphorylation was not observed in CD45 cells, where a CD44-induced round spreading occurs. These +  results suggest that early and transient activation of PLCyl was associated specifically with CD44-induced elongated cell spreading. Moreover, the regulation of CD45 seems to be at the early stage of CD44 signaling since the divergence of signaling in CD45 and +  CD45" cells lies at P L C y l phosphorylation, which is an early event. In other words, the point where CD45 is exerting its inhibitory effect should be proximal in CD44 signaling. It has been observed that the activity of Src-family kinase (SFK), Lck, was associated with P L C y l tyrosine phosphorylation (316). To determine i f SFK activity is required for CD44-mediated tyrosine phosphorylation of P L C y l , CD45" T cells were pretreated with specific inhibitor PP2 (317) before the cells were incubated on immobilized CD44 antibody. Results from figure 3.9B show that CD44-mediated tyrosine phosphorylation of P L C y l was dependent on SFK activity. Veri et al. also showed that SFK-dependent phosphorylation of P L C y l was secondary to its recruitment to the plasma membrane (316). In T cells, PLCyl recruitment to the membrane is dependent upon its binding to phosphorylated L A T . However, L A T phosphorylation was not observed in CD44-mediated signaling (section 3.2.8 and figure 3.13B).  58  A  PLCyl IP CD45  CD45"  +  pTyr PLCyl PLCyIP Time (min) 0 B  + 0  + 5  + 10  + + L 30 120 0  + 5  + 10  + + L 30 120  PLCyl IP DMSO control  Time (min) 0 C  + 0  5  10 30 B  PP2  L  0  5  10 30  PLCyl IP  175  pTyr  175  PLCyl  Time (min)  Figure 3.9  10 M  10 10 D LY  Transient tyrosine phosphorylation of PLCyl is associated  with elongated cell spreading of CD45" BW5147 T cells. (A) Induction of tyrosine phosphorylation of PLCyl was observed with CD45" BW5147 T cell spreading on KM81; however, this was absent in C D 4 5 T cell spreading. (B) The induced tyrosine phosphorylation of PLCyl was inhibited by pre-treatment of cells with 20 u.M PP2. (C) Pre-treatment of CD45" BW5147 T cells with 20 uM LY294002 did not inhibit tyrosine phosphorylation of PLCyl. "L" denotes the lysate control of the immunoprecipitation. "B" is the sample collected from cells incubated on immobilized BSA, instead of immobilized KM81. "M" denotes cell spreading medium control. "D" is the DMSO control. "LY" is the sample with cells pre-treated with LY294002. The amount of time of cells incubated on immobilized KM81 is indicated in minutes. +  59  Another possibility of P L C y l recruitment to the membrane is through its interaction with PI3K product, PIP (318, 319). To determine i f PI3K activity was required for CD443  mediated tyrosine phosphorylation of P L C y l , specific inhibitor LY294002 was utilized to pre-treat cells before incubation on immobilized antibody and immunoprecipitation. The results of immunoprecipation of P L C y l and tyrosine phosphorylation blot shown in figure 3.9 demonstrate that CD44-induced phosphorylation of P L C y l was not dependent on PI3K. This result suggests that recruitment of P L C y l to the membrane in CD44 signaling may be via a novel mechanism.  3.2.6  CD44-inducedPyk2phosphorylation  requires extracellular calcium, PI3K, PLC,  and actin polymerization As mentioned above, CD44-mediated elongated cell spreading was associated with tyrosine phosphorylation of pi20/130 and one of these proteins was identified as Pyk2 (251). This signaling protein contains several potential tyrosine phosphorylation sites. Tyrosine 402 is an autophosphorylation site of Pyk2 and phosphorylation at this site requires increase in intracellular calcium level (reviewed in 320). Upon phosphorylation of this site, a binding site for Src-family kinase SH2 domains is created, and SFK recruitment is required for further activation of Pyk2. To determine i f this calcium-dependent tyrosine phosphorylation occurs in CD45 or CD45" T cells with +  CD44-induced cell spreading, a phospho-specific antibody from Biosource was utilized. Results in figure 3.10 show that CD44-induced phosphorylation at tyrosine 402 was minimal in CD45 T cells; however, the induction of tyrosine phosphorylation in +  CD45" T cells was apparent. In more than three experiments, CD44-induced phosphorylation at tyrosine 402 peaked at 30 minutes. Therefore, the phosphorylation of tyrosine 402 of Pyk2 was associated with CD44-induced elongated T cell spreading. To determine if the requirements for the induction of tyrosine phosphorylation of pi20/130 were also applicable to Pyk2 phosphorylation, CD45" T cells were pre-treated with extracellular EGTA, U73122, LY294002, or latrunculin A for 30 minutes at 37°C before incubation on immobilized KM81 and immunoprecipitation of Pyk2. Figure 3.11  60  A  Pyk2 IP with CD45+ BW5147 T cells  175 — pY402 83 175 Pyk2 83 — Pyk2IP Time (min)  + 0  B Pyk2 IP with CD45" BW5147 T cells  pY402  Pyk2  Figure 3.10 Tyrosine phosphorylation of Y402 was observed with elongated cell spreading of CD45" BW5147 T cells. (A) Pyk2 precipitated from samples incubated on immobilized CD44 antibody with various amount of time (indicated in minutes). Phosphorylation at Y402 (pY402) of Pyk2 was detected with phosphospecific antibody. Minimal tyrosine phosphorylation induction at Y402 of Pyk2 was observed with C D 4 5 T cells. (B) More intense phosphorylation of Y402 of Pyk2 was consistently observed with CD45" BW5147 T cells, which peaked at 30 minutes. "L" denotes cell lysate control and "B" is IP sample from cells incubated on immobilized BSA instead of KM81. +  61  shows the results of Western blotting with 4G10 antibody and subsequently with stripping and re-probing for Pyk2. The results indicate that CD44-induced phosphorylation of Pyk2 requires extracellular C a  2+  (fig. 3.11 A), P L C activity (fig.  3.1 IB), PI3K activity (fig. 3.11C), and actin polymerization (fig. 3.1 IC).  3.2.7  Cas/HEF is transiently phosphorylated during elongated cell spreading Crk-associated substrate (Cas) is an adaptor protein of 130 kDa that has been  shown to interact with F A K (321, 322) and Pyk2 (323-325). Its phosphorylation has been shown to associate with cell spreading and migration (reviewed in 32). Moreover, the molecular size of Cas falls into the same molecular size range of the tyrosine phosphorylated proteins (pi20/130) associated with CD44-mediated elongated cell spreading. Therefore, I sought to determine if this adaptor was also phosphorylated in CD45" T cells incubated on CD44 antibody. To determine i f Cas was tyrosine phosphorylated during CD44-mediated cell spreading, CD45 and CD45" BW5147 T cells were incubated on immobilized CD44 +  antibody. The cells were lysed after 30 or 120 minutes of incubation and immunoprecipitation of Cas was carried out with subsequent Western blotting with 4G10 and re-probing with anti-Cas antibody. Figure 3.12 shows that Cas of two molecular weights was immunoprecipitated. Moreover, transient phosphorylation of Cas was observed with only CD44-mediated spreading of CD45" T cells, but not in CD45 T cells. +  These results show that CD44-mediated elongated cell spreading is associated with tyrosine phosphorylation of Cas with the lower molecular weight. In other cell systems, tyrosine phosphorylated Cas has been shown to interact with other adaptors, including Crkll (326), CrkL (327), and Nek (328), to mediate actin rearrangement. However, co-immunoprecipitation experiments did not show association of these adaptors with Cas in anti-CD44-stimulated BW5147 cells (data not shown). Moreover, the tyrosine phosphorylated Cas in CD44 signaling is the low molecular weight isoform, which is possibly an isoform of Cas that is known as HEF (327). The downstream effectors of this Cas isoform are yet to be identified.  62  A  Pyk2 IP with CD45" BW5147 T cells  mw (kDa)  pTyr  Pyk2 M Pyk2IP + Time 0 (min) B  M E + + 30 30 0  NL L +  Pyk2 IP with CD45" BW5147 T cells  C  Pyk2 IP with CD45" BW5147 T cells  mw (kDa)  mw (kDa) 175-  175pTyr  pTyr  83175-  83 175  Pyk2  Pyk2 83— Time (min)  M 0  M 343 122 30 30 30  83 —1_ Time (min)  II 0  M 30  D 30  LY 30  LA 30  Figure 3.11 CD44-induced Pyk2 tyrosine phosphorylation requires extracellular C a , PLC, PI3K, and actin polymerization. (A) Pre-treatment of CD45" BW5147 T cells with 5 mM EGTA (E) inhibited phosphorylation of Pyk2 upon incubation on immobilized CD44 antibody. (B) Pre-treatment of CD45" T cells with PLC specific inhibitor, 0.5 u.M U73122 (labeled as 122), significantly reduced Pyk2 phosphorylation. "343" denotes negative treatment control with an analog of U73122, U73343. (C) CD44-induced Pyk2 phosphorylation was inhibited by pre-treatment of cells with 20 u.M LY294002 (LY) or 1 u.M latrunculin A (LA). "M" denotes sample from cells suspended in normal cell spreading medium. "NL" was no lysate immunoprecipitation control. "L" is lysate control. "D" is the DMSO control. 2 +  63  Cas IP with BW5147 T cells  mw (kDa)  CD45+  CD45"  175 pTyr 83175-  ms  83Time (min)  30  120  0 L  mmm> no o IP Ab Control  30  120  Cas  0 L  Figure 3.12 Transient tyrosine phosphorylation of Cas is associated with CD44-mediated elongated cell spreading. Immunoprecipitation of Cas was carried out with BW5147 T cells incubated of immobilized CD44 antibody for varying amounts of time. Two isoforms of Cas was precipitated. Tyrosine phosphorylation was observed with only the lower molecular weight isoform of Cas (indicated by the arrowhead) in CD45" T cells. "L" is the lysate control.  64  3.2.8  CD44 signaling is distinct from CD3 signaling A l l of the signaling molecules identified thus far in CD44-mediated Pyk2  phosphorylation and elongated cell spreading overlap with those involved in CD3 signaling. Contrary to its role in CD3/TCR signaling, CD45 exerts a negative regulatory effect on this CD44 signaling pathway. To determine i f CD44 signaling is a unique pathway, compared to CD3/TCR signaling, whole-cell lysate samples stimulated with immobilized anti-CD3 (2C11) or anti-CD44 (KM81) were resolved in 10% SDS-PAGE. Western blotting with 4G10 was carried out to determine the overall tyrosine phosphorylation profiles. With CD44 stimulation, tyrosine phosphorylation of p i 20/130 was apparent only in CD45" T cells (fig. 3.13A). This band was also observed in CD3 stimulation in the CD45" cells and to a lesser extent in the CD45 cells. Two more bands +  were observed with CD3 stimulation: one at around 42-44 kDa, another one around 3638kDa. There bands were presumably phosphorylated E R K and L A T , respectively. To confirm that the 42-44 kDa was ERK, the blot was stripped and re-probed with phosphospecifc antibody that recognizes phosphorylated E R K (fig. 3.13A). Thus, the results showed that CD3 stimulation of CD45 and CD45" T cells led to phosphorylation +  of E R K , but that CD44 signaling did not. To confirm that the 36-38 kDa phosphorylation band was L A T , immunoprecipitation was carried out. Surprisingly, although the intensities of the 3638kDa bands in CD45 and CD45" T cells were similar, the level of phosphorylation of +  immunoprecipitated L A T was lower in CD45" T cells. This is consistent with the literature that CD45" T cells are impaired with CD3/TCR signaling. As suggested by the results of the tyrosine phosphorylation blots (fig. 3.13 A , B), L A T phosphorylation was not induced by CD44 stimulation. With the results from tyrosine phosphorylation profiles, E R K phosphorylation, and L A T phosphorylation, CD44 signaling is clearly distinct from CD3 signaling. Of special interest are the opposite roles of CD45 in these signaling pathways: CD45 is a positive regulator of CD3 signaling, while it plays a negative regulatory role in CD44 signaling.  65  A  Phosphotyrosine and phosphorylated Erk blots of whole-cell lysate from CD45 and CD45" BW5147 T cells +  mw (in kDa) 175 — 8362-  pTyr  47.5-  32.5-  47.5-  pERK  32.5CD45 BSA B  2C11  KM81  LAT IP with BW5147 T eels CD45  +  CD45" pTyr LAT  Time 0 (min)  30 120 30 120 0 2C11 KM81  30 120 30 120 no 2C11 KM81 IPAb  Figure 3.13 CD44 and CD3 signaling pathways are different. (A) The phosphotyrosine induction patterns are different with CD3 (2C11) or CD44 (KM81) stimulation. With CD3 stimulation, phosphotyrosine bands of 120, 42-44, and 36-38 kDa were observed. With CD44 stimulation, phosphotyrosine band of 120kDa was observed. The phosphorylated band at 42-44kDa was found to be ERK. The phosphorylation of ERK was absent in CD44 signaling. (B) Tyrosine phosphorylation of LAT was only observed with CD3 stimulation, but not with CD44 stimulation. Moreover, the absence of CD45 reduced CD3-mediated phosphorylation of LAT.  66  3.2.9  CD44 and LFA-1 signaling pathways are similar To determine i f CD44 signaling is similar to signaling by other adhesion  molecules, a comparison to LFA-1 was conducted. LFA-1 was chosen because this signaling molecule has also been shown to play important roles in T cell migration and activation. Similar to CD44, anti-LFA-1 induced elongated cell spreading morphology in CD45" T cells, but not in CD45 T cells (fig. 3.14B). Same results were obtained when +  BW5147 cells were incubated on immobilized soluble mouse ICAM-1 (data not shown). These observations suggest that CD45 maybe a negative regulator of adhesion-related signals. To determine if LFA-1 signaling, leading to elongated T cell spreading, is also associated with Pyk2 phosphorylation, immunoprecipitation was carried out with CD45  +  and CD45" T cells incubated on LFA-1 antibody. Figure 3.15 shows that LFA-1-induced phosphorylation of Pyk2 was also associated with elongated cell spreading, which was undetectable in CD45 T cells. +  Similar to CD44 signaling, preliminary data suggest that extracellular Ca , PI3K, and actin polymerization were also required for induced tyrosine phosphorylation of pi20/130 and elongated cell spreading in CD45" T cells (data not shown). Together, these observations suggest that LFA-1 signaling leading to elongated cell spreading is similar to that of CD44. In addition, CD45 seems to play a negative regulatory role on adhesion-related signaling pathway that leads to an elongated, or directionally biased, cell spreading morphology in T cells.  67  A  LFA-1 expression levels measured by flow cytometry LFA-1  2° Ab control  c 3 o o "55 o  {  CD45-  Cells only  s\  LFA-1 2° Ab control  0 10 10 10 Fluorescence Intensity 1  B  2  CD45+  Cells only  3  Light microscopy photographs  C  Phosphotyrosine blot of whole-cell lysate mw (kDa) 175 —"I  CD45* BW5147 T cells on TIB213  B  K T  CD45"  1  CD45" BW5147 T cells on TIB213  B K T CD45"  Figure 3.14 CD45 also inhibits LFA-1-mediated elongated cell spreading in BW5147 cells. (A) Flow cytometry results show that C D 4 5 BW5147 and CD45" BW5147 T cells express comparable levels of LFA-1. (B) No apparent cell spreading was observed when C D 4 5 BW5147 T cells were incubated on immobilized TIB213, while elongated cell spreading was observed in CD45" BW5147 T cells. Cells shown were incubated on immobilized TIB213 for 2 hours. (C) Elongated cell spreading was also associated with tyrosine phosphorylation of proteins of 120-130 kDa in size. No tyrosine phosphorylation was observed when cells were incubated on BSA (B). Whole-cell lysate samples were collected the cells had been incubated for 2 hours on BSA, KM81, orTIB213. "K" was samples incubated on immobilized KM81. "T" was samples incubated on immobilized TIB213. +  +  68  Pyk2 IP with BW5147 cells  mw (kDa)  CD45+  CD45"  175 — pTyr  83 175 Pyk2  83 Time (min)  30  30  Figure 3.15 LFA-1 induces tyrosine phosphorylation of P y k 2 with elongated cell spreading. Immunoprecipitation of Pyk2 indicates that LFA-1  induced Pyk2 phosphorylation is a s s o c i a t e d with elongated cell  spreading on C D 4 5 " T cells. Tyrosine phosphorylation of Pyk2 is absent in C D 4 5  +  T cells incubated on immobilized TIB213 or C D 4 5 " T cells  incubated on B S A alone (B).  69  3.3  Discussion  3.3.1  Data Summary In this chapter, the ability of CD44 to mediate cell spreading in T cells has been  demonstrated. CD44-mediated cell spreading occurs in both CD45 and CD45" T cells; +  however, the cell spreading morphologies are different. TheCD45 BW5147 T cells display a round or non-directionally biased spreading while the CD45" T cells display an elongated spreading on immobilized CD44 antibody. These observations suggest that the transmembrane tyrosine phosphatase CD45 may act as a regulator of CD44-mediated cytoskeletal rearrangement, leading to different outcomes of cell spreading. The cytoplasmic domain of CD44 is indispensable in mediating cell spreading in T cells, suggesting that it requires CD44-mediated signaling. Previously, it has been demonstrated that CD44-mediated elongated cell spreading in CD45" T cells is associated with tyrosine phosphorylation of pl207130 (251). In this study, the observation has been extended, identifying that this cell spreading morphology and tyrosine phosphorylation are dependent on extracellular calcium, PLC, PI3K, and actin polymerization. Although these are also signaling components in CD3/TCR signaling, CD44 signaling pathway is unique since it does not involve L A T or E R K . Interestingly, CD44 signaling seems to be similar to LFA-1 signaling in terms of the signaling components examined thus far. In both cases, Pyk2 was tyrosine phosphorylation phosphorylated. In the next chapter, how CD45 exerts its negative effect on CD44 signaling and under what circumstance that the regulatory effect of CD45 is relieved are examined.  