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Investigating the function and stability of CD45-associated protein, a protein implicated in T cell activation Wong, David W. 2002

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INVESTIGATING T H E FUNCTION AND STABILITY OF CD45-ASSOCIATED PROTEIN, A PROTEIN IMPLICATED IN T C E L L ACTIVATION  by D A V I D W.  WONG  B. Sc., The University of British Columbia, 1999 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N TO FT H E REQUIREMENTS FORTHE DEGREE OF  MASTER OFSCIENCE  In T H E F A C U L T Y O FG R A D U A T E STUDIES  Department of Microbiology and Immunology We accept this thesis as conforming to the required standara  T H E UNIVERSITY O FBRITISH C O L U M B I A  October 2002 © David Wong, 2002  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  ABSTRACT  Recently, a novel lymphocyte-specific transmembrane protein, CD45 associated protein or CD45ap, was identified due to its constitutive association with CD45, a transmembrane protein tyrosine phosphatase essential to the activation of T cells. Although little is known about the role of CD45ap in T cell activation, its potential importance in the T cell response has been demonstrated by the fact that the expression of CD45ap is lymphocyte specific rather than being present in all cells that express CD45 and that CD45ap-null mice have been shown to have reduced T cell proliferation upon stimulation. It was also shown that CD45ap could associate with kinases essential to the T cell response such as Lck and ZAP-70, stimulating suggestions that CD45ap may function as an adaptor molecule. Expression of CD45ap as a bacterial fusion protein yielded a highly degraded and unstable protein suggesting that the mechanism responsible for CD45ap degradation in eukaryotic cells may be conserved in E. coli. Subsequent deletion analysis demonstrated that a 40 amino acid residue deletion from the C-terminus of CD45ap could stabilize its expression. However, the same 40 amino acid residue deletion from CD45ap did not alter the half life of the molecule in eukaryotic L fibroblast cells. The stable recombinant form of CD45ap was shown in vitro to outcompete CD45 for binding to Lck. This correlates with other in vitro findings that demonstrated CD45ap significantly decreased the rate at which CD45 could dephosphorylate the F505 Lck mutant (believed to be constitutively active) but only slightly decreased the rate at which CD45 could dephosphorylate the F394 Lck mutant (believed to be constitutively inactive). This suggests that CD45ap can serve as an effective modulator of the CD45-  ii  Lck interaction as well as a promoter of T cell activation by its ability to favour the CD45-mediated activation rather than deactivation of Lck. In further support of CD45ap serving as a promoter of T cell activation, B W 5147 T cells overexpressing CD45ap demonstrated prolonged and increased phosphorylation of various T cell proteins upon CD3 stimulation.  In addition, the W W domain of  CD45ap was also shown to interact with a 50 kDa tyrosine phosphorylated protein providing another possible route through which CD45ap may promote T cell activation. Narrowing the potential roles that CD45ap may play in T cell signaling, CD45ap was found not to affect that rate of CD45 transport to the cell surface or the turnover rate of CD45 in L cells. However, CD45ap expression in L cells resulted in increased total but not cell surface levels of CD45, implicating CD45ap in the maintenance subcellular pools of CD45.  in  of  T A B L E OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  ix  List of Figures  x  List of Abbreviations  xii  Acknowledgments  xv  1. Introduction  1  1.1 A n overview of T cell activation  3  1.2 The immunological synapse  5  1.3 CD45 and lipid rafts  8  1.4 Initial identification of CD45ap  9  1.5 CD45ap knock out mice  10  1.6 Interactions between CD45ap and other T cell proteins  13  1.7 CD45ap structure  15  1.8 CD45ap and W W domains  17  1.9 CD45ap expression in CD45 deficient cells and PEST sequences  20  1.10 CD45ap phosphorylation sites  22  1.11 Intracellular proteolysis  23  1.12 Thesis objectives  27  2. Materials and Methods  29  iv  2.1  2.2  Materials  30  2.1.1 Antibodies  30  2.1.2 Cell culture  31  2.1.3 Bacterial strains and vectors  31  Methods  32  2.2.1 D N A constructs  32  2.2.1.1 P C R generation of the cytoplasmic and W W domain of CD45ap  32  2.2.1.2 Generation of the fusion protein expression constructs  33  2.2.1.3 Generation of the C-terminal deletions of GST-CD45ap..34 2.2.1.4 Restriction digests  36  2.2.1.5 P C R reactions  36  2.2.1.6 Transformation of bacterial cells  36  2.2.2 Expression of recombinant proteins  37  2.2.2.1 Preparation of whole cell lysates  37  2.2.2.2 Purification of M B P fusion proteins  37  2.2.2.3 Purification of GST fusion proteins  38  2.2.2.4 Purification of 6-His fusion proteins  39  2.2.3 Immunization and preparation of serum from rabbits WW1 and WW2  40  2.2.4 GenePorter2 transfection  41  2.2.5 Electroporation  42  2.2.6 S pulse chase labeling  42  35  v  2.2.7  14  C pulse chase labeling  2.2.8 CD3 stimulation of B W 5147 cells  44 46  2.2.9 GST fusion protein pull down assay from pervanadate treated T cell lysates  46  2.2.10 Flow cytometry  47  2.2.11 CD45 immunoprecipitations  48  2.2.12 Binding assay of recombinant CD45 to Lck in the presence or absence of CD45ap  48  2.2.13 SDS-PAGE  49  2.2.14 Immunoblotting  49  2.2.15 Densitometric analysis  50  3. Results  51  3.1 Investigation of CD45ap degradation  52  3.1.1 Generation and purification of degraded recombinant M B P - , GST-, and cmyc-CD45ap  52  3.1.2 Identification of a degradation signal in the cytoplasmic domain of CD45ap by mutational analysis in E. coli  ...54  3.1.3 No significant difference between the half life of CD45ap and CD45ap (del 157-197) in L cells  58  3.2 Generation of antisera against CD45ap  63  3.3 CD45ap and its effects on CD45 expression, half life, and transport  65  vi  3.3.1 Expression of CD45ap results in increased total but not cell surface levels of CD45 in L cells  65  3.3.2 No significant difference in the half life of CD45 in the presence or absence of CD45ap in L cells  71  3.3.3 No significant difference in the transport time (T1/2 to Endoglycosidase H resistance) of CD45 in the presence or absence ofCD45ap in L cells  71  3.4 CD45ap affects T cell signaling  73  3.5 The W W domain of CD45ap interacts with a 50 kDa tyrosine phosphorylated protein  77  3.6 CD45ap modulates the CD45-Lck interaction  79  4. Discussion  82  4.1 CD45ap degradation  83  4.1.1 Achieving stable expression of CD45ap as a recombinant fusion protein in bacteria  83  4.1.2 Assessment of the stability of CD45ap with a C-terminal 40 amino acid deletion in eukaryotic cells 4.2 Potential roles for CD45ap in T cell signaling  85 91  4.2.1 CD45ap overexpression studies  91  4.2.2 CD45ap and its effects on the CD45-Lck interaction  93  4.2.3 Novel interactions of CD45ap with other T cell proteins  95  4.3 CD45ap and its effects on CD45 expression, transport, and turnover  vn  96  4.3.1 CD45ap and its effect on cell surface and total CD45 levels  96  4.3.2 CD45ap and its effect on CD45 transport  97  4.3.3 CD45ap and its effect on CD45 half life  99  5. Conclusion  102  6. References  106  viii  LIST OF T A B L E S  Table 1.1:  Summary of CD45 ap mouse knock out models  ix  LIST OF FIGURES  Figure 1.1:  T cell receptor signal transduction  Figure 1.2:  Sequence alignment of mouse CD45ap and human L P A P  11  Figure 1.3:  CD45ap structure  16  Figure 1.4:  Yap65 W W domain and W W domain sequence alignment  18  Figure 3.1:  CD45ap recombinant proteins and mutants generated  53  Figure 3.2:  Recombinant CD45ap fusion proteins  55  Figure 3.3:  Mutational analysis of CD45ap in E. coli  57  Figure 3.4:  Sequence alignment of the C-terminus of CD45ap and L P A P  59  Figure 3.5:  Further mutational analysis of CD45ap in E. coli  60  Figure 3.6:  Half lives of CD45ap and CD45ap (del 157-197) in L cells  62  Figure 3.7:  Testing of antisera generated against CD45ap  64  Figure 3.8:  Transfection of L cells expressing CD45 with CD45ap and CD45ap (del 157-197)  6  67  Figure 3.9:  Demonstration of an association between CD45 and CD45ap in L cells..68  Figure 3.10:  Cell surface CD45 levels in L cells transfected with CD45, CD45 and CD45ap, and CD45 and CD45ap (del 157-197)  Figure 3.11:  69  Total CD45 levels in L cells transfected with CD45, CD45 and CD45ap, and CD45 and CD45ap (del 157-197)  70  Figure 3.12:  Half life of CD45 in the presence or absence of CD45ap in L cells  72  Figure 3.13:  CD45 transport time in the presence or absence of CD45ap in L cells  74  Figure 3.14:  Effects of CD45ap overexpression in B W 5147 murine T cells  76  Figure 3.15:  GST-WW pull down assay from T cell lysates  78  x  Figure 3.16:  Effect of CD45ap on the CD45-Lck interaction  xi  LIST OF ABBREVIATIONS  APC  antigen presenting cell  ATP  adenosine triphosphate  ATTC  American Type Culture Collection  bp  base pair  (3-Me  beta-mercaptoethanol  CD45ap  CD45 associated protein  CRAC  calcium release activated calcium  DIG  detergent insoluble glycolipid-enriched membranes  DMEM  Dulbecco's Modified Eagle Medium  DNA  deoxyribonucleic acid  DRM  detergent-resistant membranes  DTT  dithiothreitol  EDTA  ethylenediamine tetra-acetic acid  Endo H  Endoglycosidase H  ER  endoplasmic reticulum  FACS  fluorescence activated cell sorter  FCS  fetal calf serum  FITC  fluorescein isothiocyanate  Fyn  p59  GEM  glycolipid enriched microdomains .  GSH  glutathione  GST  glutathione-S-transferase  fyn  xii  His  histidine  HRP  horse-radish peroxidase  IL-2  interleukin 2  IP  inositol- 1,4,5-triphosphate  3  IPTG  isopropylthio-P-D-galactoside  IS  immune synapse  IT A M  immunoreceptor tyrosine-based activation motif  kb  kilobase  kDa  kiloDalton  LAT  p40  Lck  p56  LPAP  lymphocyte phosphatase-associated phosphoprotein  Lys  lysine  MBP  maltose binding protein  MHC  major histocompatibility complex  OD  optical density  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PE  phycoerythrin  PIP2  phosphatidylinositol 4,5-bisphosphate  PKC  protein kinase C  PLC-y  phospholipase C-y  LAT  lck  xiii  PMSF  phenylmethylsulfonyl fluoride  PTK  protein tyrosine kinase  PVDF  polyvinylidene difluoride  RIPA  radioimmunoprecipitation assay  RPM  revolutions per minute  SDS  sodium dodecyl sulphate  Ser  serine  SH2  Src homology 2  SH3  Src homology 3  SLP-76  SH2 domain containing leukocyte phosphoprotein of 76 kDa  SMAC  supramolecular activation cluster  Src  p60  TTBS  Tris buffered saline with Tween-20  TCR  T cell receptor  Thr  threonine  Tris  Tris (hydroxymethyl) amino methane  TX-100  Triton X-100  Tyr  tyrosine  ZAP-70  p70  src  ZAP  xiv  ACKNOWLEDGEMENTS  I would like to thank my family and friends for their continued support throughout my studies.  