3.3.2  CD44 engagement and cell spreading Although CD44 has been shown to interact with a variety of ligands, much of the  effort in studying the effect of CD44 engagement has been performed with its principal ligand H A or with CD44 antibody. Ariel et al. observed that activated human peripheral T lymphocytes, in the presence of cytokines or chemokines, displayed an elongated cell spreading morphology on immobilized H A (191). Fanning et al. (193), however, failed to observe the elongated cell spreading of activated human peripheral T lymphocytes incubated on immobilized HA. This discrepancy may be explained by the absence of cytokines or chemokines during incubation. However, Fanning et al. observed T cell  70  polarization and migration on immobilized CD44 antibody instead. Interestingly, the authors observed elongated spreading and migration of T cells in three-dimensional matrix incorporated with H A . Two reasons were suggested by the authors to explain the observations. First, the ability of CD44 to mediated cell spreading is dependent on the activation state of the cell, thus influencing the activation threshold of CD44 signaling. Second, the presentation of H A is different in a two-dimensional planar surface, as opposed to H A incorporated in a three-dimensional matrix. Since H A is a component of the extracellular matrix, it is conceivable that the three-dimensional presentation of H A is more reflective of the physiological situation in tissues. Therefore, presentation of H A in a two-dimensional surface may not provide stimulation that reaches the signaling threshold. However, the observation in this study suggests that the signals generated by CD44 engagement with immobilized antibody mimics that with H A in a threedimensional matrix. This may be because the high affinity-interaction between CD44 and the antibody provides enough stimulation to reach signaling threshold, which can only acheived by H A in a three-dimensional matrix. The round cell spreading of CD45 T cells on immobilized CD44 antibody +  observed in this study is similar to the observation made by K i m et al. (329). However, the elongated cell spreading morphology of CD45" T cells resemble the spreading of T cells on immobilized H A in the presence of cytokines or chemokines (191). The spreading morphology of CD45" T cells also resembles the spreading of T cells in threedimensional matrix in which H A is incorporated (193). The difference in cell spreading morphology of CD45 and CD45" T cells on CD44 antibody may be reflective of +  different activation thresholds present in these cells, which is modulated by CD45 - this will be further investigated in the next chapter.  3.3.3  CD44 and lipid rafts The existence of lipid rafts was first described by Brown and Rose through  biochemical means (330). Lipid rafts are special membrane compartments defined by resistance to Triton X-100 extraction at 4 °C. They are thus also known as detergentresistant microdomains (DRM). These specialized membrane compartments are enriched with glycophosphatidyl inositol-linked proteins. Since then, efforts have been made to  71  show the existence of these hypothesized special membrane compartments. Due to the limitation of imaging technology, lipid rafts have still not been directly observed (331). However, several lines of evidence do suggest their existence. First, limited movement of lipid and protein components within cholesterol-dependent microdomains was observed with chemical and biophysical methods. Second, using a milder detergent, Brij98, lipid rafts were isolated at physiological temperature, rather than at 4 °C. Third, spontaneous formation of compartmentalized membrane structure, similar to lipid rafts, was observed with defined lipid mixtures (reviewed in 150). A percentage of CD44 has been shown to be associated with lipid rafts (249, 251, 332). Upon ligation of CD44, partitioning of a higher proportion of CD44 into lipid rafts was reported (250). Moreover, depletion of cholesterol with methyl |3-cyclodextran inhibited CD44-mediated cell spreading of T cells on immobilized CD44 antibody, suggesting a role of lipid rafts in CD44-mediated events (250). However, the observation with the cytoplasmic deletion mutant of CD44 (section 3.2.2) suggests that CD44induced aggregation of lipid rafts, or mobilization of CD44 into lipid rafts, is not sufficient to mediate cell spreading. These results imply that interactions between the cytoplasmic domain of CD44 and other components may play a role in mediating cell spreading. The cytoplasmic domain of CD44 is known to interact with adaptor proteins that link transmembrane molecules to the actin cytoskeleton. These adaptor proteins include the E R M family proteins and ankyrin (331) (153). The cytoplasmic domain of CD44 may also interact with the ERM-related adaptor, merlin, which does not interact with actin (333). Moreover, the cytoplasmic domain of CD44 can associate with the signaling proteins, including Lck (248, 334), Fyn (249), T-lymphoma and metastatsis-inducing protein 1 (TIAM1) (243), and P K C (158). The contribution of these interactions to CD44mediated cytoskeletal rearrangement and cell spreading has yet to be examined.  3.3.4  CD44 signaling and calcium mobilization The observation that extracellular E G T A inhibited CD44-mediated elongated cell  spreading suggests that it required calcium entry. Bourguignon et al. have observed that the interaction between CD44 and its ligand H A on BW5147 T cells leads to CD44  72  capping and an increase in intracellular calcium (247). In addition, in an aortic endothelial cell line, the interaction between H A and an isoform of CD44, CD44vlO, increases intracellular calcium (235). This is mediated through generation of inositol 1, 4, 5-triphosphate (IP3) and the calcium mobilization was associated with migration of these endothelial cells. Moreover, HA/CD44 interaction causes calcium mobilization in human keratinocytes (335). Taken together, these observations suggest that increase in intracellular calcium is a common signaling mechanism that CD44 utilizes in various cell types. Increase in intracellular calcium concentration is a signaling mechanism that has been shown to occur in different types of cells, such as muscle and neuronal cells. Various signaling pathways also utilize calcium as a secondary messenger to propagate cellular signals; examples of these are T or B cell receptor signaling (336), selectin signaling (85, 337), and integrin signaling (338-340). The universality of calcium to convey signals that lead to behavioral changes in the cells, raises the question of how this common agent brings about different functional outcomes. It has been suggested that the speed, the amplitude, and the spatio-temporal patterning of intracellular calcium increases contribute to the outcome of calcium signaling (341). Calcium signaling is involved in many cellular responses, including the contraction of skeletal muscle cells, the release of synaptic vesicular contents at the axon terminal of neurons, and activation of lymphocytes all require highly regulated calcium signaling. In T lymphocyte activation, the increase of intracellular calcium is induced by TCR signaling and is associated with two temporally distinct programs (336). The initial program is associated with cytoskeletal rearrangement and arrest of the T lymphocyte to achieve proper interaction with the A P C (51, 52). The latter program is associated with N F A T translocation and new gene transcription (342). Although how calcium regulates actin rearrangement in T cells is still largely unknown, there are two possible effectors: protein kinase C (PKC) and Pyk2. The P K C family has been shown to regulate actin cytoskeleton in a variety of cell types (343). Moreover, PKC(3 has been shown to be essential for LFA-1-induced polarization and migration of T cells (108). Finally, a recent report has also showed the importance of P K C in CD44-induced migration of T cells (193). The role of Pyk2 in actin organization will be discussed below (section 3.3.6).  73  3.3.5  PLCyl membrane recruitment, tyrosine phosphorylation, and activation By utilizing the inhibitor U73122, a requirement for PLC in CD44-mediated  elongated cell spreading and tyrosine phosphorylation was demonstrated. This may be reflective, in part, of the calcium requirement, since PLC activation leads to an increase in intracellular calcium concentration. In this study, CD44-induced activation of P L C y l was suggested by the phosphorylation of this phospholipase. Duriong TCR signaling, cytoplasmic P L C y l is recruited to the membrane through the interaction between its SH2 domain and a phosphotyrosine residue on L A T . This interaction and the subsequent activation of P L C y l lead to production of D A G and IP3. The binding of IP3 to its receptor causes the release of calcium from internal stores. This leads an initial increase in intracellular calcium level and subsequent calcium influx from extracellular sources through store-operated calcium channels in the plasma membrane (344, 345). A similar mechanism can be envisaged for CD44 signaling. However, L A T phosphorylation was not observed in this signaling pathway. Another possible mechanism of P L C y l recruitment is through the interaction of its PH domain with membrane-bound PIP3 produced through PI3K activation (319). Intriguingly, P L C y l phosphorylation was not affected by inhibition of PI 3 K. Tyrosine phosphorylation of P L C y l is required for its activation and a member of the Tec-family kinase, Itk, is required for the process during TCR signaling (346, 347). Since tyrosine phosphorylation of Itk is indicative of its activation (348, 349), Itk was immunoprecipitated and its tyrosine phosphorylation level was evaluated with Western blotting. Although Itk was immunoprecipitated from CD45" BW5147 cells after incubation on immobilized CD44 at various time points, no induction of tyrosine phosphorylation of Itk was observed (data not shown). Therefore, at present, it is unclear how P L C y l is recruited to the membrane and tyrosine phoshporylation, leading to activation of this enzyme in CD44 signaling. One possibility is that a yet-to-be identified adaptor protein is responsible for recruiting PLCyl during CD44 signaling.  74  3.3.6  The role of the cytoskeleton in CD44-induced cell spreading, polarity, and signaling in T cells Pre-treatment of CD45" T cells with an inhibitor of microtubule disassembly,  taxol, significantly hampered the elongated cell spreading on CD44 antibody. This result suggests that microtubule disassembly is a pre-requisite of cell spreading. This further implies that CD44 signaling leads to microtubules dissembly to enable cell spreading. This result is consistent with the inhibition of fMLP-induced neutrophil polarization by taxol pre-treatment (350). The tyrosine phosphorylation of pl20/130 induced by CD44 engagement was not inhibited with the pre-treatment of taxol, however, suggesting that this signal is either independent of microtubule disassembly or upstream of this event. It has been shown that Rac activation is required for the dissolution of the microtubule network. Moreover, engagement of CD44 activates this small Rho GTPase (241, 250). It is likely that this is how CD44 signaling exerts its effect on the microtubules. It was expected that pre-treatment of CD45" T cells with latrunculin A , an inhibitor of actin polymerization, inhibited CD44-mediated elongated cell spreading. However, it was surprising to observe the inhibition of pi20/130 phosphorylation. This indicates that actin polymerization is required to effectively deliver CD44 signals. The possible mechanism of how an intact actin cytoskeleton may contribute to CD44 signaling is explored in chapter 4. The cytoskeleton of a cell is mainly composed of three systems: the actin filaments, the microtubules, and the intermediate filaments. It has long been known that the shape of a given cell is largely dictated by these cytoskeletal systems. In T lymphocytes, much effort has been put into understanding the regulation of the actin and microtubule systems. Not much about the intermediate filaments of T lymphocyte is known. It is known, however, that the intermediate filament vimentin provides the rigidity of the T lymphocyte and that the collapse of this network is required for T cell polarization (351). Therefore, the elongated cell spreading of CD45" T cells plated on anti-CD44 antibody suggest that CD44 signaling is capable of mediating the collapse of the vimentin network. It will be, thus, of interest to identify this signaling pathway. In T lymphocytes, the microtubule-organizing center (MTOC) is localized at the perinuclear region when the cells are at rest. When a T lymphocyte is polarized during  75  directed migration, the M T O C is localized at the trailing edge, or uropod, of the cell (352, 353). The position of the M T O C is re-oriented when the T lymphocyte comes into contact with an APC. The M T O C will move from the back of the cell to the "front", being positioned at the proximity of the T/APC interface (354). Although the biological significance of M T O C reorientation during T/APC interaction is unknown, it has been suggested that it may serve as a site where signaling molecules localize and act as a signaling complex (355). On the other hand, microtubule dynamics seems to play an integral role in cell polarization and migration. Previously, disruption of microtubules by colchicine and nocodazole was shown to induce neutrophil polarization and crawling (356, 357). Moreover, a recent observation suggests that an intact microtubule is necessary to preserve the "backness" of migrating cells, thereby ensuring effective directional migration (358). The elongated spreading of CD45" T cells on immobilized CD44 antibody suggests that CD44 is capable of delivering a cell polarization signal and this is consistent with the recent observation made by Fanning et al. (193). During directed cell migration, a similar elongated morphology is observed. As mentioned before, the microtubules are recruited to the uropod of a migrating lymphocyte. As for actin, active polymerization is observed at the leading edge (lamellipodium) of the migrating cell. The importance of the actin cytoskeleton during migration is demonstrated by the abolishment of cell motility with pre-treatment of cells with an actin-disrupting reagent, such as cytochalasin D. However, recent observations have led to the suggestions that the functions of the actin cytoskeleton may be more than structural support of the cell. This cytoskeletal network may provide a signaling scaffold (359), taking part in mediating signal diversification and magnification, anchoring specific signaling components at subcellular sites, preventing cross-talk between certain signaling molecules, and re-localizing active molecules (122).  3.3.7  PI3K and cell polarization Using the specific inhibitor LY294002, the requirement of PI3K for CD44-  induced elongated cell spreading was demonstrated. The results suggest that PI3K activation is upstream of phosphorylation of pl207130, Pyk2, and elongated cell  76  spreading. In Dictyostelium and in neutrophils, the role of PI3K in cell polarization and directed migration in response to chemotactic cues has clearly been demonstrated (360, 361). Moreover, PI3Ky was shown to be essential for directed neutrophil migration by using cells from knockout mice (362, 363). In T cells, the classes IA and IB PI3K have been shown to play a role in directed migration in response to the chemokine SDF-1 (364). In CD44 signaling, we are yet to identify which PI3K family member is responsible for mediating cell elongation. Moreover, it is unclear at the moment how PI3K is activated in CD44 signaling. This will be addressed in further detail in chapter 4.  3.3.8  Pyk2 activation and effector function Pyk2 phosphorylation at Y402 was observed in CD45" T cells when incubated on  CD44 antibody. On the contrary, only minimal phosphorylation was observed in CD45  +  T cells. Phosphorylation at Y402, the autophosphorylation site of this kinase, is induced by increases in intracellular calcium. However, the exact mechanism that causes such phosphorylation is still unknown. The tyrosine phosphorylation at Y402 is thought to be a docking site for Src-family kinases, leading to physical interaction and full activation of Pyk2 through phosphorylation by SFK at Y579/580 (365). In this study, CD44-induced Pyk2 phosphorylation was shown to require calcium mobilization, PLC, PI3K, and an intact actin cytoskeleton. Pyk2 phosphorylation has also been shown to be downstream of PI3K activation in (31 integrin signaling in CD34  +  cells (366) and in platelet activation (367). Although TCR-induced phosphorylation of Pyk2 has been reported, its exact function in T cell biology remains elusive. The development of T cells in the Pyk2 knockout mouse is largely normal; however, no analysis of T cell function with Pyk2-defective T cells has been published yet. In the Pyk2 knockout mouse, the development of marginal zone B cell is impaired (41). Moreover, migration of Pyk2 knockout macrophages is also defective (42). Since Pyk2 phosphorylation is induced by SDF-1 in T cells (291), Pyk2 may play a role in cell migration in response to chemotactic signals. Interestingly, several characteristics of Pyk2 " macrophages coincide with those _/  of the pi3kl/2 " Dictyostelium. First, the cells displayed multiple cytoplasmic extensions _/  in response to chemotactic stimulations. Secondly, the cells exhibit defects in  77  microtubule-associated retraction. Third, these cells have a clear defect in cellular migration. The similarities between the phenotypes of Pyk2 " macrophages and pi3kl/2" _/  A  suggest that these two signaling molecules may take part in the same signaling pathway or network that mediates cellular migration. In particular, the role of PI3K in directional sensing has clearly been established. It raises the question as to whether Pyk2 also determines the directionality of a cell. The same question was indeed raised by Ostergaard and Lysechko for different reasons (368).  3.3.9  Cas/HEFl activation and effector function Immunoprecipitation of pl30Cas (Crk-associated substrate) revealed that two  isoforms were expressed in murine BW5147 T cells. Moreover, the lower molecular weight isoform is transiently phosphorylated in CD44-induced elongated cell spreading. Cas belongs to a family of docking proteins, whose members include HEF1 (human enhancer of filamentation 1) and Efs/Sin (embryonal Fyn substrate/Src-interacting) (reviewed in 369). Since the antibody utilized for immunoprecipitation does not crossreact with Efs/Sin, the lower molecular weight isoform precipitated was most likely HEF1. HEF1 has an apparent molecule mass of 105 kDa and it is mainly expressed in hematopioetic cells. It has been shown to interact with F A K and Pyk2 (327, 370). Upon B C R or (31 integrin stimulation, tyrosine phosphorylation of HEF1 is induced and this phosphorylation regulates association with CrkL (327, 371). CrkL is a member of an adaptor family that mediates a wide range of cellular behavior, including cell adhesion and migration (372). HEF1 was first identified as a protein that takes part in the transition of S. cerevisiae from vegetative to pseudohyphal growth (370), a process that is accompanied by a change in cell morphology and polarity. It was, thus, reasonable to postulate that mammalian HEF1 might regulate cell morphology. Indeed, overexpression of HEF1 in cells caused changes in cell shape of MCF-7 cells, a breast cancer cell line (369). Tyrosine phosphorylation of HEF1 is mediated by Pyk2 upon B C R and |31 integrin stimulation (373). A similar pathway may exist in CD44 signaling since Pyk2 phosphorylation, specifically Y402 phosphorylation of Pyk2, was observed in  78  conjucntion with HEF1 phosphorylation. Although HEF1 phosphorylation was shown to induce its association with CrkL, this was not observed upon CD44 engagement (data not shown). However, HEF1 may exert its regulation through other adaptors or downstream effectors. A n example is a recently identified adaptor Chat (Cas/HEFl-associated signal transducer) (374, 375). Further experimentation is required to determine if HEF1 was directly phosphorylated by Pyk2 in CD44 signaling and what downstream effectors HEF1 interacts with to cause actin reorganization.  3.3.10 The role of CD45 in CD3, CD44, and LFA-1-mediated cell spreading and random migration The CD44 signaling pathway associated with elongated cell spreading is similar to that of L F A - 1 . In both cases, the elongated cell spreading is associated with tyrosine phosphorylation of p i 20/130 and Pyk2. Interestingly, the ability to undergo elongated cell spreading was observed only in CD45" BW5147 T cells with immobilized CD44 or LFA-1 antibody. This observation implies that CD45 may play a negative regulatory role in adhesion-related signaling in T cells. Using bone marrow macrophages, Roach et al. observed that CD45 plays a role in regulating adhesion (293). The authors suggest that CD45 exerts its effects through dephosphorylation of the tyrosine residue in the kinase domain of Src-family kinases, leading to inactivation of the kinase. Moreover, CD45 has been shown to negatively regulate a5|31 adhesion to fibronectin in T cells (292). In the same study, the researchers showed that the transmembrane and the phosphatase-active cytoplasmic domains of CD45 are required to restore normal adhesion. Together with the results with CD44- and LFA-1 adhesion and cell spreading, these observations suggest that CD45 plays a negative regulatory role in adhesion-related events and signaling. Although CD45 plays a paramount role in T cell activation, it is still unclear how CD45 activity is regulated. It has been shown in a CD45 forced dimerization model that TCR signaling events can be down-regulated (376). Moreover, pre-treatment of leukocytes with CD45 Ab leads to homotypic aggregation that is dependent on L F A 1/ICAM-l interaction (377). These observations suggest that down-regulation of CD45 activity leads to LFA-1 activation. Thus far, all LFA-1-mediated cell spreading, migration, and signaling have been observed with lymphoblasts derived from peripheral  79  blood lymphocytes (93, 106, 109, 340). However, it is unclear if the differences in CD45 activities exist between these two cell stages. The observations with tyrosine phosphorylation of total cell lysate and the adaptor protein L A T , as well as the phosphorylation of E R K , suggest that CD3 signaling is different from CD44 signaling. With CD3 stimulation, L A T has been shown to play an essential role in actin polymerization induced in this signaling pathway (378). However, L A T phosphorylation was not observed in CD44 signaling, suggesting its function is not required for CD44-mediated actin rearrangement and cell spreading. In this chapter, the proximal components of CD44 signaling, leading to elongated cell spreading and Pyk2 phosphorylation, were identified. These include SFK, P L C y l , PI3K, and increase in intracellular calcium level. Moreover, an intact actin cytoskeleton is required for this signaling to occur. A final summary of CD44 signaling network is presented in chapter 5. In the next chapter, the mechanism through which CD45 exerts its negative regulatory role on CD44 signaling is explored.  80  Chapter 4  CD45 is a regulator of CD44-induced actin rearrangement  through its regulation of Src-family kinases 4.1  Introduction In the previous chapter, a CD44-mediated signaling pathway was identified,  leading to elongated spreading of T cells when CD45 is absent. In this chapter, the mechanism of how CD45 regulates this signaling pathway was explored. This was first carried out by further establishing the regulatory effect of CD45 on CD44-mediated signaling and elongated cell spreading by utilizing activated thymocytes and cells transfected with a CD45-like phosphatase. Moreover, fluorescent labeling and confocal microscopy were employed to study the temporal and spatial distribution of the signaling molecules in CD44 pathway in both CD45 and CD45" T cells. This goal was to decipher +  how CD45 exerts its regulatory effect on CD44 signaling. Moreover, the activation state of a key Src-family kinase, Lck, in both CD45 and CD45" T cells was examined with a +  phosphospecific antibody that recognizes the kinase-active phosphotyrosine 394 (pY394), a requisite for Lck activation. Finally, a physiological situation of how the negative regulatory effect of CD45 on CD44 may be removed was explored.  4.2  Results  4.2.1  Activated thymocytesfromC57/B6 and CD45 knockout mice also display different cell spreading morphologies To determine if the regulatory role of CD45 is observed in cells other than the  BW5147 T cells, thymocytes harvested from C57/B6 (CD45 ) and CD45 knockout +/+  mice (266) were utilized in cell spreading experiments. Harvested thymocytes were stimulated with ionomycin, P M A , and IL-2 for three days before experimentation (see section 2.1). Upon stimulation, the thymocytes increased in cell size and proliferated. Cytoplasmic extensions were observed from activated thymocytes from both mice while these cells were in suspension. As expected, no cell spreading was observed when these cells were incubated on immobilized B S A . However, when these cells were incubated on immobilized CD44 antibody, elongated cell spreading was observed in CD45 knockout thymocytes. This elongated cell spreading, however, was absent from thymocytes of  81  C57/B6 origin (figure 4.1). This further strengthens our hypothesis that CD45 is exerting a negative regulatory role on CD44 signaling and prevents elongated cell spreading.  4.2.2  Receptor tyrosine phosphatase-alpha (PTPa) influences CD44-mediated cell spreading Previous efforts have been made to transfect CD45 into CD45" BW5147 T cells  without success. However, a member of our laboratory has successfully transfected the CD45" T cells with a CD45-like transmembrane tyrosine phosphatase P T P a (reviewed in 379). This phosphatase is similar to CD45 with regard to its cytoplasmic domain that it also contains two tandem phosphatase-like domains, D l and D2. In both phosphatases, the majority of the catalytic activity lies in D l . Moreover, PTPa has been shown to regulate Src and Fyn activities (380-382). We hypothesized that the effect of PTPa on CD44 signaling should be similar to that of CD45. To test this hypothesis, cell spreading experiments were carried out to determine if PTPa also inhibits elongated cell spreading. Moreover, in this series of experiments, a mutant of PTPa (Y789F) was also included. Tyrosine phosphorylation of Y789 was found to be necessary for the activation of Src in cells. Therefore, this mutant was included to determine if the Src-activating (possibly Lck or Fyn) function of PTPa influences cell spreading. Figure 4.2 shows that CD45" T cells, PTPa-transfected CD45" T cells (CD45" PTPa), and Y789F-PTPa-transfected CD45" T cells (CD45" Y789F) showed no difference in CD44 expression. Moreover, CD45" PTPa and CD45" Y789F cells showed comparable level of PTPa over-expression. The extracellular domains of PTPa and Y789F were tagged with Vaccinia stomatitis virus glycoprotein (VSVG) antigen. Therefore, flow cytometry was carried out with anti-VSVG to determine the expression levels of these phosphatases. The results of the cell spreading experiment indicate that PTPa slightly inhibits elongated cell spreading (figure 4.3A). PTPa CD45" T cells generally did not spread to the same extent as CD45" T cells, as is evident with the comparison of the cell spreading lengths (figure 4.3B). Surprisingly, Y789F exerted a slightly greater inhibitory effect on CD44-mediated elongated cell spreading (figure 4.3A and 4.3B). Although the exact mechanism of how the Y789F mutant exerts a larger inhibitory effect on CD44-induced 82  Figure 4.1 Activated thymocytes from CD45 knockout mice also displayed elongated cell spreading on immobilized CD44 antibody.  After  activation with ionomycin, P M A , a n d IL-2 for 3 days, activated thymocytes from wildtype a n d C D 4 5 knockout mice displayed similar cell morphology: s o m e cells s h o w e d round cell s h a p e while s o m e s h o w e d cytoplasmic extensions. W h e n incubated on immobilized B S A , the activated thymocytes do not s p r e a d or adhere to the underlying substratum. However, w h e n these activated thymoctyes were incubated on immobilized C D 4 4 antibody, thymocytes from C D 4 5 knockout mice s h o w e d more elongated cell spreading. T h e s c a l e bar d e n o t e s the length of 10 urn. T h i s pictures were taken after the cells were incubated on B S A or KM81 for 2 hours.  83  Expression levels of CD44 and PTPcc by flow cytometry  VSVG tag  V  anti-mouse IgG control St  V  CD44  CD45- BW5147 T Y789F-PTPa  anti-rat IgG control Cells Only VSVG tag  J  anti-mouse IgG control CD44  J  CD45- BW5147 T PTPcc  anti-rat IgG control Cells Only  J  VSVG tag anti-mouse IgG control  A  CD44  V  CD45" BW5147 T  anti-rat IgG control Cells Only  10  1  10  2  10  3  Fluorescence Intensity  Figure 4.2 CD45" T cells, PTPa, and Y789F-PTPa transfectants expressed comparable levels of CD44. The expression levels of CD44 of the three cell lines were checked with flow cytometry. The PTPa and Y789F-PTPa contructs were tagged with VSVG, the expression levels of which was indicative of the levels of PTPa and Y789F-PTPa.  84  A  Light microscopy photographs CD45" RPTPa  CD45  B  CD45" RPTPaY789F  Histogram of mean cell length with standard deviation p<0.01 i  1  p<0.01  BSA  KM81 BSA KM81 BSA KM81  CD45"  CD45" RPTPa  CD45" RPTPa Y789F  Figure 4.3 Overexpression of CD45-like phosphatase, PTPa, in CD45" BW5147 T cells reduced CD44-induced elongated spreading. (A) Over-expression of RPTPa or its mutant Y789F did not change the overall morphology of the cells. Similar to CD45" T cells, they did not spread on immobilized BSA. When these cells were incubated on immobilized CD44 antibody (KM81), reduced cell spreading length was observed. The photographs were taken after cells had been incubated on BSA or KM81 for 2 hours. The scale bar denotes the length of 10 urn. (B) Overexpression of PTPa or mutant Y789 did not change the size of the cells, as indicated by the length of the cells after they had been incubated on immobilized BSA. A significant decrease of the cell spreading lengths was observed in cells over-expressing PTPa (p<0.01, n=288) or Y789 mutant (p<0.01, n=351).  85  elongated cell spreading is not known, the results suggest that the Src-activating activity of PTPa may not be associated with CD44-induced elongated cell spreading. To determine i f PTPa and the Y789F mutant also affected the tyrosine phosphorylation of pl20/130 that is associated with CD44-induced elongated cell spreading, Western blotting for tyrosine phosphorylation signals from whole-cell lysate samples was analyzed. When CD45", CD45" PTPa, or CD45" Y789F T cells were incubated on BSA, no induction of tyrosine phosphorylation at 120-130 kDa was observed (figure 4.4A). The phosphorylated bands, p56 and p59, were hyperphosphorylated Lck and Fyn in CD45" cells, which were considerably dephosphorylated in the presence of PTPa and slightly dephosphorylated in the presence Y789F-PTPa (figure 4.4B). The dephosphorylation of Lck and Fyn does not correlate with the elongated cell spreading. This is because the CD45" Y789F cells showed the shortest spreading length, yet the Lck/Fyn phosphorylation levels were higher than those of CD45" PTPa (figure 4.3). The phosphorylated band p80 was not a consistently observed band (figure 4.4A). The induced tyrosine phosphorylation of pl20/130 was observed when CD45" BW5147 T cells were incubated on immobilized anti-CD44 antibody, as expected; however, a reduced pi20/130 phosphorylation was observed in CD45" P T P a and CD45" Y789F cells (figure 4A). To determine if the extent of cell spreading correlated with CD44-induced Pyk2 phosphorylation, these cells were incubated on immobilized CD44 antibody and Pyk2 immunoprecipitation was carried out. Figure 4.4C shows that the level of Pyk2 phosphorylation correlated with the cell spreading length. CD45" T cells, displaying the longest spreading length, showed the strongest Pyk2 phosphorylation. CD45" PTPa and CD45" Y789F cells showed intermediate and lowest Pyk2 phosphorylation levels, respectively, which correlated with the spreading lengths of these cells.  