Much gratitude is also extended to the members of the Johnson  lab, both past and present, for their support, stimulating discussions, and friendship. In addition, I would like to thank my committee members, Dr. Gerry Weeks and Dr. Francois Jean, for their useful suggestions and guidance as well as the Natural Sciences and Engineering Research Council of Canada for their support. Finally, I am grateful to my supervisor, Dr. Pauline Johnson, for giving me the opportunity to do my degree in her lab and for her perpetual support and mentorship.  xv  1. Introduction  1  Prologue Immunology's constantly evolving nature has both intrigued and perplexed me in my past two years of study. One of the aspects of immunology that attracted me to its study was the beauty in which cell signaling events in immune cells are so masterfully orchestrated. As a classical music enthusiast, I find T cell activation much akin to a well executed and interpreted score. A well executed piece of work entails the woodwinds and the brass entering at their specific cues with the correct tempo and dynamics. Instruments are held at ready, participate when appropriate, and cease at the appropriate time. In T cell signaling, specific molecules respond to outside cues in the proper fashion and terminate at the proper moment. The fact that the function of CD45ap remains unknown to date has been both a blessing and curse. I have enjoyed being involved in exploring its potential functions, but at the same time, having less support and input from the research community has been trying at times. There remains much to be learned about this molecule. The beginning of this thesis summarizes the current thinking in T cell signaling with some highlights of the major players involved, followed by an overview of CD45ap, its structure, and its potential roles in T cell signaling. The results and discussion sections follow detailing the work done in this thesis which includes the deletion analysis of CD45ap, examination of the effect of CD45ap on CD45 expression, and the use of CD45ap overexpression studies and CD45ap recombinant proteins to examine the potential roles of CD45ap in T cell signaling. The initial introduction to T cell signaling does not mention the involvement of CD45ap as the exact role of CD45ap in T cell  2  signaling has yet to be elucidated. However, proposals of how CD45ap may fit into the signaling cascade, along with supporting evidence, will be provided in various instances.  1.1 An overview of T cell activation T cells play an invaluable role in the adaptive immune response.  T cells  constantly sample their environment through their receptors (TCR) and upon recognition of foreign peptides displayed by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs), T cells can initiate the lysis of infected cells, the production of antibodies by B cells, and activate various mechanisms to kill intravesicular bacteria and parasites.  When the T C R encounters foreign antigen and a  decision by the T cell is made to respond, a cascade of events known as T cell signaling is triggered. Ultimately, the triggering of this cascade, which involves a rapid protein tyrosine phosphorylation signal, leads to the activation and proliferation of the T cell response. The ligand recognition components of the T C R which engage the MHC-peptide complex, the a and p chains, interact with other (CD3-y, -5, -s, and - Q integral T C R components  (1).  Each of these interacting molecules  contains  a cytoplasmic  immunoreceptor-based tyrosine activation motif (ITAM), consisting of a Yxxl/Lx( . 6  8)YxxL/I motif (2, 3) which is both necessary and sufficient for T cell activation (4, 5). Each CD3-y, -8, and -s molecule bears one I T A M and each CD3-<^ molecule contains three. Protein tyrosine phosphorylation events mediated by protein tyrosine kinases (PTKs) are critical to the initiation of the signaling cascade (6).  3  The T C R and its  associated CD3-<^ subunits have been implicated in the recruitment of two classes of cytoplasmic PTKs to regulate the T cell response, the Src and the Syk/ZAP-70 family of PTKs.  Upon engagement of the TCR, the first biochemical events known to occur  involve the activation of the Src family kinases Lck and Fyn (7-9). It has been proposed that CD45, a leukocyte-specific transmembrane protein whose intracellular domain exhibits protein tyrosine phosphatase activity shown to be necessary for efficient T cell signaling (10-12), mediates the T cell response upon the binding of foreign antigen by dephosphorylating the inhibitory tyrosine residue on Lck and Fyn which leads to their activation (13-16).  Activated Lck and Fyn (although to a lesser degree) are then  responsible for the phosphorylation of tyrosine residues within the ITAMs (17-19). Subsequently, the PTKs ZAP-70 and Syk are recruited through their binding to doubly phosphorylated ITAMs through their tandem Src-homology 2 (SH2) domains (17, 18, 2022). Once bound to the T C R complex, ZAP-70 and Syk are activated by phosphorylation of a tyrosine residue in their kinase domain activation loops (23, 24).  Although the  activation of Syk is primarily effected through autophosphorylation, ZAP-70 activation is carried out by the Src-family kinases.  Syk/ZAP-70-family kinases continue to  phosphorylate several other downstream signaling molecules such as the adaptors L A T (25) and SLP-76 (26-28). SLP-76 binds phospholipase C-y (PLC-y) which leads to its phosphorylation, recruitment to the membrane, and activation (29). then cleaves  phosphatidylinositol 4,5-bisphosphate  Activated PLC-y  (PIP2) generating the  second  messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). D A G generation subsequently leads to the activation of the Ras pathway via RasGRP, a guanine nucleotide exchange factor.  Upon the binding of IP3 to receptors in the endoplasmic  4  reticulum (ER), intracellular calcium (Ca ) z+  stores are released, which together with  D A G is responsible for the activation of protein kinase C (PKC) (30).  Activated P K C  ultimately leads to the phosphorylation of IKB and its release from the transcription factor NFKB  which is then able to enter the nucleus and effect gene transcription (31).  In  coordination with IP3 receptor binding and release of intracellular C a , calcium release 2+  activated calcium (CRAC) channels are opened to allow the influx of extracellular C a into the cell (32). while C a  2+  Intracellular C a  2+  release is critical to the early rise in C a  2+  2+  levels,  influx through the C R A C channels is responsible for the sustained rise in  intracellular free C a . 2+  Increased free C a  2+  levels leads to the dephosphorylation and  activation of the transcription factor N F - A T and its subsequent translocation to the nucleus (33) where the AP-1 transcription factor also accumulates as a result of the Ras pathway activation (34, 35). Ultimately, the convergence of all of these events results in IL-2 gene transcription (34) and the proliferation of the T cell response.  A diagram  summarizing T cell signal transduction is provided in figure 1.1.  1.2 The immunological synapse The biochemical pathways activated during T cell signaling have been fairly well characterized, but much remains to be known about how the physical interaction between T cells and APCs initiate T C R signaling. It has been known for some time that the plasma membrane is not homogenous (36, 37) and over the last decade, there have been modifications made to the classic lipid bilayer description of the organization of molecules within the membrane (38-40). The heterogeneous distribution of glycolipids and cholesterol has been proposed to form microdomains distinct from the more abundant  5  T cell signal transduction  i  PLC^  PIP  2  - » IP  NF-AT, NFKB,  I  3  + DAG  AP-1  IL-2 transcription & T cell activation Figure 1.1. T cell receptor signal transduction. Upon binding of foreign antigen displayed by M H C molecules on APCs by the TCR, CD45 dephosphoryles the inhibitory 6  tyrosine residue on Lck and Fyn leading to their activation (13-16). Activated Lck and Fyn (although to a lesser degree) are then responsible for the phosphorylation of tyrosine residues (i) within the ITAMs of the CD3 subunits (CD3-5, -s, -y, and - Q (17-19). The phosphorylated CD3-<^ homodimer subsequently recruits the P T K ZAP-70 through the binding of ZAP-70 (via tandem SH2 domains) to doubly phosphorylated ITAMs (17, 18, 20-22). Once bound, ZAP-70 is activated by Lck-mediated phosphorylation of a tyrosine residue in its kinase domain activation loop (23, 24). ZAP-70 continues to phosphorylate several other downstream signaling molecules such as the adaptors L A T (25) and Slp-76 (26-28). SLP-76 binds phospholipase C-y (PLC-y) which leads to its phosphorylation, recruitment to the membrane, and activation (29). Costimulatory signals from CD28 are also important for the induction of specific pathways and second messengers. Activated PLC-y then cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating the second messengers inositol-l,4,5-triphosphate (IP3) and diacylglycerol (DAG). D A G generation subsequently leads to the activation of the Ras pathway via RasGRP, a guanine nucleotide exchange factor. Upon the binding of IP3 to receptors in the endoplasmic reticulum (ER), intracellular calcium (Ca ) stores are released, which together with D A G is responsible for the activation of protein kinase C (PKC) (30). Activated P K C ultimately leads to the phosphorylation of I K B and its release from the transcription factor N F K B which is then able to enter the nucleus and effect gene transcription (31). In coordination with IP3 receptor binding and release of intracellular C a , C R A C channels are opened to allow the influx of extracellular C a into the cell (32). Increased free C a levels leads to the dephosphorylation and activation of the transcription factor N F - A T and its subsequent translocation to the nucleus (33) where the AP-1 transcription factor also accumulates as a result of the Ras pathway activation (34, 35). Ultimately, the convergence of all of these events results in IL-2 gene transcription (34) and the proliferation of the T cell response. 2+  2+  2+  2+  7  glycerolipids. Such domains have been termed DRMs (detergent-resistant membranes), GEMs (glycolipid enriched microdomains), DIGs (detergent insoluble glycolipidenriched membranes), or rafts.  