4.2.3  CD44-mediated cell spreading of CD45 and CD45' T cells is associated with +  different actin rearrangement Cytoskeletal rearrangement is an absolute requirement for cell spreading. To determine what kind of actin rearrangement is associated with the two forms of CD44mediated cell spreading with CD45 and CD45" T cells, Alexa488-conjugated phalloidin +  86  A  Phosphotyrosine blot of whole-cell lysate  mw(kDa)  &  &  &  <f  <F ~* p120/130 p80 ^ p56 + p59  BSA B  KM81  Phosphotyrosine and Lck blots of whole-cell lysate  mw(kDa)  cP  cP  cj  3  62 — pTyr Lck C  Pyk2 IP mw(kDa)  C D 4 5  -  CD45" p  T  P  a  Y  CD45" 789F  175pTyr 83175—1 Pyk2  Figure 4.4 Over-expression of PTPa inhibited CD44-induced tyrosine phosphorylation of p120/130 and Pyk2. (A) Whole-cell lysate of the cells were collected 2 hours after incubation on either immobilized BSA or KM81. Several tyrosine phosphorylation bands were noticeable: p56, p59, and p80. When cells were incubated on KM81, p120/130 was induced in CD45" cells, but the intensities of the bands were reduced in PTPa and Y789F-PTPa transfectants. (B) Over-expresson of PTPa and Y789F also reduced the hyperphosphorylation of Lck. (C) The levels of tyrosine phosphorylation of Pyk2, upon stimulation by immobilized CD44 Ab, were reduced in CD45" PTPa, and CD45 Y789F. "L" denotes the lysate control. -  87  was utilized to label F-actin in spreading and non-spreading cells. Generally, the F-actin content was low in both CD45 and CD45" T cells, so faint staining of F-actin labeling +  was often observed. However, upon stimulation with immobilized CD44 antibody, specific F-actin structures were observed in CD45 and CD45" T cells. Confocal microscopy was utilized to capture the fluorescent images at different levels of the cell. In CD45 T cells, a specific F-actin ring structure was observed at the level of the cell +  that is close to the interface between the cells and the immobilized antibody (fig. 4.5 A). This F-actin ring structure was no longer observable, as the image level is further away from the interacting surface between the cell and the immobilized antibody. In CD45" T cells, the F-actin ring was not observed. Instead, new F-actin structures were observed as fiber-like structures at the sides and the ends of the spreading cells. With either CD45 or +  CD45" T cells, no new F-actin structure was formed close to the interacting surface between the cells and the slide when these cells were incubated on immobilized B S A . Therefore, these data showed that different CD44-induced cell spreading morphologies of CD45 and CD45" T cells were indeed associated with different F-actin structures, +  suggesting different actin rearrangements.  4.2.4  SFK activity is required for CD44-mediated actin polymerization in CD45 or +  CD45' T cells, but Lck activity is sustained in CD45' T cells CD45 is a major regulator of the SFK Lck and Fyn in T cells (258, 275). To determine if these kinases are required for any form of the CD44-induced actin rearrangement, CD45 and CD45" BW5147 T cells were pre-treated with PP2, a specific +  SFK inhibitor, before incubation on immobilized CD44 antibody. The cells were then labeled for F-actin and observed with confocal microscopy. Figure 4.6A showed that PP2 treatment inhibited both the formation of actin ring in CD45 T cells and the actin +  fibers in CD45" T cells. The results suggest that both forms of actin rearrangement in CD45 or CD45" T cells require SFK activity. This also implies that CD44 engagement +  leads to Lck and/or Fyn activation. The kinase activity of Lck depends on the phosphorylation of tyrosine 394 in the kinase domain (383). The laboratory of Dr. Andrey Shaw has developed a phosphospecific antibody that recognizes phosphorylated tyrosine 394 of Lck (384).  88  A  F-Actin CD45  CD45-  +  BSA  KM81  Figure 4.5 CD44-mediated cell spreading of C D 4 5 and CD45" T cells is accompanied by different actin rearrangement. F-actin staining with Alexa 488-conjugated phalloidin was shown in the figures. The cells were fixed after 2 hours of incubation on immobilized KM81. Images shown are the F-actin staining of cells close to the immobilized antibody. (A) Faint and punctate F-actin staining was observed in C D 4 5 or CD45" BW5147 T cells when these cells were incubated on BSA. However, when C D 4 5 or CD45" BW5147 were incubated on immobilized KM81, round (63%, n=136, >3 experiments) and fiber-like actin structures (68%, n=81, >3 experiments) were observed, respectively, in C D 4 5 and CD45" cells. (B) A three-dimensional projection of C D 4 5 BW5147 cells with F-actin staining is shown (see section 2.2.7 for details). The staining indicates that the F-actin is localized close to the interface between the cell and the slide. The scale bar represents the length of 10 urn. +  +  +  +  +  89  A  F-actin CD45+  CD45"  •  \  J  s  ••«  ^' J '  no PP2 CD44 mAb  1 20uM PP2 CD44 mAb  B pY394-Lck CD45  CD45"  +  Figure 4.6 SFK activity is required for CD44-induced rearrangement in either CD45 or CD45" T cells. (A) After C D 4 5 T treated with 20 u.M PP2, an SFK inhibitor, actin ring formation was inhibited. An example of C D 4 5 T cells were included in the figure, where minimal F-actin staining was observed (n=77, 3 experiments). PP2 pre-treatment of CD45" T cells also inhibited CD44-induced F-actin re-arrangement (n=39, 3 experiments). The cells were fixed and labeled after a 2-hour incubation. (B) Labeling of phosphorylated Y394-Lck (pY394-Lck) showed that minimal staining was observed in C D 4 5 T cells (n=147, 3 experiments) after 30 minutes of incubation on immobilized KM81. Cytoplasmic and microcluster staining of phosphorylated Y394-Lck staining was observed in CD45" T cells (n=276, 3 experiments), however. The scale bars in the diagram indicates the length of 10 LLITI. +  +  +  +  90  We received this antibody from Dr. Shaw and utilized it to examine the activity of Lck during CD44-mediated actin re-arrangement. CD45 or CD45" BW5147 T cells were +  incubated on immobilized KM81 antibody for 30 minutes before these cells were fixed with paraformaldehyde. After the cells were permeabilized with 0.1% Tx-100, they were labeled with pY394-Lck antibody and Alexa488-conjugated secondary antibody. The cells were then observed with confocal microscopy. Figure 4.6B shows representative figures of three independent experiments. CD45 T cells showed minimal staining with +  pY394-Lck antibody, while CD45" T cells showed cytoplasmic and microcluster staining. Together with the PP2 inhibition results, these data suggest that SFK, Lck in particular, were activated during CD44-mediated actin rearrangement and cell spreading. However, by 30 minutes, sustained activation of Lck was only observed in CD45" cells.  4.2.5  CD44 is recruited into microclusters during CD44-mediated cell spreading in an actin-dependent manner Since cellular signaling is an event that requires tight control of spatial and  temporal recruitment of signaling molecules, confocal microscopy and fluorescent labeling was utilized to study the spatial relationships between the critical signaling components involved in CD44-mediated signaling. First, an antiserum generated in the laboratory, J1WBB, was used to label the cytoplasmic domain of CD44 (251). The use of this antiserum was particularly advantageous. First, this antiserum does not compete for CD44 binding sites with the immobilized CD44 antibody. Second, the antiserum was generated in rabbits, so the Alexa dye-conjugated secondary antibody would not crossreact with the immobilized rat anti-CD44 antibody. The results of CD44 labeling were indeed quite surprising. Figure 4.7 shows the typical staining of CD44 cytoplasmic domain (CD44cyt). Originally, CD44 staining was expected to be uniform at the interacting surface between the T cells and the immobilized antibody on the slide. However, specifically stanined clusters of CD44 were observed.  91  Confocal images of CD44  Figure 4.7 CD44 is recruited to microclusters when BW5147 T cells were incubated on immobilized CD44 antibody. These microclusters were not observed when cells were incubated on BSA. However, these microclusters were readily observable after the cells were incubated on immobilized KM81 for 5 minutes. The recruitment of CD44 into microclusters were observable in both C D 4 5 and CD45" T cells. By 30 minutes, the CD44 microclusters were still observable in both cell types (CD45 cells, 80%, n=152, 3 experiments; CD45" cells, 89%, n=175, 3 experiments). Shown above are the images close to the interface between the cell and the immobilized Ab. The scale bar respresents the length of 10 pirn. +  +  92  The clustering of CD44 was not observed when cells were incubated on BSA. However, this CD44cyt clusters was observable in both CD45 and CD45" T cells after 5 minutes of +  incubation of cells on immobilized K M 8 1 . The CD44 clusters were sustained and were still observable by 30 minutes, which is the time when the majority of the CD45" T cells displayed elongated cell spreading. These results indicate that the presence of CD45 did not inhibit recruitment of CD44 into these specific clusters; further strengthening the hypothesis that the dichotomy of CD44-induced actin rearrangements in CD45 and +  CD45" T cells was a result of signaling difference. Moreover, the results suggest that an underlying mechanism exists in the recruitment of the membrane-bound CD44 into these specific clusters. The cytoplasmic domain of CD44 has been shown to interact with actin-interacting molecules of the E R M family of proteins (ezrin, radixin, moesin) and ankyrin. Therefore, CD44 recruitment into these specific clusters may be an actin-dependent process. To test this hypothesis, BW5147 T cells were treated with an actin polymerization inhibitor, latrunculin A , before incubation on immobilized CD44 antibody. Two readouts are indicative of whether CD44 recruitment depends on actin: the percentage of cells showing microclusters and the size of the CD44 clusters upon latrunculin A treatment. Figure 4.8 shows the CD44cyt staining of latrunculin A-treated BW5147 cells. The CD44 clusters in both CD45 and CD45" T cells showed a significant decrease in size (Table 4.1) with +  Latrunculin A treatment. Moreover, in both CD45 and CD45" T cells, unusual +  cytoplasmic extensions were observed when these cells were incubated on immobilized KM81. Also, a decrease in the percentage of cells displaying CD44 clusters was also observed (Table 4.1). When comparing the CD44 cluster size, it was noticed that CD45  +  T cells showed a significantly smaller cluster size than those in CD45" cells; moreover, the cluster size in both cells was reduced with latrunculin A pre-treatment. These results suggest that recruitment of CD44 into microclusters is an actin-dependent process and it is negatively regulated by CD45.  93 \  Figure 4.8 Clustering of CD44 is an actin-dependent process. BW5147 T cells displayed significaly smaller CD44 microclusters (please refer to Table 4.1.1) after incubation on immobilized KM81 with latrunculin A pre-treatment. Moreover, unusual cytoplasmic extensions were observed from latrunculin A-treated cells. The cells were fixed and labeled after a 2-hour incubation on immobilized KM81. Above images are sections close to the interface between the cells and the immobilized antibodies. The scale bar denotes the length of 10 Lim.  94  Table 4.1  CD44 clustering is an actin-dependent process CD45  % cell showing CD44 microclusters  CD45"  +  80% (n=83, 3 exp)  92% (n=71, 4 exp)  59% (n= 105, 4 exp)  75% (n=106, 4 exp)  2.0 (n=98, 4 exp)*  2.9 (n=l 59, 4 exp)*  1.6 (n=l34, 4 exp)  1.9 (n=l 80, 4 exp)  - no Lat A % cell showing CD44 microclusters - Lat A pre-treatment Microcluster size (urn ) 2  #  +  - no Lat A Microcluster size (urn ) 2  #  - Lat A pre-treatment m  Comparisons between different pairs are indicated by * (pO.Ol) is observed in all the pair-wise comparison.  95  jj  +  "  . Significantly difference  +  4.2.6  Src-family kinases are differentially recruited into microclusters during CD44-  mediated cell spreading in CD45  +  versus CD45' T cells  As mentioned before, CD44 does not possess any intrinsic catalytic ability and its capability to initiate cellular signals has largely been attributed to its interacting SFKs, Lck and Fyn. In order to determine if these kinases were also recruited into microclusters during CD44-mediated actin rearrangement and cell spreading, these kinases were labeled with antibodies and Alexa488-conjugated secondary antibody for confocal microscopy. Figure 4.9 shows representative labeling of Lck in both CD45 and CD45" T +  cells. The recruitment of Lck was specific to CD44 stimulation, since no clustering of Lck was observed when cells were incubated on B S A . The recruitment was readily observable in both CD45 (84%, n=210, 4 experiments) and CD45" T (88%, n=l 12, 3 +  experiments) cells only 5 minutes after incubation of these cells on immobilized KM81. By 30 minutes, the Lck clusters were still visible (CD45 cells, 87%, n=109, 4 +  experiments; CD45" cells, 96%, n=91, 3 experiments). The results suggest that CD44induced Lck recruitment was not affected by the presence of CD45. As opposed to Lck recruitment, Fyn recruitment in CD45 cells was delayed +  (figure 4.10). Five minutes after incubating the cells on immobilized K M 8 1 , most of CD45" T cells displayed recruitment of Fyn into specific clusters (74%, n=84, 3 experiments), while only about 25% of CD45 T cells (n=68, 3 experiments) displayed +  Fyn recruitment. There was an increase in recruitment over time, however. By 30 minutes, 91% of the CD45" T cells (n=67, 3 experiments) displayed Fyn recruitment while only 48% of CD45 T cells (n=93, 3 experiments) showed Fyn clusters. These data +  show that the difference in actin rearrangements in CD45 versus CD45" T cells lies not +  in the recruitment of CD44cyt or Lck; however, the delayed Fyn recruitment may be associated with the difference of CD44-induced actin rearrangement observed in CD45 T cells.  4.2.7  CD45 inhibited the accumulation of tyrosine phosphorylation at CD44 microclusters  Since SFK are tyrosine kinases, the recruitment and activation of these kinases will lead to propagation of cellular signals through tyrosine phosphorylation. To  96  +  Confocal imges of Lck CP45+  CD45 '  5' on BSA  5' on KM81  30' on KM81  Figure 4.9 CD44-induced Lck recruitment into microclusters is independent of CD45. When BW5147 T cells were incubated on immobilized  CD44 antibody, Lck recruitment into microclusters were readily observable in a high percentage of cells by 5 minutes (CD45 , 84%, n=210, 4 experiments; CD45", 88%, n=112, 3 experiments). By 30 minutes, Lck microclusters were still present (CD45 , 87%, n=109, 4 experiments; CD45", 96%, n=91, 3 experiments). Lck recruitment was not observable when cells were incubated on immobilized BSA. The images are sections close to the interface between the cells and the immobilized antibody. The scale bar denotes the length of 10 Lim. +  +  97  Confocal images of Fyn  Figure 4.10 Recruitment of Fyn into microclusters is hampered in the presence of CD45. When BW5147 T cells were incubated on immobilized CD44, Fyn was recruited into microclusters, similar to Lck. However, by 5 minutes, only 25% (n=68, 3 experiments) of the C D 4 5 T cells showed Fyn recruitment while 74% (n=84, 3 experiments) of CD45" T cells showed Fyn recruitment. By 30 minutes, although the percentage of C D 4 5 T cells showing Fyn recruitment increased to 48% (n=93, 3 experiments), the percentage increased to 91% (n=67, 3 experiments) among CD45" T cells. The images shown above are sections close to the interface between the cells and the immobilized antibodies. The scale bar represents the length of 10 um. +  +  98  determine if tyrosine phosphorylation is resulted at the CD44 microclusters, co-labeling of CD44 and phosphotyrosine was carried out with J l W B B and 4G10 antibodies and secondary antibodies that are conjugated with the Alexa dye. Figure 4.11 showed representative figures of the co-labeling. As reported in the section 4.2.5, clustering of CD44 was observed in CD45 and CD45" T cells at 5 and 30 minutes after incubation on +  immobilized KM81. Only minimal tyrosine phosphorylation signal was observed in CD45 T cells at both time points; however, increasing intensities of phosphotyrosine +  signals were observed in CD45" T cells from 5 minutes to 30 minutes of incubation. These results suggest that CD44-induced SFK activity is down-regulated by CD45, resulting in failure to accumulate phosphotyrosine signals.  4.2.8  CD45 co-localizes with CD44 microclusters Since CD45 regulates integrin-mediated adhesion in T cells (292) and  macrophages (293), it has been hypothesized that CD45 may temporarily translocate into integrin-mediated focal adhesions to exert its inhibitory effect on integrin-induced signaling (385). To determine the location of CD45 during CD44-mediated cell spreading, Alexa488-conjugated 13/2 antibody, which recognizes the extracellular domain of CD45, was utilized. Figure 4.12A shows representative labeling of CD45 at 5 and 30 minutes after incubation on immobilized CD44 antibody. CD45 was observed at clusters and in a ring after 5 minutes of incubation on immobilized CD44 antibody; the clustering pattern was not observed when cells were incubated in BSA. By 30 minutes, CD45 rings were more evident, which were absent when cells were incubated on BSA. To determine i f CD45 co-localizes with CD44 at the 5-minute time point, colabeling of these transmebrane molecules were carried out. Figure 4.12B shows the clear co-localization of these two transmembrane molecules at the microclusters after a 5minute incubation of CD45 BW5147 T cells on immobilized CD44 antibody. +  4.2.9  Inhibition of PI3K leads to F-actin ring formation in CD45" T cells As reported in sections 3.2.3 and 3.2.6, the activity of PI3K is required for CD44-  induced elongated cell spreading and Pyk2 phosphorylation. To determine if PI3K activity is required for CD44-induced actin rearrangements in CD45 and CD45" T cells, +  99  A  Confocal imges of CD45+ BW5147 T cells CD44  pTyr  Merge  5' CD44 mAb  •  •  30' CD44 mAb B  Confocal imges of CD45- BW5147 T cells CD44  pTyr  Merge  m 30' BSA •  5' CD44 mAb  *  30' CD44 mAb  *  Figure 4.11 Co-localization of tyrosine phosphorylation and CD44. Shown above are images of CD44 and phosphotyrosine (pTyr) labeling close to the interface between the cells and the immobilized antibodies or BSA. Minimal pTyr was observed with C D 4 5 BW5147 cells (A) while pTyr accumulation was evident with CD45" BW5147 T cells (>150 cells observed in each case, 3 experiments). +  100  BSA  KM81  Figure 4.12 CD45 translocates transiently to the CD44 microclusters. (A) CD45 staining was visible at microclusters after C D 4 5 BW5147 T cells were incubated on KM81 for 5 minutes. CD45 was also observed in a ring structure in some cells. By 30 minutes, most of the CD45 was found in the ring structure. (B) Labeling of CD44 and CD45 suggests that they co-localized at the microclusters 5 minutes after incubation of the C D 4 5 T cells on immobilized KM81. The scale bar indicates the length of 10um. +  +  101  these cells were treated with LY294002 before incubation of immobilized CD44 antibody and labeled for confocal microscopy. Figure 4.13 shows a typical observation of LY294002-treated CD45 and CD45" +  T cells with incubation on K M 8 1 . The results showed that PI3K activity is not required for F-actin ring formation in CD45 T cells. However, PI3K activity is required for the +  F-actin fiber formation in CD45" T cells. More interesting, the inhibition of PI3K led to formation of actin ring structures in CD45" T cells. This suggests that the difference in CD44-induced actin rearrangement in CD45 and CD45" T cells lies in the activity of +  PI3K. In other words, CD45 may be preventing the activation of PI3K in CD44 signaling.  4.2.10 Recruitment of the p85 subunit of PI3K is not inhibited in the presence of CD45 To determine if the inhibitory effect of CD45 on PI3K activation lies in the recruitment of PI3K to CD44 clusters, co-labeling of p85 of PI3K, which is the regulatory subunit of this enzyme (386), and CD44 was carried out and observed with confocal microscopy. From previous sections, the data suggest that CD44 clustering leads to formation of signaling complexes, including Lck, Fyn, and in the case of CD45" cells, other proteins containing phosphotyrosine residues. Therefore, it is likely that PI3K recruitment is hampered in the presence of CD45 due to the lack of tyrosine phosphorylated proteins in CD44 clusters, since the Src-homology domain 2 (SH2) of p85 is thought to recruit PI3K to specific sites. The results from the co-labeling of p85 of PI3K and CD44 suggest that the recruitment of PI3K was not inhibited in the CD45 T cells (figure 4.14). By 30 minutes +  of incubation of CD45 and CD45" T cells on immobilized K M 8 1 , a small population of +  p85 of PI3K was recruited to CD44 clusters. One noted observation was that p85 staining in CD45" T cells localizes also at perinuclear regions; however, at present, the significance of such localization is unknown. Since the recruitment of p85 of PI3K was not hampered in CD45 T cells, I set +  out to determine if PI3K pathway is more activated in the CD45" T cells. Phosphorylation of Akt/PKB was utilized as readout of PI3K activity. Whole-cell lysate samples of spreading and non-spreading cells were resolved with SDS-PAGE. Western  102  F-actin staining CD45"  CD45"  1  i \  w  no treatment  . s 20nM LY294002  Figure 4.13 Inhibition of PI3K in CD45 T cells leads to CD44-induced actin ring fromation. Cells were fixed and labeled after 2 hours of incubation on immobilized anti-CD44 antibody. Pre-treatment of CD45" T cells with 20 uM LY294002 inhibited actin re-arrangement at the sides of the cells, with 60% of the observed cells (n=71, 3 experiments) showing actin rings. On the other hand, CD44-induced ring formation in C D 4 5 cells was not inhibitied, with 75% of the observed cells (n=48, 3 experiments) showing actin rings. The cells shown are images close to the interface between the cells and the immobilized antibodies. The scale bar denotes the length of 10 urn. -  +  103  Confocal images of p85 (PI3K) and CD44 double labeling  p85  CD44  Merge  Figure 4.14 PI3K is recruited to CD44 microclusters in both CD45 and CD45" BW5147 T cells during CD44-mediated cell spreading. BW5147 T cells were incubated on KM81 for 2 hours before fixation and labeling. Images shown aboves are the ones that are close to the interface between the cells and the immobilized antibodies. Results from labeling of the p85 subunit of PI3K and CD44 suggest that PI3K is recruited to CD44 microcluters in a CD45independent mechanism. The scale bar denotes the length of 10 um. +  104  blotting was then carried out with antibody specifically directed against phosphorylated serine 473 of Akt (pS473-Akt), which is indicative of upstream PI3K activation (figure 4.15). The results of the Western blot show that pS473-Akt increases in both CD45 and +  CD45" T cells, which suggests that PI3K was activated in both cell lines. However, the intensity of pS473-Akt is much higher in CD45" cells than that in CD45 cells. One +  intriguing observation was that pS473-Akt was also induced when CD45 or CD45" T +  cells were incubated on BSA, suggesting PI3K was activated through a CD44independent mechanism. Pre-treatment of cells with PP2 inhibited induction of pS473Akt, suggesting PI3K activation depends on SFK activity. At present, the possibility that PI3K activation can be induced by CD44 signaling cannot be ruled out, since this has also been shown in cancer cells (387-389). However, more experiments are required to confirm if PI3K is activated by CD44 signaling in T cells.  4.2.11 Pyk2 recruitment to CD44 microclusters is independent of CD45 The induction of Pyk2 phosphorylation was induced in CD44-mediated elongated cell spreading in CD45" T cells. To determine if the failure of Pyk2 phosphorylation in CD45 cells is associated with the absence of recruitment to CD44 microclusters, co+  labeling of Pyk2 and CD44 was carried out and analyzed with confocal microscopy. Results in figure 4.16 show the co-localization of Pyk2 and CD44 in either CD45 or +  CD45" T cells after a 30-minute incubation on immobilized K M 8 1 , suggesting recruitment of Pyk2 to CD44 microclusters was not inhibited in CD45 T cells. Although +  Pyk2 is recruited to CD44 microclusters in both cases, Pyk2 phosphorylation was observed only in CD45" T cells.  4.2.12 The chemokine SDF-1 induces segregation of CD45 and CD44 The results thus far suggest that CD45 exerts its inhibitory effect by physically translocating into CD44 clusters, down-regulating Lck activation, affecting Fyn recruitment and further propagation of tyrosine phosphorylation signals. One pending question that remains to be explored is under what circumstances is the inhibitory effect of CD45 removed. The regulation of CD45 has long been an interest of immunologists.  105  Western blot of whole-cell lysate CD45  CD45'  +  w  Time 0 (in min)  10 30 BSA  10 30 KM81  0  «...  10 30 BSA  pS473-Akt  10 30 KM81  0  10 30 KM81 20  PP2  Figure 4.15 Induction of Akt phosphorylation is CD44-independent but SFK-dependent. Whole-cell lysate samples were collected after BW5147 T cells had been incubated on BSA or KM81 for varying amounts of time. When C D 4 5 and CD45" BW5147 T cells were incubated on immobilized KM81, phosphorylation of serine 473 of Akt was induced. The intensity of induction was stronger in CD45" T cells. Also, the induction was dependent on SFK, as the induction was inhibited by PP2 treatment. However, incubation of cells on immobilized BSA also induced serine 473 phosphorylation to the same magnitude. +  106  Confocal images of CD44 and Pyk2 double labeling CD44  Pyk2  Merge  CD45+ BSA  CD45 KM81  +  m m •  CD45 KM81  Figure 4.16 Pyk2 is recruited to CD44 microclusters in CD45+ and CD45" BW 5147 T cells. Co-labeling of CD44 and Pyk2 was done with Alexa-conjugated secondary antibodies. Co-localization Pyk2 at CD44 microclusters was observed in both C D 4 5 and CD45' cells after the cells were incubated on immobilized KM81 for 30 minutes. Shown above are images close to the interface between the cells and the immobilized antibodies.The scale bar indicates the length of 10 urn. +  107  Different models have been postulated; however, the exact mechanism of regulation is still unclear. The results in section in 4.2.9 suggest that the regulatory effect of CD45 lies in its physical presence at signaling complexes. Earlier work with neutrophils showed that f-MLP, a chemotactic cue, induces cellular polarization and segregation of CD45 and CD44 at different ends of the cells (390). Moreover, CD44 has been shown to localize at the trailing ends of migrating lymphocytes, called the uropods, upon SDF-1 stimulation (391). To determine if physically segregation of CD45 and CD44 can be induced by the chemokine SDF-1, CD45 BW5147 T cells were treated with 100 n M of SDF-1 for +  various amounts of time. Labeling of CD44 and CD45 was carried out with KM81,13/2 and Alexa-conjugated secondary antibodies after fixation of treated cells. The results were observed with confocal microscopy. Figure 4.17 shows the results of some of the CD45 T cells in the presence or +  absence of SDF-1 for 5 minutes. Dramatic change of cell shape is observed along with segregation of CD45 and CD44. However, not all the cells observed showed such dramatic change of shape and segregation of CD45 and CD44 (Table 4.2). To determine if segregation of CD45 and CD44 upon SDF-1 stimulation is observed in primary cells, thymocytes and splenic C D 4 T cells were also examined with the same stimulation and +  labeling conditions. Similar to the observations with CD45 BW5147 T cells, a +  population of thymocytes and splenic T cells also displayed segregation of CD45 and CD44 (figure 4.18, table 4.2). These results show that SDF-1 can induce segregation of CD45 and CD44 in T cells (14-17%).  108  Confocal images of CD44  CD44  and C D 4 5 double labeling Merge  CD45  \ SDF-1  t  100mM SDF-1  Figure 4.17 CD45 and CD44 segregation in BW5147 T cells upon SDF-1 stimulation. Images shown above are projections of all confocal images. In general, distribuiton of CD44 and CD45 was uniformacross the cell surface. Upon SDF-1 stimulation, CD44 and CD45 polaization was observed, creating segregation of these surface molecules. Moreover, an elongated cell morphology was observed with the segregation of CD44 and CD45.  1 0 9  A  Confocal images of thymocytes CD44  Merge  CD45  100nM SDF-1  Figure 4.18 Segregation of CD44 and CD45 in thymocytes and splenic T cells from Balb/c mice upon SDF-1 stimulation. When freshly isolated thymocytes (A) and splenic T (B) cells from Balb/c mice were stimulated with SDF-1, segregation of C D 4 4 and C D 4 5 was also observed. The images are projections of all confocal images. The scale bar denotes the length of 10 urn.  110  Table 4.2  Percentage of cells showing segregation of CD44 and CD45  Cells\Treatment  No SDF-1  100 n M SDF-1,  100 n M , SDF-1,  2' or 5'  10'  CD45 BW5147 T cells  0% (n=148, 4 exp)  14% (n=98, 5 exp)  18%(n=85,4exp)  Balb/c thymocytes  2% (n=46, 3 exp)  17%(n=53, 5 exp)  5% (n=86, 3 exp)  Balb/c CD4+ splenic T  2% (n=82, 4 exp)  21%(n=63, 4 exp)  13% (n=87, 3 exp)  +  111  4.3  Discussion  4.3.1  Data Summary The negative regulatory role of CD45 in CD44-mediated signaling and cell  spreading was supported by the observations with the activated thymocytes from wildtype and CD45" mice. Similar to the observations with CD45" BW5147 T cells, CD45" A  A  thymocytes exhibited cell spreading on anti-CD44 antibody with much elongated morphology, when compared to wild-type thymocytes. The results with PTPa suggest that the transmembrane phosphatase activity may play a role in regulating CD44mediated signaling and cell spreading. This is implicated by the shorter cell spreading lengths of CD45" PTPa and CD45" Y789F-PTPa transfectants as well as the accompanied reduction in pi20/130 and Pyk2 phosphorylation. Moreover, the intensity of tyrosine phosphorylation of p i 20/130 and Pyk2 correlates with the spreading lengths of these cells on immobilized CD44 antibody. The underlying actin structure correlates with the spreading morphology of CD45 and CD45" T cells upon CD44 engagement. A n actin ring was observed with +  round cell spreading of CD45 T cells, while long actin fibers were observed at the sides +  of the cells, aligning with the longitudinal axis of the cell body, in CD45" T cells. Moreover, a meshwork of actin filaments was also observed at the ends of the CD45" cells. SFK's are required for the formation of both forms of actin structures in CD45  +  and CD45" cells. When PI3K was inhibited, actin ring formation was observed in CD45" T cells. Upon engagement of CD44, clustering of this adhesion molecule is observed. The experimental results with latrunculin A pre-treatment suggest that CD44 clustering was an actin-dependent process. With CD44 clustering, Lck and Fyn were also recruited into specific clusters. In both CD45 and CD45" T cells, the recruitment of Lck was the +  same; however, the recruitment of Fyn was delayed and reduced in CD45 compared to +  CD45" cells. Recruitment of PI3K and Pyk2 were observed at the CD44 microclusters in both CD45 and CD45" T cells. Since SFK is required for both forms of actin +  polymerization in the CD45 and CD45" cells, CD44 clustering must bring about SFK +  activation. The labeling with phosphospecific antibody that recognizes pY394 of Lck, which is indicative of Lck activity, showed that Lck was more active in CD45" cells,  112  when compared with CD45 cells, after 30 minutes of incubation on immobilized anti+  CD44 antibody. This suggests the presence of CD45 results in down-regulation of Lck activity. The differences in Fyn recruitment and duration of Lck activity may explain the absence of phosphotyrosine residues in CD45 cells at the CD44 microclusters. +  Moreover, labeling of CD45 indicates that it is recruited to CD44 microclusters and presumably dephosphorylates Lck and possibly other recruited proteins. At the moment, it is unclear how CD45 is regulated. However, one possibility of CD45 regulation is the cellular location of CD45 and its potential substrate. Upon SDF-1 stimulation, CD45 and CD44 segregates to different ends of the same cell, creating a local absence of CD45. This presents a situation where CD45 may not exert its regulatory effect on CD44 signaling.  4.3.2  Role of protein tyrosine phosphatase alpha (PTPa) in cell spreading and migration The overexpression of PTPa or Y789F-PTPa significantly reduced the spreading  length of CD45" T cells upon CD44 engagement. This reduction in eel! spreading length was associated with reduced phosphorylation of pi20/130 and Pyk2. Moreover, in the absence of CD45, hyperphosphorylation of SFK, Lck and Fyn, is usually observed in T cells. This is also the case in BW5147 T cells utilized in this study. With the overexpression of PTPa or Y789F-PTPa, the levels of hyperphosphorylation of these kinases were decreased. However, the inhibitory action of PTPa or Y789F-PTPa did not correlate with their influence on the phosphorylation states of the SFK. Therefore, even though the effect of PTPa on CD44-mediated cell spreading was consistent with our hypothesis, the mechanism involved may be different from that of CD45. This is suggested by the observation that Y789F-PTPa CD45" T cells show the shortest spreading length while the phosphorylation levels of SFK was lowest in PTPa CD45" T cells. One explanation of this observation is that the SFK phosphorylation states observed were reflective of the overall SFK population since whole-cell lysate was utilized. There may exist specific populations of Lck and Fyn that are utilized by CD44 signaling. This question may be addressed by evaluating the phosphorylation states of Lck and Fyn that co-immunoprecipitate with CD44. Moreover, the possibility that PTPa  113  and Y789F-PTPa may display different substrate specificities to other downstream signaling molecules involved in CD44 signaling cannot be denied. P T P a has been shown to play a role in epidermoid carcinoma cell adhesion (380). Expression of PTPa in these cells inhibited EGF-induced rounding, leaving the cells adherent and spread on the underlying substratum. The effect is correlated with increased focal adhesion kinase (FAK) phosphorylation and Src activation. Moreover, PTPadefective fibroblasts displayed reduced migration on fibronectin when compared to wildtype cells. This was due to reduced Src-FAK and Fyn-FAK association and F A K activation, which was reverted by transfection of these cells with functional PTPa (392). These observations suggest that P T P a plays a positive regulatory role in adhesion, spreading, and migration of adherent cells; however, the observations made in this study suggest that P T P a plays a negative regulatory role in spreading of suspension cells. Phosphorylated tyrosine 789 (pY789) in PTPa was found to associate with the adaptor molecule Grb2. The SH2 domain of Src interacts also with pY789; therefore, a competition between Grb2 and Src for interaction with PTPa exists (393, 394). Generally, Grb 2 displays a higher affinity for pY789. However, upon phosphorylation of membrane-proximal serine residues (SI 80 and S204) of PTPa, the affinity of pY789 to Grb2 is lowered, encouraging interaction between PTPa and Src (395). Nonetheless, pY789 has an overall effect of encouraging the interaction between PTPa and Src, leading to Src dephosphorylation, since Y789F mutation abrogated the interaction between P T P a and Src (394). Co-immunoprecipitation of Grb2 with PTPa has also been observed in PTPa CD45" T cells while this interaction is absent with Y789F mutation (Wang Y and Johnson P, unpublished data). Previously, a constitutive interaction between Grb2 and CD44 in BW5147 cells was also observed (Li R and Johnson P, unpublished data). Therefore, PTPa may interact with CD44-associated Grb2, bringing P T P a in close proximity of CD44-associated Lck or Fyn. This may lead to enhanced access of PTPa to Lck or Fyn, resulting in lower phosphorylation states of these kinases in PTPa CD45" T cells, comparing to those in Y789F-PTPa CD45" T cells. As mentioned before, the spreading length of PTPa CD45" T cells and Y789F-PTPa CD45" T cells did not correlate with the phosphorylation levels of Lck and Fyn. It may be due to  114  the possibility that PTPa and Y789F-PTPa possess different substrate specificities to downstream substrates in CD44 signaling. One possbility is that PTPa may directly dephosphorylate Pyk2 or HEF1 (section 3.2.7) and the Y789F mutation encourages the interaction between the phosphatse and these substrates.  