Recently, there has been evidence that there is an  enrichment in the number of certain signaling proteins such as the Src-family kinases, L A T , CD4, and CD8 (39, 41-45) in lipid enriched rafts and that these microdomains may serve to effect efficient T cell signaling. It has been proposed that an immunological synapse (IS) forms during T cell-APC contact and that this IS serves to cluster TCRs and MHC-peptide complexes, as well as lipid rafts and specific signaling and adhesion proteins in order to facilitate T cell signaling (46-48).  The IS is proposed to be  comprised of two distinct zones, the c-SMAC (supramolecular activation cluster) containing the T C R and CD28, and a second zone surrounding the c - S M A C known as the p - S M A C which is enriched in the integrin LFA-1 (46, 49-51). Up until very recently, the current dogma was that T cell signaling and activation was a result of IS formation, but data has now been presented that challenges this model by suggesting that T cell signaling occurs before mature ISs form (52).  Hypotheses have thus been put forward  that IS formation may be to allow polarized secretion from T cells (53) or to attenuate or sustain the T cell response (54).  1.3 CD45 and lipid rafts To date, conflicting evidence exists as to whether CD45 resides in or out of the rafts. Although the function of CD45 is mainly stimulatory due to its ability to activate Src family kinases, CD45 has also been shown to have an inhibitory role on T cell signal transduction with some CD45 deficient T cells lines displaying elevated basal levels of  8  hyperactive  Src-family  kinases  probably  due to  a  lack  of  CD45-mediated  dephosphorylation of the activating tyrosine on Src-family kinases (55, 56). Due to the ability of CD45 to dephosphorylate both the inhibitory and activating tyrosines of Srcfamily kinases, there must be a method by which the two functions of CD45 are differentially regulated. One possibility is that CD45 may dephosphorylate and activate inactive Src-family kinases but dephosphorylate and inactivate active Src-family kinases and that temporal sequestering of dephosphorylated active Src kinases away from CD45 is necessary to maintain a strong activating signal in the cell.  In support of this  hypothesis, CD45 has been found to be excluded from the IS (57). However, other groups have demonstrated that CD45 is found in the T cell-APC contact zone (58) while another group has demonstrated the movement of a discreet pool of CD45 that is initially excluded from the area of T C R - A P C contact back into the contact zone (59). Thus, whether CD45 plays an activating or inhibitory role in T cell signaling may depend on its temporal localization and proteins in close proximity.  1.4 Initial identification of CD45ap Although it has been demonstrated that CD45 is critical in lymphocyte development and response to foreign antigen (10, 12, 60, 61), the mechanism for regulating the protein tyrosine phosphatase activity of CD45 remains unclear.  In  addition, it is unclear how CD45 targets and dephosphorylates specific substrates rather than impartially dephosphorylating any tyrosine phosphorylated residue on any tyrosine phosphorylated protein in its vicinity. The cloning of a gene encoding a novel 30 kDa lymphocyte specific transmembrane protein due to its constitutive association with CD45  9  on the surface of T cells (62-64) led to the suggestion that other molecules may modulate the activity of CD45.  This molecule was aptly named CD45 associated protein or  CD45ap. Its predicted molecular weight based on its primary sequence is 19 kDa, but curiously, it migrates to an apparent molecular weight of 30 kDa when subjected to SDSP A G E probably due to post translational modifications. Studies have demonstrated that the majority of CD45 molecules (70%) in lymphocytes exist in a complex with CD45ap (65) with an estimated stoichiometry of 1:1 (64-66). The interaction between CD45ap and CD45 is known to occur through their respective transmembrane domains and this interaction does not require other T cell proteins such as Lck, ZAP-70, or Fyn (67-69). To date, the role of CD45ap in T cell activation remains unknown, however, its potential importance in the T cell response has been demonstrated by the fact that the expression of CD45ap is specific to lymphocytes rather than being present in all leukocyte subsets like CD45. Strong CD45ap expression can be detected in T, preB, and B cells whereas weak expression of CD45ap is seen in mast cells and not detected at all in plasma cells or cells from the monocyte/macrophage lineage (66, 70).  A human  homologue of CD45ap, lymphocyte phosphatase-associated phosphoprotein (LPAP), was cloned near the same time as the cloning of CD45ap and was shown to have similar expression patterns in leukocyte subsets (63, 66, 70, 71). A manual alignment between the primary sequences of CD45ap and L P A P is provided in figure 1.2.  1.5 CD45ap knock out mice The data obtained from three different CD45ap knock out mice is controversial. The discovery of CD45ap led to the proposal that it might play an adaptor-like role,  10  CD45ap and LPAP Manual Alignment leader  I  CD45ap  ,  EX  |  JM  ^  4  MALPGTLRFGVLMALPGALASGADPEDGVGSSWTIVLLLLLLLLLVTALALAW  LPAP  MALPCTLGLGMLLALPGALGSGGSAEDSVGSSSVTWLLLLLLLLLATGLALAW 1 55  CD45ap  108  RRLSHASGGYYHPARLGAALWGHTCRLLWASPAGRWLRARTELESPEESGP-PE  LPAP  RRLSRDSGGYYHPARLGAALWGRTRRLLWASPPGRWLQARAELGSTDNDLERQE 55  109  !09  CD45ap  CYT  DEEDAE-DFVIDGGPEEAAAKEEEQRCQAEQTRDP  LPAP  ^  5  3  RDTDSDGGLGLS  DEQDTDYDHVADGGLQADPGEGEQQCGEASSPEQVPVRAEEARDSDTEGDLVLG 110  164  154  197 1  CD45ap  SQGPVGS GS S A E A L L SDLHAF SGSAAWDD SAGGAGGQGLRVTAL  LPAP  S  PGPASAGGSAEALLSDLFLAFAGSAAWDDSARAAGGQGLHVTAL  165  206  Figure 1.2. Sequence alignment of mouse CD45ap and human L P A P . CD45ap consists of a 23 amino acid residue N terminal leader sequence (leader), a 9 amino acid extracellular domain (EX), a 22 amino acid transmembrane domain (TM), and a 143 amino acid cytoplasmic domain (CYT).  11  perhaps in coordinating and mediating the interactions of CD45 with other signal transduction molecules. In support of this hypothesis, the first CD45ap knock out model isolated by Matsuda et. al. (72) showed that although such mice exhibited normal T cell development, they showed a decrease in the proliferation of thymocytes and splenic Tcells in response to T C R stimulation as well as a decrease in T cell mediated cytolysis of infected cells.  In addition, it was demonstrated that T-cells from these mice showed  decreased CD45 protein expression and a reduction in the amount of Lck that could be coimmunoprecipitated with CD45, implicating CD45ap as a direct or indirect mediator of the CD45-Lck interaction. At this point, this was strong evidence that CD45ap was a critical player in T cell signaling. Nevertheless, studies on two subsequent CD45ap mice demonstrated that CD45ap deficient and normal mice performed similarly in response to various mitogens or antigen receptor crosslinking. One of the groups, Ding et. al. (73), also found that there were no significant difference in CD45-Lck complex assembly in the absence of CD45ap as proposed by the first model by Matsuda et. al, but found a decrease in CD45 expression in the absence of CD45ap in accordance with the first model. This group also observed increased cellularity of the lymph nodes suggesting a possible role of CD45ap in the down regulation rather than potentiation of the T cell response. The third CD45ap knock out mouse isolated by Kung et. al. (74) demonstrated even sharper contradictions with the knock out model by Matsuda et. al, showing that CD45 expression and the CD45-Lck interaction was unaffected by the ablation of CD45ap in their mouse model. In support of the latter two knock out models, human Jurkat T cells deficient in L P A P expression did not show any differences in the CD45Lck interaction compared to L P A P expressing Jurkats (75). Possible explanations of the  12  controversy between the CD45ap knock out models include the use of different methods to inactivate the CD45ap gene (the first model used a neomycin cassette inserted in an anti-sense direction while in the latter two models, it was inserted in the opposite direction) and genetic variability in the different mouse strains. Finally, in the latter two models, the possibility of the inserted targeting vector affecting the closely linked gene encoding for the actin binding Coronin-2 protein was ruled out in the latter two models but not in the first. A clear role for CD45ap defined by mouse knock out models has yet to be established. A summary of the CD45ap mouse knock out models is provided in table 1.1.  1.6 Interactions between CD45ap and other T cell proteins As CD45ap associates specifically with CD45, a known modulator of Src-family P T K activity, and that T cell responses to stimulation are potentially impaired in CD45ap knock out mice, it is foreseeable that CD45ap plays an adaptor like role in coordinating the interaction between CD45 and its substrates.  Recently, it was shown that CD45ap  could associate directly with Lck and to a lesser extent with ZAP-70, in the absence of all other lymphoid-specific components (76, 77). CD45ap was also shown to associate with the TCR, CD4, and CD8 coreceptors, but not with Csk and Fyn. It is also interesting to note that the ability of CD45ap to interact with and associate with ZAP-70 and Lck was increased upon T C R crosslinking and stimulation (76) and that Lck binding to CD45ap increased with the catalytic activation of Lck (77). Taken together, this provides a model for the functioning of CD45ap as an adaptor molecule that associates directly and selectively with Lck and ZAP-70 in response to T C R mediated signaling events in order  13  Murine CD45ap Knock-Out Models  Lymphocyte activity  Matsuda et. al.  Kung et. al.  Ding et. al.  Decreased T cell proliferation in response to stimuli  Normal T cell response and proliferation in response to stimuli  Normal T cell response and proliferation in response to stimuli  Impaired cytotoxic T cell function CD45 expression  Decreased  Normal  Decreased  CD45-Lck interaction  Decreased  Not examined  Normal  Notes  Possible Coronin-2 gene perturbation  Lck kinase activity normal  Increased cellularity of lymph nodes  Table 1.1. Summary of CD45ap mouse knock out models. Summary of lymphocyte activity, CD45 expression, CD45-Lck interaction, and other facts from CD45ap knock out models by Matsuda et. al. (72), Kung et. al. (74), and Ding et. al. (73).  14  to coordinate the interactions between various signaling components.  Veillette et. al.  propose that CD45ap may bring activated Lck in close proximity to the TCR, facilitating I T A M phosphorylation by Lck and subsequent T cell signal amplification (77). It is also possible that CD45ap may act as a negative regulator of T cell signaling, by bringing activated Lck closer to CD45 which, in addition to being responsible for the activation of Lck, is also responsible for its inactivation. Clearly, CD45ap shows promise in playing an adaptor molecule-like role in T cell signal transduction and further elucidation of its role is critical as one of the fundamental issues in signal transduction is how specificity is maintained in the various interactions between the various signaling molecules.  1.7 CD45ap structure Analysis of mouse CD45ap cDNA sequences predicts a protein of 197 amino acid residues. Utilizing molecular cloning techniques and protease susceptibility assays, the majority of the protein was shown to reside intracellularly (68).  CD45ap was shown to  contain a 23 amino acid ER-targeting leader sequence that is most likely cleaved in mature forms of CD45ap (66, 70) and a short extracellular domain of 9 amino acids. The extracellular domain is followed by a 22 amino acid transmembrane region which as mentioned above, mediates the CD45-CD45ap interaction, and a cytoplasmic domain of 143 residues (63, 64, 70). A schematic of the structure of CD45ap is provided in figure 1.3. The short extracellular domain of CD45ap coupled with a much larger cytoplasmic domain is reminiscent of other transmembrane proteins with short extracellular and large intracellular domains involved in T cell signaling, namely, the CD3-C, chains and the adaptor protein L A T . Both proteins are critical to effective T cell signaling, with the C,  15  Figure 1.3. CD45ap structure. CD45ap consists of a 23 amino acid residue N terminal leader sequence (leader), a 9 amino acid extracellular domain (EX), a 22 amino acid transmembrane domain (TM), a 44 amino acid W W domain (WW) whose first 7 residues overlap with the last 7 residues of the transmembrane domain, and potentially two PEST sequences (PEST 1 and PEST 2). The residue numbers at the beginning and end of the various regions are marked.  16  chains providing ITAMs that are phosphorylated to effect binding of other molecules and L A T , in conjunction with SLP-76, interacting with Vav, Nek, and PLC-y to ultimately activate Ras, cytoskeletal changes, and IL-2 transcriptional activity necessary for T cell activation (78-83).  1.8 CD45ap and W W domains The cytoplasmic tail of CD45ap can be divided into two domains: a W W domain spanning amino acid residues 48 through 92 (67, 84) (see figure 1.3) and an acidic region located downstream of the W W domain. It is interesting to note that the first seven residues of the predicted W W domain overlap the last seven residues of the predicted transmembrane domain. W W domains are small protein modules that consist of about 40 amino acids (85). Each W W domain folds as a stable antiparalell triple stranded P-sheet and derives its name from two highly conserved tryptophan residues spaced 20-22 residues apart (85). Such domains are found in proteins of diverse functions including: dystrophin, a part of the multimeric complex linking the actin cytoskeleton to the extracellular basal lamina; utrophin, a dystrophin like protein of unknown function; Yes kinase-associated protein (YAP65), which binds to the SH3 domain of the Yes protooncogene via a proline rich region; Nedd4, a ubiquitin ligase involved in embryonic development and differentiation of the central nervous system in mice; and E s s l , a protein implicated in yeast cell division (85, 86). For illustration, the crystal structure of the Yap65 W W domain is provided in figure 1.4 (87).  W W domains have been  implicated in binding proline rich sequences and have thus been compared to the prolinerich region binding Src homology domain 3 (SH3 domain) (reviewed in 84). One group  17  Yap65 WW domain  "CD45ap loop"  B  WW domain Alignment 01  YAP65 Dystrophin CD45ap  62  33  VPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRKAMLS TSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELY TALALAWCR  LSHASGGYYHPA*TRRLLWASPAGRWLR  / \  " (8  RLGAALWGH amino acid insertion)  Figure 1.4. Yap65 W W domain and W W domain sequence alignment. (A) Structure of the Yap65 (Yes kinase-associated protein) W W domain complexed with a proline-rich peptide reproduced from Macias et. al. (87). Here, the three antiparallel pstrands ( p i , P2, and P3) can be seen together with the two interconnecting loops. Due to the fact that the W W domain of CD45ap contains an 8 amino acid insertion between the p2 and P3 strands not seen in any other W W domain, the interconnecting loop between the P2 and P3 strands has been dubbed the "CD45ap loop". (B) Manual alignment of the W W domains of human Yap65 (87), human Dystrophin (93), and murine CD45ap (67, 84). The locations of the P-strands solved by crystal structure analysis for Yap65 and Dystrophin are indicated. Note the insertion of 8 amino acids ( R L G A A L W G H ) between the second and third P strand in CD45ap which has not been observed in anv other W W domain to date. 18  of W W domains has been found to prefer a PPxY motif (Group I) while Group II W W domains prefer PPLP motifs embedded in long stretches of prolines (88). Group III W W domains have been implicated in binding unique poly-P motifs with adjacent R or K residues and Group IV W W domains bind to phosphorylated serine or threonine residues followed by a proline residue in a phosphorylation dependent manner (88).  Recently,  there has been much interest in W W domains as some of the signaling complexes moderated by W W domains have been implicated in diseases such as muscular dystrophy, Alzheimer's disease, Huntington's disease, and cancer (88, 89).  In  Alzheimer's disease, the W W domains in P i n l , a protein necessary for cell cycle progression, has been implicated in the binding and activation of the microtubule assembly activity of Tau, a protein involved in the formation of the neurofibrillary tangles found in the brains of Alzheimer's patients (90-92). Identification of a W W domain within the cytoplasmic domain of CD45ap provides a possible binding site for the interaction of CD45ap with other proteins, and may allow for its functioning as an adaptor molecule. It is interesting to examine the alignment between the W W domain in CD45ap with other W W domains and note that the CD45ap W W domain seems to be variant, containing an insert of eight amino acids between the second and third p strand not present in any other W W domain. A sequence alignment between the W W domains of CD45ap, Yap65 (87), and Dystrophin (93) is provided in figure 1.4.  It is also interesting to note that the predicted W W domain of  CD45ap overlaps the predicted transmembrane domain by seven amino acids. It is thus foreseeable that the binding of a ligand to the W W domain of CD45ap could disrupt the  19  CD45-CD45ap interaction and function as a "binary switch" to modulate signal transduction (84).  1.9 CD45ap expression in CD45 deficient cells and P E S T sequences In CD45 deficient Jurkats, L P A P expression was not detected, while L P A P expression could be detected in the CD45 positive parental Jurkat cell line and a revertant CD45 deficient Jurkat cell line that regained CD45 expression (63). Since L P A P m R N A levels were similar between the three cell lines, this suggested that the L P A P protein was being degraded in absence of its binding partner CD45 (63, 66). Similarly, in pulse-chase studies, it was observed that CD45ap was degraded within 1-2 hours in a CD45 negative murine B W 5147 T cell line, whereas the level of labeled CD45ap remained constant to the latest timepoint tested (6 hours) in CD45 positive cells (N. Gill and P. Johnson, unpublished data). Taken together, this data suggests that CD45 may mask a degradation signal in the cytoplasmic tail of CD45ap and that CD45 is required for stable expression ofCD45ap. Numerous proteins such as metabolic enzymes, kinases, transcription factors, and cell cycle regulators have been shown to be rapidly degraded (94).  Often, such rapidly  degraded proteins including for example, c-Jun, c-Fos, c-Myc, and IkBoc, are protooncogenes and any deregulation of their degradation could potentially lead to unchecked cell division (95).  Sequence analysis of various rapidly degraded proteins including  those just mentioned have revealed that they share a similar protein motif, the PEST sequence, implicated in rapid protein degradation (96).  Sequence analysis of the acidic  region downstream of the W W domain of CD45ap has indicated the possible presence of  20  two PEST sequences within the cytoplasmic tail of CD45ap.  Interestingly, another  plasma membrane protein, a specific Ca -ATPase was found to be degraded upon 2+  cytoplasmic exposure of PEST sequences (97). PEST sequences are enriched in proline (P), glutamate (E), serine (S), and threonine (T) and uninterrupted by positive charges. A more complex publicly available computer algorithm, PEST-FIND, aimed to define potential PEST sequences was developed by Rechsteiner et. al. (94) and defines PEST sequences as hydrophilic stretches of 12 or greater amino acid residues that contain no positively charged residues and at least one P, one E or D, and one S or T, flanked by lysine (K), arginine (R), or histidine (H) residues. PEST-FIND produces scores ranging from +50 to -50, with values above zero denoting possible PEST sequences and values above +5 indicating sequences of true interest. The first (amino acid residues 94-127) and second (amino acid residues 142-172) (see figure 1.3) potential PEST sequences of CD45ap return PEST-FIND scores of+19.02 and +0.74, respectively. For comparison, i Fos and IKBCC have PEST-FIND scores of 10.1 and 5.9, respectively. Significant evidence exists to implicate PEST sequences in ubiquitin mediated degradation by the 26S proteasome (reviewed in 94) although a few examples of ubiquitin independent 26S proteasome degradation of PEST sequence containing proteins exist (98).  The current view is that PEST sequences are minimum consensus  phosphorylation sites recognized by several protein serine kinases (99-102).  The  subsequent ability of an E3 ubiquitin-protein ligase to recognize such sequences could be dependent on the presence of binding motifs, possibly phosphorylated W W domains (103).  There still remains other details to be examined as there is still no clear  phosphorylation pattern that targets proteins for ubiquitination. In addition, it has been  21  observed that phosphorylation can lead to the inhibition of ubiquitination of some proteins such as c-Fos or c-Jun (103). With the presence of potentially one or two PEST sequence in CD45ap, it is temping to suggest that this sequence is involved CD45ap degradation in T cells and that this sequence may be masked by the presence of CD45, leading to longer CD45ap half lives when CD45 is present.  