4.3.3  Role of CD45 in CD44 and CD3/TCR signaling through its regulation on SFK CD45 co-localized with CD44 microclusters when CD45 BW5147 T cells were +  incubated on immobilized CD44 antibody. Moreover, pre-treatment of BW5147 T cells with a specific inhibitor of SFK led to inhibition of F-actin formation in both CD45 and +  CD45" cells. Phosphorylated Y394-Lck, which is indicative of activity, was observed only in CD45" cells after 30 minutes of incubation. In addition, accumulation of tyrosine phosphorylation was observed at CD44 microclusters only in CD45" cells. Together, these observations suggest that CD44 engagement leads to activation of SFK in both CD45 and CD45" T cells. However, in CD45 T cells, CD45 was also recruited to these +  +  microclusters. The presence of this protein tyrosine phosphatase may then dephosphorylate and inactivate Lck, resulting in failure to further propagation of cellular signals through tyrosine phosphorylation. Although CD45 has long been thought to activate Lck through dephosphorylation of the inhibitory C-terminal tyrosine (Y505) (283) , there is increasing evidence that CD45 may also negatively regulate Lck activity. Hyperphosphorylation of Lck and Fyn has been observed in CD45-defective cell lines (reviewed in 396). Tyrosine 505 and, to a lesser extent, Y394 were hyperphosphorylated (284) . Lck immunoprecipitated from CD45-defective thymocytes showed higher kinase activity, when compared to Lck from wild-type thymocytes (286). CD45 has also been shown to be capable of directly dephosphorylating Y394 of Lck in vitro (287). Therefore, it is possible that CD45 exerts its effect on CD44 signaling by dephosphorylating pY394. CD45 was observed as clusters at 5 minutes after CD44 engagement; however, it was no longer visible by 30 minutes in the majority of the cells. This observation is similar to the recruitment of CD45 to c S M A C upon CD3 engagement (60). The authors observed that CD45 was associated with CD3 early during T/APC interaction in conjunction with Lck co-localization. By 7 minutes, CD45 was already excluded from  115  c S M A C and later localized at p S M A C . The authors suggested that the early recruitment of CD45 into c S M A C was to dephosphorylate TCR-associated proteins, including Lck, to reset the phosphorylation states. This is thought to be required for effective T cell activation even in the presence of small number of pMHC/TCR interaction. However, this does not seem to be the case in CD44 signaling, since the recruitment of CD45 resulted in dephosphorylation of pY394-Lck and prevented accumulation of tyrosine phosphorylation. CD45 was observed at the microclusters and ring strucutre, with 13/2 (296) antibody labeling, after incubating the BW5147 cells on immobilized anti-CD44 antibody for 5 minutes. By 30 minutes, most of the CD45 staining was observed at the ring structures (section 4.2.8). Labeling with another antibody, R02.2 (397), that recognizes the cytoplasmic domain of CD45 was able to observe the presence of CD45 at the microclusters at both 5- and 30-minute time points (Lai J and Johnson P, unpublished data). Moreover, further experimentation with 13/2 antibody at a higher gain setting (higher sensitivity) with the confocal microscopy was able to detect the presence of CD45 at the microclusters at the 30-minute time point, abeit not to the same extent as with the R02.2 Ab (Lai J and Johnson P, unpublished data). There are two possibilities that explain these observations. First, CD45 may be cleaved upon recruitment to CD44 microclusters, leaving the cytoplasmic domains at the sites, allowing R02.2 labeling. Second, CD45 may be internalized after the recruitment to CD44 microclusters, limiting access of 13/2 for labeling of CD45. More experiments will be required to determine which possibility is the case. Although the roles of CD45 seem to be different in TCR/CD3 and CD44 signaling pathways, a common observation is that CD45 is recruited to specific signaling complex. It will be of interest to determine how the movement of CD45 is controlled in both cases. A possible mechanism is the association of CD45 with the actin cytoskeleton through its interaction with fodrin. It has previously been observed that capping of CD45 with monoclonal antibody is associated with partial capping of fodrin and actin (398). Moreover, CD45 physically interacts with this actin-binding protein and a related-protein, spectrin (399-401). As mentioned before, CD44 interacts with the actin cytoskeleton through its interaction with ankyrin (153, 162, 402) and E R M proteins (151). The  116  recruitment of surface molecules to specific cellular locations through their interaction with the actin cytoskeleton has been demonstrated. Wulfing et al. have observed that inhibition of myosin motor reduced re-distribution of ICAM-1 to the immunological synapse (403). The directed recruitment may make use of the actinomyosin network in the cell (reviewed in 404). Upon engagement of the TCR, CD43 is excluded from the immunological synapse. This specific translocation requires the loss of interaction and re-association between CD43 and meosin, which belongs to the E R M family of adaptors (405). It has been proposed that shifting of actin filaments (403) or temporary concentration and subsequent depolymerization of actin may bring about the specific translocation of surface molecules (378, 406). Similar mechanisms might exist that bring about the translocation of CD45 to CD44 microclusters. It is unclear i f CD45 is required to enter lipid rafts in order to exert its effect on CD44 signaling, since CD44 has been observed to reside in lipid rafts and interact with Lck and Fyn (299). Conflicting observations have been reported in terms of whether CD45 is found in the lipid rafts. This may be reflective of differences in experimental procedures and cell types examined. CD45 was reported to reside in lipid rafts of human leukemic cell lines and granulocytes (407). However, CD45 was only slightly partitioned into lipid rafts of murine thymocytes and T cell line (251, 408, 409). Recently, CD45 has also been reported to associate with lipid rafts in a dynamic manner, leaving the lipid rafts upon TCR stimulation (290). Although the nature of association between CD45 and lipid rafts is uncertain at the moment, CD45 may not be required to enter lipid rafts to exert its effect on CD44 signaling since lipid rafts may exist as very small compartments (410).  4.3.4  CD45 translocation Upon stimulation of BW5147 T cells, Balb/c thymocytes, and C D 4 splenic T +  cells, a population of cells showed segregation of CD45 and CD44 at opposite ends of the cells. This observation is consistent with that of Seveau et al., which they observed segregation of CD45 and CD44 at opposite ends of human neutrophils upon stimulation with f-MLP (390). Moreover, upon SDF-1 stimulation of T cells, CD45 associates with  117  CXCR4 (291), which has been found to localized at the leading edge of migrating T cells (411,412). The physiological importance of this translocation is suggested by the observation made by Fernandis and co-workers. These authors discovered that association of CD45 with CXCR4 is required for effective signaling of SDF-1 in T cells that is required for the chemokine-induced migration (291). The observations of CD45 translocation during T/APC interaction and chemoattractant stimulation suggest significance of this process, for this may be the regulatory mechanism on CD45 activity. For years, immunologists and cell biologists have been trying to decipher the regulatory mechanism of CD45. One of the prevailing ideas is that CD45 activity is regulated by dimerization, either with the same isoform or with different CD45 isoforms (413). This was first proposed by the observation that forced dimerization of CD45 led to defective TCR signaling (376). This model was supported by the crystal structure of a very similar transmembrane phosphatase P T P a (414), in which case the dimer formation was observed. However, the recently reported crystal structure of CD45 casts serious doubt as to whether CD45 dimerization can occur, since the reported structure does not allow dimer interaction (415). Moreover, the authors failed to observe CD45 dimers even at high protein concentration. Another possibility of how CD45 activity is regulated is the cellular location of this tyrosine phosphatase. As reported in this study, specific localization of CD45 can exert specific effects with local signaling complexes. The chemoattractantinduced segregation of CD45 and CD44 observed, in this study and by others, may create a local absence of CD45 where the strength and outcome of CD44 signaling may result in a different type of actin rearrangement and cell spreading. In this study, only about 14-21% of cells showed CD45 and CD44 segregation in response to SDF-1 (table 4.2). The reason for the low percentage of responding cells is unclear. One possibility is the difference in experimental procedures. The chemoattractant-induced segregation of CD45 and CD44 observed by Seveau et al. was performed with neutrophils adhered to fibronectin-coated surface. Rey et al. observed the localization of CXCR4 with cells plated on fibronectin-coated surface also. Fibronectin is an E C M component that is recognized by various integrins (416) and integrin engagement has been reported to influence the signaling threshold of other  118  pathways (67, 417). Thus, it is likely that integrin engagement in these cases lowered signaling threshold of these cells, leading to effective response to the chemoattractants.  4.3.5  The role of Lck and Fyn in CD44 signaling and T cell activation Upon engagement of CD44, Lck and Fyn were recruited into microclusters. The  recruitment of Lck was not dependent on CD45 since this occurred in CD45 and CD45" +  T cells with comparable efficiency. Fyn recruitment was delayed, comparing to Lck recruitment. Moreover, Fyn recruitment was more efficient in CD45" T cells. In this study, CD44 clustering and recruitment of Lck seemed to occur with similar efficiency, this is suggested by the comparable percentage of cells showing these microclusters after 5-minute incubation of cells on CD44 antibody. This is readily explicable by the CD44/Lck interaction. Previously, CD44 was reported to associate with SFK in lipid rafts (249). As CD44 is recruited into microclusters, CD44-associated Lck was likely to be recruited as a secondary consequence. When Monks et al. first described the formation of c-SMAC and p-SMAC, they noted the recruitment of Lck and Fyn to c-SMAC (56). In the paper, the authors showed Lck recruitment to the c-SMAC by 5' while the figure with Fyn recruitment was captured by 13'. However, the authors did not address if there is a significant temporal difference with the recruitment of these two kinases. Ilangumaran et al. observed that more Lck than Fyn was co-immunoprecipited with CD44 from lipid rafts (249). It has previously been reported that Lck and Fyn displayed different subcellular localization. Lck was observed mainly at the plasma membrane while Fyn was associated with centrosomes and microtubules (418). It is possible that the recruited Fyn observed was not the CD44-associated population. Lck and Fyn involvement in TCR signaling requires recruitment and activation of these kinases. Lck is recruited through its association with either CD4 or CD8 (419, 420). However, how Fyn is recruited to the TCR signaling complex is currently unknown, although Fyn is capable of interacting specifically with CD3£ at low stochiometry (421). Similarly, it is uncertain how Fyn is recruited during CD44 signaling.  119  Although similarities between Lck and Fyn led to the suggestions that they may play redundant roles in T cell activation (422), there is convincing evidence that they play different roles in T cell biology. First, targeted gene disruption of Lck resulted in arrest in thymocyte development (423), while this defect in thymocyte development was not observed in Fyn " mice (424, 425). Second, unique substrates of Fyn have been _/  identified (426-429). Third, recent observations suggest the Fyn activation was due to Lck mobilization into lipid rafts upon T C R and CD4 engagement, indicating there exists a hierarchy of SFK activation in TCR signaling (430). Indeed, the delayed recruitment of Fyn to specific complexes in CD44 signaling also suggests such hierarchy in activation. Impairment of TCR signaling has been observed with Fyn-defective T cells (424, 425, 431), suggesting a role of Fyn in this signaling pathway. Recent identification of Fyn-specific substrates suggests that Fyn may participate in T cell activation, in part, through its regulation of the actin cytoskeleton (432). The focal adhesion kinase Pyk2 was found to be a specific substrate of Fyn in TCR signaling (426) and Pyk2 has been implicated in cytoskeletal regulation (35, 42, 433, 434). Moreover, the specific Fynbinding protein (Fyb, also known as SLAP-130 or A D A P ) (435) has been shown to associate with various adaptors and effectors that cause actin rearrangement (428). Furthermore, engagement of co-stimulatory molecule CD28 leads to Fyn-dependent tyrosine phosphorylation of Vav (436), which is a guanine-nucleotide exchange factor that is involved in actin rearrangement (437-439). Therefore, the specific function of Fyn may be associated with cytoskeletal rearrangement in T cells. Given the fact that tyrosine phosphorylation of Pyk2 is induced in CD44 signaling, Fyn activation in this signaling pathway is likely. Moreover, the specific downstream substrates of Fyn, mentioned above, were shown to mediate cytoskeletal rearrangement, these effectors may also be involved in CD44 signaling that result in reorganization of cytoskeleton.  