In addition, the fact that CD45ap also contains a W W  domain, which can potentially mediate interaction with ubiquitin ligases, fuels further speculation.  1.10 CD45ap phosphorylation sites It is clear that CD45ap can be phosphorylated in vivo (63, 64, 68) but no phosphorylation of CD45ap tyrosine residues has ever been detected in vivo or in vitro (68, 104) suggesting that the phosphorylation of CD45ap occurs on serine or threonine residues. L P A P was shown to be heavily serine phosphorylated and threonine phosphorylated to a lesser extent in human T cells (63, 105). However, L P A P could be tyrosine phosphorylated by Lck in vitro (105) and in a pervanadate treated human T cell line (63). Pervanadate is a potent inhibitor of protein tyrosine phosphatases. Thus, it is possible that CD45ap may be tyrosine phosphorylated in vivo but not detected because it is dephosphorylated rapidly by a tyrosine phosphatase. While it has been suggested by several groups that CD45ap does not contain any consensus tyrosine phosphorylation sites (64, 106), it may contain a phosphorylation site for calmodulin-dependent kinase II (Thr ) and glycogen synthase kinase-3 (Ser ) (106). 83  87  In contrast, L P A P was predicted by one group to have one potential tyrosine  22  phosphorylation site (Tyr ), one phosphorylation site for cAMP-dependent serine kinase (Ser ), and six other potential phosphorylation sites for casein-kinase 2 (Ser , Thr , 58  24  100  Thr , Ser , Ser , and Thr ) (63). Interestingly, NetPhos 2.0 (107), an automated 113  138  153  155  sequence and structure based predictor of eukaryotic protein phosphorylation sites (107), predicts six potential serine phosphorylation sites (amino acid positions 9, 87, 91, 134, and 150), three potential threonine phosphorylation sites (amino acid positions 86, 126, and 132), and one potential tyrosine phosphorylation site (amino acid position 52) for CD45ap.  For L P A P , NetPhos 2.0 predicts 11 potential serine phosphorylation sites  (amino acid positions 24, 28, 31, 58, 61, 99, 139, 153, 163, 168, and 172), one potential threonine phosphorylation site (amino acid positions 113), and two potential tyrosine phosphorylation sites (amino acid position 64 and 115). From the above, one can hypothesize that serine and threonine phosphorylation may occur in the PEST sequence of CD45ap, possibly leading to the ubiquitin dependent or independent degradation of CD45ap.  1.11 Intracellular proteolysis As the degradation of CD45ap is of interest, it seems appropriate to present a brief outline of the regulation of protein degradation within eukaryotic cells. This by no means intends to be a comprehensive review of protein degradation. Intracellular proteolysis is not only for the regulation of the levels of various proteins but also for their quality control and regulation of function. As alluded to above, such regulation of proteins is key to various biological events including cell cycle regulation, signal transduction, oncogenesis,  23  and the control of gene transcription  (reviewed in 108). There are three major routes through which protein degradation is achieved in the cell: the endolysosomal, calpain, and proteasome-ubiquitin systems. The degradation of proteins can take place in the ER, cytoplasm, nucleus, or lysosomes. The endolysosomal system is responsible for bulk, non-specific proteolysis and is comprised of a series of membrane bound compartments where endocytosed materials (acquired via endocytosis, autophagy, or phagocytosis) are hydrolysed (109, 110) and reviewed in 111).  Current thinking suggests that the organelles in which this type of degradation  occurs contain inactive hydrolases that are activated by endoproteases to degrade substrates once they enter the lysosomal compartments (112-114). A known signal that targets proteins for lysosomal proteolysis is the K F E R Q motif (115). With regards to non-lysosomal protein degradation, the calpain-cathepsin degradation pathway involves cytoplasmic proteases known as calpains which are activated by calcium. Calpains recognize structural determinants rather than motifs and besides being very substrate specific, will cleave their substrates only at a few specific sites (116, 117). Few calpain substrates have been identified but potentially include cFos, c-Jun (118-120), and p53, the tumour suppressor protein (121). The third system of proteolysis in eukaryotic cells is the ubiquitin-proteasome pathway and is the major player in intracellular proteolysis. It is a very elaborate and selective protein degradation system which encompasses two steps: the covalent attachment of a polyubiquitin chain to the target protein and the targeting of this marked protein for degradation by the 26S proteasome (122, 123). The ubiquitin protein is small, highly conserved, present and functionally similar in all eukaryotic cells (124-131). In the presence of A T P , the ubiquitin-activating enzyme (El) initiates the ubiquitination  24  process by promoting ubiquitin to a high energy thiol ester intermediate which it then transfers to one of the E2 ubiquitin-conjugating enzymes. E2 then initiates ubiquitin to undergo a trans-thiol esterification and form a thiol ester linkage between a cysteine residue in the active site of E2 and ubiquitin.  Subsequently, ubiquitin-protein ligases  (E3s) and/or E2s bind one ubiquitin molecule to specifically targeted proteins by forming an isopeptide bond between the s-amino group of a lysine residue in the target protein and the C-terminal glycine residue of ubiquitin. This is followed by the addition of more ubiquitin molecules to the target protein, usually added to the L y s  48  residue of an already  attached ubiquitin molecule, by E3s and/or E2s. A fourth enzyme, E4, has been recently characterized in yeast, and has been proposed to be involved in the formation of very long ubiquitin chains (132). In order for a substrate to be targeted for degradation, it must have at least four ubiquitin moieties bound to it (133).  Marked proteins are  degraded by the 26S proteasome, which is a complex of two 19S regulatory complexes and one 20S proteasome.  Specific signals known to target proteins for ubiquitin  mediated-degradation include: PEST sequences (96), the cyclin destruction box (122), the N-end rule (134, 135), K E K E and pentaleucine signals (136, 137), and misfolded, oxidized, and mutant proteins (102, 122).  Interestingly, W W domains have also been  implicated in targeting proteins for ubiquitination, but such proteins are ultimately degraded in lysosomes (138, 139). The 26S proteasome degrades proteins into peptides and except when such peptides are presented by M H C molecules on APCs, these peptides must be hydrolysed further into amino acids (140). Further understanding of degradation systems and subsequent antigen presentation is important and may allow for better understanding of how certain proteins are degraded and presented by APCs for  25  recognition by immune cells. A motif that targets proteins for degradation may have practical applications as well. For example, the fusion of a specific degradation motif to proteins used in vaccines may ensure enhanced antigen presentation by M H C molecules and potentially improve the immune response against the antigens immunized against. Other recent developments in the field of proteolysis include the development of specific proteasome inhibitors to inhibit the degradation of cyclins or transcription factors that are undergoing human trials for the treatment of some cancers (141). In addition to degrading ubiquitin tagged proteins, the proteasome has also been implicated in the degradation of proteins passing through the ER (142). The E R is a compartment important for membrane proteins, sometimes with the aid of chaperone proteins, to acquire their tertiary and quaternary structures.  To ensure the quality of  proteins produced and protein complex maturity, ER degradation is important in the cell to remove misfolded, unassembled or incompletely assembled membrane proteins and protein complexes from continuing through the secretory pathway. For example, T C R a chains not complexed with the various CD3 molecules are rapidly degraded.  The two  charged residues present in the transmembrane domain of T C R a are thought to be important in E R retention and subsequent degradation (143). Proteins targeted for degradation by ubiquitin dependent (142, 144, 145) or independent (146,  147) pathways within the ER must be transported back to the  cytoplasm before degradation occurs. The Sec61p complex, involved in translocating proteins into the ER, may also be responsible for removing various proteins from the E R for degradation (148-150). Other proteins involved in importing proteins into the E R  26  such as BiP (Kar2p) and Sec63p have also been found to be responsible for exporting proteins from the E R for degradation (149). The proteolytic system by which CD45ap is degraded is unknown. Clearly, the presence of one or two PEST sequences, along with a W W domain in the cytoplasmic domain of CD45ap leads to speculation that CD45ap could contain signals that mediate its rapid degradation. In addition, the increased turnover rate of CD45ap in the absence of CD45 suggests that this signal may be masked in the presence of CD45.  Taken  together, the investigation into factors mediating the degradation of CD45ap should prove to be interesting.  1.12 Thesis objectives Although the differing phenotypes of three CD45ap knock out mice have lead to controversy as to the importance of CD45ap in T cell signaling, the potential importance of CD45ap in the T cell response has been demonstrated by the fact that most CD45 molecules are found associated with CD45ap and that the expression of CD45ap is lymphocyte specific rather than being present in all cells that express CD45. In addition, the functioning of CD45ap as an adaptor molecule has emerged from demonstrations of both in vivo and in vitro associations of CD45ap with key molecules involved in signaling such as CD45, Lck, CD3-£, and ZAP-70. CD45ap is expressed as a highly degraded recombinant bacterial protein (D. Wong, unpublished observations) while CD45 expression is required for stable CD45ap expression (63, 66 and N . Gill and P. Johnson, unpublished data).  Many proteins  involved in signaling are highly degraded to achieve a regulated response.  27  Little is  known about the regulation of CD45ap turnover, and the presence of potential PEST sequences in the cytoplasmic domain of CD45ap remains intriguing. Mutational analysis of CD45ap was carried out in order to gain insight into factors controlling the degradation of CD45ap and to allow stable expression of recombinant CD45ap for in vitro protein binding assays. The intimate relationship between CD45ap and CD45 is evidenced by their quick association in the E R which occurs within minutes of CD45 biosynthesis (151) and their constitutive association at the cell surface (62-64). Thus, the possibility that CD45ap may act as a chaperone to aid CD45 expression and transport to the cell surface and/or increase CD45 half life was investigated. Finally, CD45ap overexpression studies as well as studies to examine novel interactions of the W W domain of CD45ap, a putative protein-protein interaction domain, with other T cell proteins were undertaken to gain further insight into the responsibilities of CD45ap in T cell signaling. The results obtained in this thesis, further solidify the importance of CD45ap in T cell signaling as well as narrow the number of possible roles for CD45ap in T cell signaling. A better understanding of how CD45ap may serve to modulate the immune response may one day lead to better methods of controlling the T cell response, which would be desirable in disease states such as autoimmunity and leukemia.  28  2. Materials and Methods  29  2.1 Methods  2.1.1 Antibodies Anti-phosphotyrosine antibody (4G10) (Upstate  Biotechnology),  anti-actin  antiserum (Sigma), anti-CD3s antibody (145-2C11 from American Type Culture Collection (ATCC)), anti-TCR antibody (H57-597 from A T C C ) , anti-cmyc antibody (9E10 from A T C C ) , anti-6-His antibody (Amersham Pharmacia Biotech), anti-MBP antisera (New England Biolabs), and the anti-GST antibody (Santa Cruz Biotechnology) were acquired through their respective sources. CL-4B sepharose beads (Sigma), Protein A sepharose beads (Amersham Pharmacia Biotech), Glutathione (GSH) Sepharose 4B beads (Amersham Pharmacia Biotech), nickel-NTA-agarose beads (Qiagen), and amylose resin (New England Biolabs) were also purchased though their respective suppliers. Horse-radish peroxidase (HRP) conjugated protein A was purchased from Biorad Laboratories and the HRP conjugated goat anti-mouse IgG was from Southern Biotechnology Associates.  Fluorescein isothiocyanate (FITC) labeled goat anti-rat  antibody was purchased from Pierce and FITC labeled goat anti-hamster antibody was purchased from Southern Biotechnology Associates.  Phycoerythrin (PE) labeled goat  anti-mouse antibody was purchased from Becton Dickinson. Anti-CD45 antibodies used included 13/2, a rat antibody that recognizes all isoforms of murine CD45 (a gift from I. Trowbridge), and R02.2 rabbit antisera raised against the cytoplasmic domain of CD45 (prepared by J . Felberg).  Anti-CD45ap antibodies included rabbit antisera developed  against the cytoplasmic domain of CD45ap obtained from Dr. A . Veillette and from immunizations of rabbits WW1 and WW2.  30  2.1.2 Cell culture Murine L fibroblasts (ATTC) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (Hyclone), 100 units/ml of penicillin, 100 p-g/ml of streptomycin, 2 m M L-glutamine (Calbiochem), and 1 m M sodium pyruvate (Gibco). L cells transfected with CD45ap and CD45ap (del 157197) were maintained as above except for the addition of 10 (J.g/ml puromycin (Sigma). B W 5147 CD45 positive and CD45 negative mouse T lymphoma cells (ATCC) were transfected with CD3-5 and CD3-<^ (a gift from Dr. B. Malissen) and were maintained in D M E M supplemented with 10% horse serum (Hyclone) 100 units/ml of penicillin, 100 p,g/ml of streptomycin, 2 m M L-glutamine, 1 m M sodium pyruvate, and 3 m M L histidinol (Sigma). B W 5147 cells transfected with CD45ap were maintained as above except for the addition of 1.5 mg/ml G418 (Gibco). A l l cells were incubated at 37°C with 5% CO2. Routine checks by flow cytometry were used to ensure similar T C R and CD3 levels between the B W 5147 cell lines.  2.1.3 Bacterial strains and vectors The Escherichia coli (E. coli.) D H 5 a , UT5600 (New England Biolabs) and B L 21 (DE3) strains were used for the cloning of D N A constructs and the expression of recombinant proteins. M B P , GST, and 6-His recombinant fusion proteins were expressed in the D H 5 a and UT5600 strains while cmyc fusion proteins were expressed in the B L 21 (DE3) strain. M B P fusion protein constructs were cloned into the p M A L - c R i vector (New England Biolabs), GST fusion protein constructs were cloned into the p G E X - 4 T l vector (Amersham Pharmacia Biotech), cmyc fusion protein constructs were cloned into  31  the pSSBS.myc2 vector (Stratagene), and 6-His protein constructs were cloned into the pET-28a  vector  (Novagen).  Eukaryotic expression  vectors  used included the  pBCMGSneo (152) (obtained from H . Karasuyama) and the pMX-pie (obtained from A . Mui) expression vectors.  2.2 Methods  2.2.1 DNA constructs The 997bp mouse CD45ap cDNA (Genebank accession number 18640745) (64, 70) had previously been cloned into the EcoRI site of the pBSKII" plasmid (Stratagene) (construct #4.18).  In addition, #4.18 was modified such as to remove the c D N A  sequence of CD45ap 3' of the W W domain and place a translational stop codon directly after the W W domain of CD45ap. This construct was subsequently named #4.18.8.  2.2.1.1 PCR generation of the cytoplasmic and WW domain of CD45ap Two D N A fragments were generated by polymerase chain reaction (PCR). One contained the complete cytoplasmic domain of CD45ap (amino acid positions 42-197) and the other contained only the W W domain of CD45ap (amino acid positions 42-93). The full-length cytoplasmic domain P C R product was generated using #4.18 as the template  and 5' - C G G A A T T C C T C C T G C T G C T G C T G G T C A C T G C C C T A - 3 '  as the  forward primer (annealing to positions 172 to 198 of CD45ap, i.e., the beginning of the cytoplasmic domain of CD45ap) and 5 ' - C A C C G C G G T G G A G C T C C A G C T T T T G T T C C  32  C T T T A G T G A G G G T T A A T T - 3 ' (annealing to pBSKII" downstream of the CD45ap c D N A insert) as the reverse primer. This resulted in the generation of an 846 bp product that encoded for the complete cytoplasmic domain of CD45ap (amino acid positions 42197). The amplified P C R product was then digested with EcoRI and Xbal to produce 5' EcoRI sites and 3' Xbal sites. The digested product was subsequently isolated from an agarose gel and purified using a Qiagen Gel Extraction Kit (Qiagen). The W W domain alone P C R product was generated using the same forward and reverse primers used above to construct the above full-length CD45ap cytoplasmic domain P C R product, but #4.18.8 was used as the template instead of #4.18. This resulted in a 178 bp product that encoded for the W W domain of CD45ap with a translational stop codon placed directly after the W W domain. The digested product was subsequently isolated from an agarose gel and purified using a Qiagen Gel Extraction Kit (Qiagen).  2.2.1.2 Generation of the fusion protein expression constructs The above P C R products were subsequently ligated in frame into EcoRI-Xbal digested and dephosphorylated (calf intestinal phosphatase, New England Biolabs) p M A L - c R i directly downstream of the M B P coding sequence, EcoRI-Sall digested and dephosphorylated p G E X - 4 T l directly downstream of the GST coding sequence, and EcoRI-Xbal digested and dephosphorylated PSSBS.myc2 directly downstream of the cmyc epitope tag coding region. The regions of the constructs generated by P C R were sequenced to ensure that no mutations had been generated.  33  The p M A L - c R i constructs and expressed M B P recombinant protein will be herein named MBP-CD45ap while p M A L - c R i (vector alone without any insert) will be herein named M B P alone. The p G E X - 4 T l constructs and expressed GST recombinant protein will be herein named GST-CD45ap while p G E X - 4 T l (vector alone without any insert) will be herein named GST alone.  The GST fusion protein containing only the W W  domain of CD45ap will be herein named GST-WW. The pSSBS.myc2 constructs and expressed cmyc recombinant protein will be herein named cmyc-CD45ap. Both the p M A L - c R i and p G E X - 4 T l vectors contain an inducible Ptac promoter to allow transcription of the fusion protein gene. The two expression vectors also contain the lacl gene encoding for the Lac repressor, which turns off transcription from P^c until q  isopropylthio-P-D-galactoside (IPTG) is added. A l l ligations were performed with overnight incubations at 16°C using T4 D N A ligase (New England Biolabs) supplemented with the supplied buffer.  2.2.1.3 Generation of the C-terminal deletions of GST- CD45ap To construct GST-CD45ap (del 142-197) (amino acid residues 142-197 deleted from the cytoplasmic domain of CD45ap), GST-CD45ap was digested with Pmll (which cut at position 470/997 of CD45ap) and NotI (which cut the p G E X - 4 T l vector 3' of the CD45ap insert), followed by the filling in of the 3' recessed ends by Klenow treatment (New England Biolabs) and religation of the blunt ends. To construct GST-CD45ap (del 157-197), GST-CD45ap was digested with PpuMI (which cut at nucleotide position 604/997 of CD45ap) and NotI (which cut the  34  p G E X - 4 T l vector 3' of the CD45ap insert), followed by the filling in of the 3' recessed ends by Klenow treatment and religation of the blunt ends. To construct GST-CD45ap (del 52-126), GST-CD45ap was digested with Styl (which cut at nucleotide positions 200/997 and 426/997 of CD45ap), followed by religation of the vector. To construct GST-CD45ap (del 176-197), a D N A fragment generated by P C R using  5 '-CGGTGTCAAGCAGAGCAGACCCGAGATCCACGTGACACAGACAGT-  3' (annealing to amino acid residue positions 132-146 of CD45ap) as the forward primer and 3 ' - G A C T C A C T G G A C G T A C G G A A A A G A - 5 '  (annealing to amino acid residue  positions 168-175 of CD45ap) as the reverse primer and #4.18 as the template was digested with Pmll and NotI and ligated into the Pmll-NotI digested GST-CD45ap D N A construct. To construct GST-CD45ap (del 186-197), a D N A fragment generated by P C R using  5 '-CGGTGTCAAGCAGAGCAGACCCGAGATCCACGTGACACAGACAGT-  3' (annealing to amino acid residue positions 132-146 of CD45ap) as the forward primer and  3 '-CGACGGACCCTACTGTCGCGACCTATCGCGGCCGCAAAAGGAAAA-5'  (annealing to amino acid residue positions 178-185 of CD45ap) as the reverse primer and #4.18 as the template was digested with Pmll and NotI and ligated into the Pmll-NotI digested GST-CD45ap D N A construct. To construct 6-His-CD45ap (del 157-197), a D N A construct encoding for cmycCD45ap (del 157-197) (constructed by a PpuMI partial digestion of the cmyc-CD45ap D N A construct followed by Klenow treatment (New England Biolabs) and subsequent relegation) was digested with EcoRI and NotI to excise the fragment containing residues  35  42-157 from the cytoplasmic domain of CD45ap.  