4.3.6  Lck and Fyn activation The observations in this study suggest that SFK activity is required in CD44-  mediated actin polymerization, in both CD45 and CD45" T cells. CD44 engagement +  leading to Lck-mediated tyrosine phosphorylation of cellular proteins has also been reported (334). However, how CD44 engagement led to SFK activation is unclear. It has  120  long been thought that Lck or Fyn activation during T/APC interaction is dependent upon CD45. However, recent observations made by Holdorf et al. challenge that idea (384). The authors observed that TCR cross-linking did not lead to Lck autophosphorylation at Y394; instead, Lck was recruited and activated through CD4- and CD28-dependent translocation. Moreover, Lck activation was independent of CD45. The observations made in this study indicate also that CD44-mediated activation of Lck and/or Fyn is CD45-independent. Moreover, the role of CD45 in this signaling pathway is inhibitory. The crystal structures of Src, Hck and pY394-Lck suggest the activity of SFK is regulated by intramolecular interactions (282). Two major intramolecular interactions were identified. One is mediated through binding between the SH2 domain and the phosphorylated tyrosine residue at the C-terminus of the kinase. The other interaction occurs between the SH3 domain and the poly-proline region that is situation between the SH2 and the kinase domains. Dephosphorylation of the C-terminal phosphotyrosine is thought to partially release the intramolecular interaction and prime SFK for activation. Another way to disrupt this intramoleculer binding is through interaction with high affinity SH2 or SH3 ligands of SFK. The Nef protein of FflV has been shown to interact with the SH3 domain of a SFK, Hck, and enhances its kinase activity in vitro and in vivo (440, 441). A similar mechanism has been proposed by Thomas and Brown under physiological circumstances (385). The authors proposed that the significance of the Cterminal phosphotyrosine would have little effect in the presence of such high affinity ligand. Indeed, a recently identified cellular adaptor Unci 19 has been shown to activate Src through its interaction with the SH2 and SH3 domains of the kinase (442). Moreover, Unci 19 has also been shown to activate Lck and Fyn and it is essential for T cell activation (443). There are also previously identified candidates that may serve as the high affinity SH2/SH3 ligand that activates Lck or Fyn. HS1 (or L c k B P l ) is an adaptor protein that contains two proline-rich regions, which have been shown to interact with Lck (444, 445). Moreover, upon TCR stimulation, HS1 is phosphorylated and is associated with the SH2 domain of Lck (446). Therefore, HS1 serves as a good highaffinity SH2/SH3 ligand candidate. Another such candidate is Sam68 (447). This protein was also shown to interact with Fyn and Lck through their SH2 and SH3 domains. Furthermore, the adaptor Grb2 has also been shown to interact with  121  phosphorylated Lck (448). As mentioned before, Grb2 is constitutively associated with CD44 in BW5147 T cells. It is likely that the SH2 domain of Grb2 interacts with the Cterminal phosphotyrosine of Lck or Fyn. This interaction may also disrupt the intramolecular binding such that the kinases are primed for transautophosphorylation and activation. This is suggested by previous observations that tyrosine 505 of Lck is hyperphosphorylated in CD45-null cells (283) and more Lck is associated with CD44 in C D 4 5 T cells (251).  4.3.7  CD44 signaling and actin rearrangement The difference in CD45 and CD45" T cell spreading on immobilized CD44 +  antibody corresponds with different actin structures. CD44 engagement resulted in Factin ring formation in CD45 T cells while F-actin fibers and meshwork organized in an +  elongated fashion was observed in CD45" T cells. Incubation of Jurkat T cells on immobilized CD3 antibody also induces formation of F-actin ring (378), similar to the actin structure induced with CD44 engagement in CD45 T cells. In both cases, actin polymerization was inhibited by PP2, a specific +  inhibitor for Src-family kinases. However, the adaptor L A T is required in CD3-induced actin ring formation, yet L A T phosphorylation is not induced in CD44 signaling (section 3.2.8). When a T cell comes into contact with an A P C , a stop signal in generated (57). Therefore, the actin rearrangement generated by CD3 engagement should correspond with immobilization of T lymphocytes. This raises the question whether CD44, in the presence of CD45, generates a cooperative signal to enhance actin ring formation during the T/APC interaction. On the other hand, the actin polymerization pattern in CD45" T cells generated by CD44 engagement resembles the actin organization of a migrating cell. This raises the question i f CD45 mediates or adjusts the threshold of a CD44 signal in T cells for "stop" or "go" signals. Another interesting piece of datum is that pre-treatment of CD45" T cells with the PI3K inhibitor led to formation of an actin ring. Similar result was obtained with inhibitor of PLC, U73122 (Lai J and Johnson P, unpublished data), where treatment of CD45" BW5147 T cells with this inhibitor and subsequent incubation of these cells on immobilized anti-CD44 antibody also led to actin ring formation. This observation  122  suggests that PI3K activity is not sufficient to cause longitudinal F-actin formation in the CD45" cells, although the PI3K activity is required. This is also supported by the obseration that CD45" cells did not spread on BSA, even though Akt phosphorylation was observed. The observed Akt phosphorylation was an SFK-dependent process (figure 4.15). In CD45" T cells, the SFK Lck is hyperphosphorylated (283) and is hyperactive (284). This may result in lowered threshold for signaling. Therefore, the elongated cell spreading and longitudinal F-actin formation in CD45" T cells, upon CD44 engagement, may be a result of the exisitence of a population of hyperactive SFK that activates P L C y l (section 3.2.5) and PI3K. Since Akt is a downstream effector of PI3K to regulate actin reorganization (449) and its serine phosphorylation at S473 is indicative of activity, pS473-Akt was utilized as an activation readout of PI3K in CD44 signaling. It was intriguing to observe that pS473-Akt was induced in CD45" T cells when these cells were incubated on either CD44 antibody or BSA. Indeed, induction of pS473-Akt was also observed in CD45 T cells +  also; however, the level of induction in CD45 cells were consistently lower than that in +  CD45" T cells. Previously, engagement of CD44, leading to PI3K activation, has been observed (244, 387, 388, 450-452). Moreover, Bates et al. observed that the CD44induced PI3K was dependent on SFK Lyn (388). This is consistent with the observation that PP2 inhibition significantly reduced pS473-Akt induction in CD45" T cells (fig. 4.15). At the moment, it is impossible to deny the possibility that CD44 engagement does lead to PI3K activation in T cells while incubation of BW5147 T cells on B S A activate PI3K with other mechanisms. Moreover, recruitment of p85 of PI3K to CD44 microclusters further suggests the involvement of PI3K in CD44 signaling. Since BW5147 T cells should not interact with the BSA-coated surface with any cellular receptor, PI3K activation may be a result of mechanical stress as the cells were sitting on a surface, experiencing the weight of the cells. This possibility is suggested by the involvement of PI3K in mechanotransduction in various cell types (453-455).  4.3.8  CD44 signalosome The results from confocal microscopy show that CD44 clustering occurs upon  engagement. Pre-treatment of BW5147 T cells with latrunculin A resulted in a  123  significant decrease in proportion of cells displaying CD44 microclusters and in the microclusters sizes. These observations suggest that CD44 clustering is an actindependent process. Since the cytoplasmic domain of CD44 interacts with adaptor proteins that interact with the actin cytoskeleton, it raises the possibility that CD44 engagement causes recruitment of CD44 through these means. Together with observations reported in 4.2.10, CD44 clustering led to formation of signaling complexes that co-localized with PI3K and Pyk2. Moreover, in the absence of CD45, accumulation of signaling molecules containing phosphotyrosine residues was observed. Furthermore, Lck and Fyn were recruited to specific clusters upon CD44 engagement. Together, these observations suggest that CD44 engagement lead to formation of signaling complexes or CD44 signalosomes. The CD44 signalosomes in CD45 T cells differ from those in CD45" T cells in a few ways: recruitment of CD45, +  delayed Fyn recruitment, and lack of phosphotyrosine accumulation. These discrepancies are associated with different outcomes of actin rearrangement and cell spreading. Formation of signaling complexes, or signalosomes, is not unique to CD44 signaling. Indeed, other signaling complexes have previously been observed. These include integrin-induced focal adhesions, TCR signaling complex, and B C R signalosomes (456-458). These signalosomes are macromolecular structures that regulate qualitative and quantitative natures of signaling (457). The organization of signaling molecules into such macromolecular structures may bring about several functional advantages. First, partial and preformed signalosomes may exist in a non-functional state. Upon stimulation, fusion of these partial signalosomes will ensure efficient delivery of signals. The observation of raft heterogeneity is consistent with this idea (459). Second, signalosome organization promotes concentration of signaling molecules, including intermediates, which may act as a means of controlling signal strength. Third, such compartmentalization may ensure directionality during cell-cell interaction (e.g. during cytotoxic T lymphocyte-target cell interaction) or cell-ECM interaction (e.g. during cell migration). Fourth, such signalosomes may result from dynamic interactions between signaling molecules, such that recycling of these participants become possible.  124  A n example of such is the formation and dissociation of focal adhesions during cellular migration.  4.3.9  Association of signal strength and duration with functional outcomes In this study, differences in CD44 signaling duration and strength are implicated  in CD45 or CD45" T cells, leading to different actin reorganization and cell spreading. +  This is suggested by the observations that SFK is required in CD44-induced actin polymerization in both CD45 and CD45" cells and accumulation of tyrosine +  phosphorylation is observed only in CD45" cells. Tyrosine phosphorylation signals may be propagated in CD45 cells; however, the level of phosphotyrosine may be below the +  sensitivity of the detection method utilized. Moreover, these observations suggest that CD45 is a regulator of signal strength and duration. In immunology, variation in strength and duration of signals, leading to different outcomes, is a common theme. First, the affinity/avidity of the TCR of maturing thymocytes to self-peptide, presented in the context of M H C , suggest the role of signaling strength and duration in positive and negative selections (460). Second, the TCR signaling strength has been implicated in ct|3 or v6 T cell lineage commitment (461). Third, the duration of TCR signals, in the presence of cytokines, has been shown to partially regulate Thl/Th2 cell polarization (462). As a widely expressed adhesion molecule, CD44 contributes to cellular behaviour mostly through its ability to interact with various ligands and its signaling function. In T cells, CD44 may also contribute to T cell biology through its signaling to actin rearrangement. In chapter 5, the possible contribution of this signaling function in antigen presentation and cell migration will be discussed. Moreover, the role of CD45 as a threshold regulator in signaling of hematopoietic cells will also be discussed.  125  Chapter 5 Summary and Perspectives 5.1  CD44 signaling pathway In this study, a CD44 signaling pathway associated with dramatic cytoskeletal  rearrangement, elongated cell spreading, and related-focal adhesion kinase (Pyk2) phosphorylation was identified. The prototypical transmembrance tyrosine phosphatase CD45 may regulate the strength and duration of CD44 signaling through its physical presence at the CD44 clusters. The signaling components identified here should serve as a framework where CD44 signaling, in association with cytoskeletal changes, can be further studied. A working model of CD44 signaling in T cells is summarized as follows. CD44 engagement leads to an actin-dependent clustering of CD44. This clustering may also recruit CD44 from the non-raft population since CD44 engagement can result in mobilization of this adhesion molecule into lipid rafts (250). CD44 clustering is also associated with Lck and Fyn recruitment. This can lead to activation of these Src family kinases, possibly through transautophosphorylation (275). When CD45 is present (figure 5.1), the CD44-induced signal is comparatively weak and brief, leading to an F-actin ring formation. In this situation, PI3K and Pyk2 phosphorylation are not involved, even though they are also recruited to CD44 signalosomes. When CD45 is absent (figure 5.2), presumably due to specific recruitment of CD45 to other cellular locations, the threshold for SFK activation is lowered and upon CD44 ligation, P L C y l is activated in an SFKdependent manner. This leads to IP3 and D A G production, resulting in mobilization of intracellular and then extracellular calcium. In response to an increase in intracellular calcium, autophosphorylation of Y402 of Pyk2 occurs, leading to the creation of SFK docking site and further activation of Pyk2 (365). Moreover, the subsequent phosphorylation and activation of Pyk2 may be mediated by Fyn in T cells (365, 426). PI3K is also activated in an SFK-dependent manner, either through engagement of CD44 or by mechanical stress. Although it is unclear how PI3K is activated at present, it is required for Pyk2 phosphorylation. Similar dependence of Pyk2 activation on PI3K has also been observed with p i integrin signaling (366). Pyk2 activation then leads to recruitment and phosphorylation of HEF1, which may regulate actin rearrangement with  126  CD44 engagement  brief and weak signal  F-actin ring formation + round cell spreading  Figure 5.1  Schematic diagram of CD44 signaling in the presence of CD45 (transient and weak signal)  127  CD44 engagement  longitudinal F-actin formation + elongated cell spreading  Figure 5.2  Schematic diagram of CD44 signaling in the absence of CD45 (sustained signal)  128  its downstream adaptors and effectors. The signals initiated with CD44 engagement does not necessarily have to be linear. Fyn activation may result in its association with Fyb, and exerts its regulation on actin rearrangement through SLP-76, V A S P , Nek, WASP, and Arp 2/3 (428, 463). This signal may work in conjunction with the signal that regulates actin rearrangement through Pyk2 and HEF1. Although the components in CD44 signaling identified in this study also participate in CD3 signaling, these two signaling pathways are distinct. This is indicated by the lack of L A T and E R K phosphorylation in CD44 signaling. Indeed, at present, the CD44 signaling pathway identified in this study involves a subset of CD3 signaling components. This suggests CD44 signaling may act synergistically with CD3 signaling to produce stronger signals and possibly different stimulation outcomes. Others have observed the involvement of Rac and P K C (|3 and 5 isoforms) in CD44-mediated cytoskeletal rearrangement (193, 250). These observations are consistent with the working model proposed above since Rac activation may result from downstream effectors of Fyn and P K C activation may resulted from P L C y l activation.  