This fragment was subsequently  ligated into EcoRI-NotI digested pET-28a. For eukaryotic expression, the D N A fragment encoding for cmyc-CD45ap was excised from the cmyc-CD45ap D N A construct by digestion with Xhol and NotI and subsequently ligated into XhoI-NotI digested pBCMGSneo and pMX-pie. In addition, the D N A fragment encoding for cmyc-CD45ap (del 157-197) was excised from the cmyc-CD45ap (del 157-197) D N A construct by digestion with Xhol and NotI and subsequently ligated into XhoI-NotI digested pBCMGSneo and pMX-pie.  2.2.1.4 Restriction digests A l l restriction digests were performed using enzymes from New England Biolabs supplemented with the supplied buffers.  2.2.1.5 PCR reactions A l l P C R reactions were carried out using Vent D N A polymerase (New England Biolabs). Denaturation temperatures of 95°C for one minute, annealing temperatures of 55°C for one minute, and extension temperatures of 72°C (with a lkb per minute extension time) were used for all reactions.  2.2.1.6 Transformation of bacterial cells Heat shock transformations (90 seconds at 42°C) of competent DH5a, UT5600, or BL-21 (DE3) E.coli. cells with the various plasmids were performed. Cells were allowed to recover in Luria broth (LB) for 30 minutes at 37°C with shaking at 180 rpm  36  before plating onto Luria agar plates with the appropriate selection (ampicillin (100 (ag/ml), ampicillin and chloramphenicol (30 p.g/ml), or kanamycin (30 (xg/ml)).  2.2.2 Expression of recombinant proteins Cells containing the various expression constructs were grown in 10 ml or 500 ml volumes of Luria broth shaking at 180 rpm at 37°C until they reached an optical density (at 600 nm) (OD600) value of 0.6-0.8. At this cell density, the cultures were supplemented with 0.1 m M IPTG, and allowed to shake at 180 rpm at 26°C for 2.5 hours or overnight. The lower incubation temperature of 26°C was utilized to allow slower rates of protein production that may aid in protein stability and proper folding.  2.2.2.1 Preparation of whole cell lysates After the addition of IPTG and subsequent incubation at 26°C, 200 u.1 of cells were taken and resuspended in 150 ul of reducing sample buffer (0.125 M Tris-HCI pH 7.5, 10% glycerol, 100 m M dithiolthreitol (DTT), 2% sodium dodecyl sulfate (SDS), and 0.2% bromophenol blue). 7.5 p.1 of the sample was subsequently electrophoresed in an SDS-PAGE gel, transferred to an Immobilon-P P V D F membrane (Millipore) according to the manufacturer's instructions (Biorad), and visualized by immunoblotting.  2.2.2.2 Purification of MBP fusion proteins After the addition of IPTG and subsequent incubation at 26°C, the 500 ml cultures were centrifuged at 4000xg for 10 minutes at 4°C. The cell pellet was then resuspended in 5 mis of ice cold lysis buffer (20 m M Tris-HCI p H 7.5, 10 m M NaCI, 0.5% TX-100,  37  and 1 m M EDTA).  Protease inhibitors (0.2 m M PMSF, 1 pg/ml aprotinin, 1 pg/ml  leupeptin, and 1 pg/ml pepstatin) were also added at this point. The resuspended cells were then frozen in a dry ice/ethanol bath for five minutes and then thawed at 37°C. The freeze/thaw cycles were repeated three times. 20 ul of DNAasel (1 mg/ml) was added after the third thaw, followed by an incubation at 37°C for five minutes. The lysate was then centrifuged at 10,000xg for 20 minutes at 4°C. The supernatant was subsequently collected and incubated (rotating end over end at 4°C for two hours) with 500 ul of 50% amylose resin previously washed three times in column buffer (20 m M Tris-HCI p H 7.5, 200 m M NaCI, 1 m M EDTA). After the incubation, the amylose resin was washed three times with column buffer and resuspended in 500 pi of column buffer. Samples for SDSP A G E were prepared by heating the amylose resin (and bound M B P fusion protein) to 100°C for five minutes in reducing sample buffer.  The sample buffer containing the  proteins liberated from the amylose resin was then electrophoresed in an SDS-PAGE gel, transferred to a P V D F membrane, and visualized by immunoblotting with M B P antiserum.  2.2.2.3 Purification of GST fusion proteins After the addition of IPTG and subsequent incubation at 26°C, the 500 ml cultures were centrifuged at 4000xg for 10 minutes at 4°C. The cell pellet was then resuspended in 10 mis of ice cold lysis buffer (20 m M Tris-HCI p H 7.5, 150 m M NaCI, and 1% T X 100). Protease inhibitors (0.2 m M PMSF, 1 pg/ml aprotinin, 1 pg/ml leupeptin, and 1 p.g/ml pepstatin) were also added at this point. The resuspended cells were then frozen in a dry ice/ethanol bath for five minutes and then thawed at 37°C. The freeze/thaw cycles  38  were repeated three times. 20 pi of DNAasel (1 mg/ml) was added after the third thaw, followed by an incubation at 37°C for five minutes. The lysate was then centrifuged at 10,000xg for 20 minutes at 4°C. The supernatant was then collected and incubated (rotating end over end at 4°C for two hours) with 250 pi of 50% GSH sepharose previously washed three times in wash buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.1%o Triton X-100 (TX-100)). After the incubation, the GSH sepharose beads were washed three times with wash buffer and resuspended in 250 pi of wash buffer. Samples for SDS-PAGE were prepared by heating the GSH sepharose (and bound GST fusion protein) to 100°C for five minutes in reducing sample buffer.  The sample buffer  containing the proteins liberated from the GSH sepharose was then electrophoresed in an SDS-PAGE gel, transferred to a PVDF membrane, and visualized by immunoblotting with GST antiserum. Alternatively, GST fusion proteins bound to GSH sepharose could be eluted by incubating the GSH beads in an equal volume of elution buffer (15 mM glutathione in 50 mM Tris-HCI pH 8.0) with end over end rotation for 15 minutes at room temperature.  2.2.2.4 Purification of 6-His fusion proteins  After the addition of IPTG and subsequent incubation at 26°C, the 500 ml cultures were centrifuged at 4000xg for 10 minutes at 4°C. The cell pellet was then resuspended in 10 mis of ice cold A l buffer (20 mM Tris-HCI pH 7.5, 150 mM NaCI, 20 mM imidazole pH 7.2, 0.5% TX-100, 1 mM EDTA, 0.025% p-Me). Protease inhibitors (0.2 mM PMSF, 1 ug/ml aprotinin, 1 pg/ml leupeptin, and 1 ug/ml pepstatin) were also added at this point. The resuspended cells were then frozen in a dry ice/ethanol bath for five  39  minutes and then thawed at 37°C. The freeze/thaw cycles were repeated three times. 20 ul of DNAasel (1 mg/ml) was added after the third thaw, followed by an incubation at 37°C for five minutes. The lysate was then centrifuged at 10,000xg for 20 minutes at 4°C. The supernatant was then collected and incubated (rotating end over end at 4°C for two hours) with 250 ul of 50% Nickel-NTA-Agarose beads (Qiagen) previously washed three times in A l buffer. After the incubation, the nickel beads were washed with A l buffer, followed by a wash in a wash buffer containing 20 m M Tris-HCI pH 7.5, 500 m M NaCl, 20 m M imidazole pH 7.2, 0.5% TX-100, 0.025% P-Me, followed by a wash in A l buffer, followed by a wash in a wash buffer containing 20 m M Tris-HCI p H 7.5, 1 M NaCl, 20 m M imidazole pH 7.2, 0.5% TX-100, 0.025% p-Me, and finally followed by a wash in A l buffer. Samples for SDS-PAGE were prepared by heating the nickel beads (and bound 6-His fusion protein) to 100°C for five minutes in reducing sample buffer. The sample buffer containing the proteins liberated from the nickel beads was then electrophoresed in an SDS-PAGE gel, transferred to a P V D F membrane, and visualized by immunoblotting with 6-His antiserum. Alternatively, 6-His fusion proteins bound to nickel beads could be eluted by incubating the nickel beads in an equal volume of B l buffer (150 m M NaCl, 1.0 M imidazole p H 7.2, 0.025% P-Me, 0.1% TX-100) with end over end rotation for 15 minutes at room temperature.  2.2.3 Immunization and preparation of serum from rabbits WW1 and WW2 A l l procedures were carried out at and in accordance with standards put forward by the Animal Care Facility located at South Campus (University of British Columbia). Before the immunization program began, a 10 ml bleed was collected from each rabbit.  40  The first injection, injected subscapularly, consisted of 1 mg of eluted GST-WW dialysed against phosphate buffered saline (PBS) (2.7 m M KC1, 150 m M NaCl, 4.3 m M N a H P 0 , 2  4  1.5 m M KH2PO4) and mixed with Freund's complete adjuvant (Gibco) per rabbit. Subsequent booster injections of 500 (j.g of GST-WW per rabbit in Freund's incomplete adjuvant (Gibco) followed at one month intervals. 30 mis of blood was collected from each rabbit 14 days after the injections.  Due to the failure of the rabbits to produce  antibodies against CD45ap with this immunization procedure, approximately one year after the initial injection, the rabbits were immunized with 500 u.g of 6-His-CD45ap (del 157-197) in Freund's complete adjuvant. In order to obtain denatured 6-His-CD45ap (del 157-197) for injection, purified 6-His-CD45ap (del 157-197) was boiled in PBS containing 0.5% SDS for 10 minutes.  The SDS was diluted to 0.1% with PBS and  adjuvant prior to injection. Four booster injections with denatured CD45ap (del 157-197) in Freund's incomplete adjuvant were carried out at one month intervals. 30 ml bleeds were taken from each rabbit 14 days following each injection. The collected blood was allowed to clot at room temperature for four hours, after which the clot was loosened, and the blood placed at 4°C overnight to allow the clot to retract. The blood was then centrifuged at lOOOxg for 10 minutes after which the serum collected and respun. The serum was then filtered though a 0.22 u M filter and stored at 20°C.  2.2.4 GenePorter2 transfection L cells were transfected with constructs encoding for cmyc-CD45ap or cmycCD45ap (del 157-197) in the pMX-pie eukaryotic expression vector. L cells previously  41  transfected with the full length CD45 isoform, CD45RABC, were transfected with the pMX-pie expression vector carrying constructs coding for CD45ap or CD45ap (del 157197).  A l l of the pMX-pie D N A constructs were purified using a Qiagen EndoFree  Plasmid Maxi Kit (Qiagen). For each construct, 5 x 10 L cells were transfected with 8 6  pg of the construct using the GenePorter2 transfection reagent (Gene Therapy Systems) according to the manufacturer's instructions. 10 ug/ml of puromycin was added 48 hours post transfection and the surviving clones were analyzed by flow cytometry over a period of one month to allow for the selection of stably transfected clones.  