5.2  CD44 as a co-stimulatory molecule Effective activation of T cells requires not only the stimulation through  interaction between TCR and pMHC, but it also requires stimulation through costimulatory molecules. The lack of co-stimulation in naive T cell stimulation may result in induction of tolerance, known as anergy. CD28 is the most studied co-stimulatory molecule. Many others have also been proposed (200-203), including CD44. As mentioned above, the cellular components involved in CD44 signaling contains a subset of CD3 signaling components. CD44 has the potential to act as a costimulatory molecule by enhancing CD3 signal strength. Indeed, recent data suggest that the action of CD28 as a co-stimulatory molecule is through amplification of TCR signal (reviewed in 199). Moreover, another adhesion molecule LFA-1 has been shown to play a role in lowering signaling threshold of T cells (67). Therefore, CD44 may act as a costimulatory molecule in a similar manner as CD28 or L F A - 1 . In addition, co-ligation of CD44 and CD3 with monoclonal antibodies has previously been shown to enhance IL-2 production (205). Furthermore, the presence of CD44-receptor globulin fusion protein  129  reduced IL-2 production from T cells stimulated through specific peptide presentation by a B cell line (206). This observation is the same as that reported with the presence of CTLA-4-Ig fusion protein (464), suggesting a similarity between CD44 and CD28 costimulatory effects. In addition, administration of anti-CD44H antibody has been shown to inhibit T cell activation in a mouse delayed-typed hypersensitivity model (465). These observations strongly suggest that CD44 may play a role in co-stimulation through its signaling function. Another mechanism that CD44 may also act as a co-stimulatory molecule is through signaling in concert with other co-stimulatory molecules. It has been shown that CD44 may function synergistically with CD2 to enhance IL-2 production (466). Moreover, in conjunction with CD28, CD44 also contribute to produce a strong mitogenic effect on peripheral T lymphocytes (204). CD28 has also been shown to play a role in mediating T/APC interaction, possibly through acting as an adhesion molecule and mediating rearrangement of actin cytoskeleton. Blocking antibody against CD28 was able to reduce T/B conjugate formation (467). Moreover, CD28 engagement has been shown to induce cytoskeletal rearrangement that is essential for T/APC interaction (403). Furthermore, CD28 crosslinking induces F-actin polymerization (199), which is mediated through activation of Vav and small RhoGTPase Cdc42 (468). Do et al. has recently reported that CD44 on T cells could mediate T/DC interaction, leading to IL-2 production and cell proliferation (208). Being an adhesion molecule and an inducer of F-actin rearrangement CD44 may contribute in the same manner as CD28 during T/APC interaction. Currently, the location of CD44 with respect to the S M A C is unknown. It is of particular interest to determine i f CD44 is present at the p-SMAC, where LFA-1 is located. Since CD45 is excluded from the p-SMAC, CD44 may signal to regulate actin rearrangement, enhancing the T/APC interaction. Although ample of evidence suggests a role for CD44 as a co-stimulatory molecule, conflicting results have also been reported. Stoop et al. demonstrated that IL-2 production and T cell proliferation in response to collagen was not affected in CD44" T /_  cells, when compared with wild-type T cells (469). Moreover, it was observed that CD44 blocking antibody only affected T cell responsiveness when the antigen-presenting dendritic cells were pre-treated, while this effect was not observed with T cells pre-  130  treated with the blocking antibody (470). Furthermore, Do et al. also reported that T cell activation in response to antigen presentation by splenic cells is CD44-independent (208). The discrepancies between these observations may be due to various factors. First, the combination of APC and T cell may determine the dependence on CD44 as a costimulatory molecule. It has been shown that different APC/T combination may determine the type of interaction in terms of duration and adhesion molecules involved (471). Second, Mempel et al. observed that naive T cells go through a three-phase program as they enter and survey a lymph node (47). The first and third phase of the program are characterized by highly mobile T cells and transient T/DC interaction. The second phase of the program, which is about 8 to 20 hours after T cell entry, is marked by immobile T cells and prolonged T/DC interaction. Concomitantly, CD44 expression increased dramatically. One possibility is that CD44 may only play a role in T/APC interaction in specific time window in vivo. It will be of interest, thus, to determine i f this interaction is dependent on CD44 functioning as both adhesion and co-stimulatory molecule. Second, the affinity or abundance of pMHC to the TCR may also play a role since this determines the dependence of co-stimulatory molecule for interaction (472, 473). Third, the presence of other co-stimulatory molecules, such as CD28, may compensate for the lack of CD44. In conclusion, CD44 does posses a few qualities of costimulatory molecules; however, under what physiological situations will CD44 act as one are yet to be determined.  5.3  CD44 and lymphocyte migration There is a large body of evidence that CD44 is involved in lymphocyte migration  in various inflammation models. These include acute peritonitis induced by superantigen (194), collagen-induced arthritis (196, 197, 469, 474), delayed-type hypersensitivity (198, 465), and experimental autoimmune encephalomyelitis (195, 475). In these models, the role of CD44 in lymphocyte recruitment to the site of inflammation may be attributed to the ability of this adhesion molecule to mediate rolling during lymphocyte extravasation (187, 188, 476). However, little is known about how recruited lymphocytes migrate within the extravascular tissue and if CD44 plays a role in this process.  131  Several lines of evidence suggest that CD44 may regulate cell motility in inflamed tissue. First, CD44 expression is up-regulated in activated T lymphocytes (47, 477). Second, the principal ligand of CD44, hyaluronan (HA), is also up-regulated at inflammatory sites (478). Third, three-dimensional matrix incorporated with H A stimulated motility of activated human peripheral T lymphoblasts (193). Furthermore, most of CD44 ligands identified are components of the extracellular matrix, such as fibronectin (168) and collagen (479). Therefore, CD44 may support lymphocyte migration through interaction with other E C M components. Other than a role in lymphocyte migration during inflammation, CD44 may also play a role in memory T cell migration to and in bone marrow. This is suggested by the observation that CD44 is also a murine memory T cell marker (480) and bone marrow is an HA-rich environment. One peculiar feature of migrating lymphocyte is the formation of uropod and the concentration of certain surface molecules to this specialized cellular compartment. CD44 is indeed one of such molecules and is referred as to a uropod marker. At present, little is known about the physiological significance of the recruitment of CD44 to uropod. However, according to the observation of this study, during lymphocyte migration, CD45 and CD44 may segregate to different ends of the cell. In the local absence of CD45, CD44 may have enhanced signaling ability that is associated with cytoskeletal rearrangement. This may act in a cooperative manner with other signals transmitted to the cells through chemokine receptors and adhesion molecules. Since CD44 is located in such strategic location of the cell, i.e. at the back of the cell, it raises the question if the signals arise from CD44 engagement indicate the "backness" of the cell. In a neutrophil migration model, the "frontness" and "backness" of the migrating cell is preserved. This is shown by the observation that an abrupt 180° change of the source of chemotactic cue led to a "U-turn" of the migrating cell (481). The authors showed that there are antagonistic signals that help develop the "frontness" and "backness" of the cell. In particular, the "backness" of the cell is dependent on activation of Rho, a Rho-dependent kinase, and myosin II. The dependence of Rho in uropod formation and CD44 localization has also been shown to occur in T cells (482, 483). However, whether CD44 may signal at the uropod and if this signal contributes to  132  maintain the uropod is yet to be determined, even though CD44/HA interaction was shown to activate Rho in a tumor cell line (244).  5.4  CD45 as regulator of signal strength and duration The F-actin ring polymerization in CD45 T cells upon CD44 engagement and the +  requirement of SFK activity for such actin rearrangement showed that CD44 signaling does exist in CD45 T cells. One possibility is that the strength and the duration of such +  signaling are too weak and short that they are beyond the sensitivity of the detection methods utilized in this study. Indeed, Descamps et al. have observed that CD45 and +  CD45" myeloma cells responded differently to insulin-like growth factor (IGF)-l (484). The CD45" myeloma cells exhibited Akt phosphorylation of greater magnitude and duration in response to IGF-1, when compared to CD45 myeloma cells. Moreover, +  CD45 was co-immunoprecipitated with IGF-1 receptor. The authors suggest that CD45 may directly dephosphorylate IGF-1 receptor or associated proteins required for activation of Akt. These observations are consistent with the ones made in this study, further strengthening the hypothesis that CD45 is a regulator of signaling strength and duration. Together with the observations made by others (293, 484) and the ones in the current study, CD45 may regulate signal strength and duration by its physical presence at the signaling complexes. As discussed in the previous chapter, this translocation may be achieved by a cytoskeletal dependent mechanism. It will be of interest then to determine how the temporal and spatial elements of this translocation are regulated. Another possibility may be related to where CD45 is compartmentalized in terms of lipid rafts since mobilization of CD45 out of the lipid rafts was observed in response to CD3 stimulation in T cells. Moreover, signal strength and duration may be affected by the overall expression level of CD45 and/or the isoforms of CD45 expressed. The observation in this study and by others suggest that CD45 may play a negative regulatory role in adhesion-related events and signaling. Its negative regulation is likely attributed to its phosphatase activity on SFK. On the other hand, CD45 has long been thought to positively regulate TCR/CD3 signaling. However, a recent observation suggests that the involvement of CD45 in T cell activation may not be as simple. First,  133  CD45 was shown to be able to dephosphorylate tyrosine 394 (Y394) of Lck, which is important for the full kinase activity. Second, artificial coupling of CD45 and CD3 inhibited T cell activation (485). This is, indeed, similar to the observations made in this study, in which case the presence of CD45 inhibited SFK activity and accumulation of phosphotyrosine signal. Therefore, the similarity between TCR/CD3 signaling is that the presence of CD45 inhibits further signaling. It has been observed that during T/APC interaction, CD45 briefly associated with the TCR cluster, yet it was later excluded from the c-SMAC (60). In other words, the brief presence of CD45 at the TCR cluster is advantageous to initiation of signals. The authors suggest that the brief presence of CD45 may be responsible for resetting the SFK prior to activation. However, this does not seem to be the case at the CD44 clusters. The co-localization of CD45 was sufficient to inhibit further propagation of CD44 signals. Therefore, the molecular composition of these signaling complexes may also determine the effect of CD45.  5.5  Different pools of SFK In the present study, CD45" T cells showed defective CD3 signaling yet enhanced  CD44 signaling. In both cases, SFK activity is required proximal to receptor engagement. In these cells, Lck and Fyn are hyperphosphorylated. Moreover, CD45initiated activation is not required for this population of SFK. In CD45 cells, the overall +  Lck and Fyn phosphorylation level is kept low. In normal T cell, it is possible that a dynamic equilibrium exists between the various phosphorylated states of Lck and Fyn. The hyperphosphorylated Lck may be preferentially associate with CD44, thus leading to enhanced CD44 signaling in CD45" T cells. On the other hand, Lck and Fyn of low phosphorylation level are reserved for CD3/TCR signaling events. It will be then of interest to determine how the equilibrium is shifted. Moreover, this shift of equilibrium may well be associated with the regulation of CD45.  5.6  Future experiments and conclusion The tyrosine phosphorylation of Pyk2 is correlated with elongated cell spreading  of CD45" BW5147 T cells on immobilized CD44 antibody. In particular, the PTPa  134  transfectant studies showed that the cell spreading length was correlated with the extent of Pyk2 phosphorylation (section 4.2.2). In order to determine if Pyk2 activity is required for elongated cell spreading, dominant-negative Pyk2, such as the kinase-dead mutant, may be transfected into CD45" BW5147 T cells and determine i f this will abolish the cell spreading morphology upon incubation on immobilized anti-CD44 antibody. Moreover, the kinase-dead Pyk2 transfectants may also used to assess i f HEF1 phosphorylation was a result of Pyk2 activation in T cells. At present, how HEF1 may regulate actin reorganization in CD44 signaling is uncertain. Therefore, pull-down assays with radioactively labeled cell lyaste and HEF1 may identify potential downstream effectors. The elongated cell spreading morphology of CD45" BW5147 T cells resemble that of migrating cells. It will be of interest to determine if the enhanced CD44 signaling in CD45" BW5147 T cells, showing this elongated cell spreading morphology on antiCD44 antibody, translates into better migrating ability, when compared to CD45  +  BW5147 T cells. The CD44-dependent migration may be assessed by performing transwell migration assay with HA-coated surface or with three-dimensional matrix incorporated with H A . As mentioned before, high affinity binding partners of SFK may disrupt the intramolecular interaction of SFK and prime the kinases for activation. Since the results form this work suggest that SFK activation in CD44 is independent of CD45, such high affinity ligand may be involved in CD44 signaling. In CD45" BW5147 T cells, Lck and Fyn are hyperphosphorylated, particularly at the C-terminal tyrosine Y505. The Cterminal domain of Lck containing phosphorylated Y505 can be utilized in pull-down assays to identify such potential interacting ligands. The activity of PI 3 K was required for the formation of longitudinal F-actin and elongated cell spreading of CD45" BW5147 T cells. However, it is unclear which PI3K is involved in CD44 signaling, as there are many classes of this kinase. One method to identify the PI3K is to utilize specific siRNA to knock down the expression level of each class of the kinase. Such systematic study should identify the specific PI3K involved in CD44 signaling.  135  In this work, CD44 has been demonstrated to possess signaling ability to mediate actin reorganization. As discussed above, CD44 is capable to act as a co-stimulatory molecule during T/APC interaction. Therefore, it will be of interest to determine the location of CD44 during such interaction and if the signaling ability of CD44 affects such interaction. This can be done by labeling CD44 for confocal microscopy studies after T/APC conjugate is formed. Moreover, whether the signaling ability of CD44 may affect such interaction can be assessed by transfectants of CD44" cells with a CD44 mutant that does not interact with Lck. The results from this work imply that when the inhibitory effect of CD45 is relieved, a sustained or strong signal can be delivered in CD44 signaling. One such possibility is the recruitment of CD45 away from CD44 upon stimulation of chemokines. To assess i f CD44 possess better signaling capability, CD45 T cells can be stimulated +  with chemokines and be allowed to interact with anti-CD44 antibody- or HA-coated beads. The cells can then be labeled for pY394-Lck or phosphotyrosine signals for confocal microscopy analysis. Comparing the stimulated versus the unstimulated cells, whether CD44 may deliver a stronger signal with chemokine stimulation can be assessed. Results from confocal microscopy suggest that CD45 is recruited to CD44 microclusters upon CD44 engagement. Moreover, recruitment of CD45 to TCR complexes and exclusion from c-SMAC at later time points were observed. However, it is unclear how CD45 localization is controled. As mentioned before, one such possibility is the interaction of CD45 with the cytoskeleton through fodrin or ankyrin. Using transfected CD45" T cells with deletion mutants of CD45 that cannot interact with fodrin and ankyrin will be able to assess such possibility. Moreover, it will be of interest to determine i f such mutants fail to initiate CD3/TCR signals due to the lack of such interaction between CD45 and the cytoskeleton. Overall, the work in this thesis has shown that CD44 engagement in T cells generates cellular signals that regulate actin rearrangement and cell spreading. The initiation of CD44 signals begins with an actin-dependent recruitment of CD44 into microclusters. In the presence of CD45, the CD44 signal is brief and weak, as indicated by the minimal phosphotyrosine signals at the CD44 microclusters. This signal is SFKdependent and results in F-actin ring formation and round cell spreading. Results from  136  confocal microscopy studies suggest that the presence of CD45 at the CD44 microclusters prevents sustained activation of Lck, providing an explanation of the brief signal. In the absence of CD45, the CD44 signal is strong and sustained, in which case accumulation of phosphotyrosine at the CD44 microclusters was observed. This strong and sustained signal is associated with the tyrosine phosphorylation of Pyk2 and the activities of PLC and PI3K, actin polymerization, and calcium mobilization are required for the process. The result of this strong and sustained CD44 signal in T cells is longitudinal F-actin formation and elongated cell spreading. The CD44 signaling pathway identified in this study is distinct from CD3 signaling since it does not involve L A T phosphorylation or E R K activation. Moreover, CD45 plays different roles in these signaling pathways: it negatively regulates CD44 signaling while it positively regulates CD3 signaling. The results from this thesis should provide a working model of CD44 signaling for further studies.  137  Chapter 6 References 1.  Imhof B A , Aurrand-Lions M . 2004. 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