2.2.5 Electroporation For the CD45ap overexpression studies, 8 x 10 B W 5147 cells were transfected 6  with 20 pg of the pBCMGSneo expression vector encoding for cmyc-CD45ap by electroporation (250V, 950 pF on the Gene Pulser II electroporation system (Biorad)). The D N A construct was purified using a Qiagen EndoFree Plasmid Maxi Kit (Qiagen) and the D N A for transfection was prepared by ethanol precipitation and resuspended in sterilized distilled and deionized water.  G418 selection was added 48 hours post-  el ectroporation at a concentration of 1.5 mg/ml and the clones were analyzed over a period of one month to allow for the selection of stably transfected clones.  2.2.6 S pulse chase labeling 35  For the CD45 transport studies, each 100 mm plate of cells to be labeled was 2  grown to 70% confluency (~5 x 10 cells) and washed with 5 mis of PBS before being 6  starved of methionine and cysteine by incubation in 5 mis of methionine and cysteine free  42  media (methionine-free, cysteine-free D M E M (ICN) supplemented with dialysed fetal calf serum (Gibco),  100 units/ml of penicillin, 100 (ig/ml of streptomycin, 2 m M L -  glutamine, and 1 m M sodium pyruvate) for one hour. The cells were then pulsed with 75 uCi/ml of EasyTag EXPRESS Protein Labeling M i x [ S] 35  (specific activity >1000  Ci/mmol, 70% methionine, 30% cysteine, Perkin Elmer) for 15 minutes. Following the pulse period, the label was removed and the cells were washed with PBS before the addition of the chase media ( D M E M supplemented with 10% fetal calf serum, 100 units/ml of penicillin, 100 ng/ml of streptomycin, 2 m M L-glutamine, and 1 m M sodium pyruvate) for 0, 15, 30, 60, or 120 minutes. At the end of each chase period, the cells were washed with PBS and then lysed in the plates with the addition of ice cold lysis buffer (35 m M Tris-HCI pH 7.5, 262.5 m M NaCl, 0.875% deoxycholic acid, 0.175% SDS, 1.75% Nonidet P-40) supplemented with 0.2 m M PMSF, 1 (ag/ml aprotinin, 1 ug/ml leupeptin, and 1 f^g/ml pepstatin. After the lysis buffer was added, the plates were left on ice for five minutes before the cells were removed by scraping. The lysed cells were then transferred to an Eppendorf tube, vortex ed, and left on ice for an additional 10 minutes. After the incubation on ice, the lysates were centrifuged at 14,000xg for 10 minutes at 4°C and the supernatants were removed to another tube. The lysates were then pre-cleared for one hour at 4°C rotating end over end with 40 uL of 50% CL-4B sepharose beads, followed by an incubation with 40 uL of 50% 13/2 antibody pre-coupled to CNBr-beads (J. Felberg and Amersham Pharmacia Biotech) rotating end over end for 2 hours at 4°C. The immunoprecipitates were washed two times in a high salt wash buffer (20 m M Tris-HCI pH 7.5, 500 m M NaCl, 0.5% deoxycholic acid, 0.1% SDS, 1% Nonidet P-40) followed by three washes in a low salt wash buffer (20 m M Tris-HCI pH 7.5, 150  43  m M NaCI, 0.5% deoxycholic acid, 0.1% SDS, 1% Nonidet P-40) and one final wash in 10 m M Tris-HCI pH 7.5. The samples were then divided equally and either treated or not treated with Endoglycosidase H (Endo H) (Boerhinger Mannheim). Endo H digestions were carried out by the addition of 12 p L of 85 m M sodium citrate p H 5.5, 0.5 p L Endoglycosidase H (2.5 mU), and 0.06 pL of 0.1 M P M S F to the immunoprecipitates. The digestion was allowed to continue for 22 hours at 37°C. Samples not treated with Endo H were also incubated in 12 pL of 85 m M sodium citrate p H 5.5, 0.5 pL distilled and deionized water, and 0.06 p L of 0.1 M P M S F to ensure consistency between the samples that were treated with Endo H and those that were not. After the digestion, reducing sample buffer was added to the immunoprecipitates before boiling and analysis by SDS-PAGE (5%). The samples were transferred over to a P V D F membrane and allowed to dry. The dried P V D F membrane was then soaked in Amplify (Amersham Pharmacia Biotech) with gentle shaking for 30 minutes before exposing it to film (Kodak BioMax) with an intensifying screen for up to one month. For the CD45 half life studies, the procedure was as above, except that the radiolabeling time was 1 hour instead of 15 minutes and the chase times were 0, 24, 48, 72, and 96 hours. There was no Endo H digestion of these samples.  2.2.7 C pulse chase labeling 1 4  Each 100 mm plate of cells to be labeled was grown to 70% confluency (~5 x 10 cells) and washed with 5 mis of PBS before being starved of leucine by incubation in 5 mis of leucine free media (leucine-free D M E M (ICN) supplemented with dialysed fetal calf serum, 100 units/ml of penicillin, 100 pg/ml of streptomycin, 2 m M L-glutamine,  44  and 1 m M sodium pyruvate) for 30 minutes. The cells were then pulsed with 5 pCi of 14  C-labeled leucine (specific activity >300 mCi/mmol, Amersham Pharmacia Biotech) for  two hours. Following the pulse period, the label was removed and the cells were washed with PBS before the addition of the chase media ( D M E M supplemented with 10% fetal calf serum, 100 units/ml of penicillin, 100 pg/ml of streptomycin, 2 m M L-glutamine, and 1 m M sodium pyruvate) for 0, 24, 48, 72, or 96 hours. At the end of each chase period, the cells were washed with PBS and then lysed in the plates with the addition of ice cold lysis buffer (1% Nonidet P-40, 20 m M Tris-HCI p H 7.5, 150 m M NaCI, 2 m M EDTA) containing 0.2 m M PMSF, 1 pg/ml aprotinin, 1 pg/ml leupeptin, and 1 pg/ml pepstatin. After the lysis buffer was added, the plates were left on ice for five minutes before the cells were removed by scraping. The lysed cells were then transferred to an Eppendorf tube, vortexed, and left on ice for an additional 10 minutes.  After the  incubation on ice, the lysates were centrifuged at 14,000xg for 10 minutes at 4°C and the supernatants were removed to another tube. The lysates were pre-cleared for one hour at 4°C rotating end over end, followed by an incubation with end over end rotation at 4°C with 3 pL of purified 9E10 anti-cmyc antibody for two hours, followed by an incubation with end over end rotation at 4°C with 20 pL of 50% protein G sepharose. Subsequently, the immunoprecipitates were washed two times in a high salt buffer (1% Nonidet P-40, 20 m M Tris-HCI p H 7.5, 500 m M NaCI, 2 m M EDTA) followed by three washes in a low salt buffer (1% Nonidet P-40, 20 m M Tris-HCI p H 7.5, 150 m M NaCI, 2 m M EDTA). After the washes, reducing sample buffer was added to the immunoprecipitates before boiling and analysis by SDS-PAGE (12.5%). The samples were transferred over to a P V D F membrane and allowed to dry. The dried P V D F membrane was then soaked  45  in Amplify with gentle shaking for 30 minutes before exposing it to film (Kodak BioMax) with an intensifying screen for up to one month.  2.2.8 CD3 stimulation of B W 5147 cells  1 x 10 cells were resuspended in 100 uL D M E M supplemented with 0.25% FCS 7  equilibrated at 37°C. Purified 145-2C11 anti-CD3 antibody was added at time zero to stimulate the cells which were incubated at 37°C.  2 x 10 cells were removed, 6  centrifuged, and lysed at the appropriate timepoints in lysis buffer (1% TX-100, 150 m M NaCl, 20 m M Tris-HCI p H 7.5, 2 m M E D T A , 5 m M sodium orthovanadate, 2 m M sodium molybdate, 0.2 m M PMSF, 1 [J.g/ml aprotinin, 1 u.g/ml leupeptin, and 1 (J.g/ml pepstatin).  After the lysis buffer was added, the cells were left on ice for 10 minutes.  After the incubation on ice, the lysates were centrifuged at 14,000xg for 10 minutes at 4°C and the supernatants were removed to another tube. Reducing sample buffer was then added to the samples before boiling and analysis by SDS-PAGE (10%). The samples were transferred over to a P V D F membrane and analyzed by immunoblotting with the 4G10 anti-phosphotyrosine antibody.  2.2.9 GST fusion protein pull down assay from pervanadate treated T cell lysates  Pervanadate was prepared fresh by mixing 10 m M of sodium orthovanadate (Sigma) solution with 10 m M of H2O2 for five minutes at room temperature. Catalase (Sigma) was subsequently added at a concentration of 200 (ag/ml to remove any residual H 0 . 2  2  1 x 10 B W 5147 T cells were resuspended in 1 ml of growth media ( D M E M 7  supplemented with 10% horse serum, 100 units/ml of penicillin, 100 |ag/ml of  46  streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate) and pervanadate was added to a final concentration of 100 pM. Following a 15 minute incubation at 37°C, the cells were pelleted and washed with PBS and subsequently lysed in ice cold lysis buffer (1% TX-100, 150 mM NaCI, 20 mM Tris-HCI pH 7.5, 2 mM EDTA, 5 mM sodium orthovanadate, 2 mM sodium molybdate, 0.2 mM PMSF, 1 pg/ml aprotinin, 1 pg/ml leupeptin, and 1 pg/ml pepstatin). Insoluble debris was removed by centrifugation at 14,000xg for 10 minutes at 4°C and the detergent soluble cell lysates were pre-cleared by incubation with 40 pL of a 50% slurry of CL-4B sepharose beads rotating end over end at 4°C for 30 minutes. After the pre-clear step, the cell lysates were incubated with 2 pg of purified GST-WW fusion protein immobilized on GSH sepharose beads rotating end over end at 4°C for 90 minutes. Subsequently, the samples were centrifuged and the pelleted GSH sepharose beads were washed three times in lysis buffer.  Samples were then  subjected to SDS-PAGE and analysis by immunoblotting with the 4G10 antiphosphotyrosine antibody.  2.2.10 Flow cytometry  2 x 10 cells were incubated with 100 pL of tissue culture supernatant containing 5  the anti-CD45 (13/2), anti-cmyc (9E10), anti-CD3 (145-2C11), or anti-TCR (H57-597) antibody for 20 minutes on ice. After subsequent washing of the cells with PBS containing 2% FCS and 2 mM ethylenediamine-tetra-acetic acid (EDTA), the cells were incubated with 100 pL of a 1/100 dilution of the appropriate FITC or PE labeled secondary antibody. Cells were then washed and analyzed on a FACScan analyzer (Becton Dickinson) using the Cell Quest software (Becton Dickinson).  47  2.2.11 C D 4 5  immunoprecipitations:  5 x 10 L cells were lysed in the plate with the addition of ice cold lysis buffer 6  (1% Brij-97, 20 m M Tris-HCI pH 7.5, 150 m M NaCl, 2 m M EDTA) containing 0.2 m M PMSF, 1 (J.g/ml aprotinin, 1 jag/ml leupeptin, and 1 |J.g/ml pepstatin. After the lysis buffer was added, the plates were left on ice for five minutes before the cells were removed by scraping.  The lysed cells were then transferred to an Eppendorf tube,  vortex ed, and left on ice for an additional 10 minutes. After the incubation on ice, the lysates were centrifuged at 14,000xg for 10 minutes at 4°C and the supernatants were removed to another tube. The lysates were pre-cleared once for one hour at 4°C rotating end over end with 40 uL of 50% CL-4B sepharose beads, followed by an incubation with 40 uL of 50% 13/2 beads rotating end over end for 2 hours at 4°C. Subsequently, the immunoprecipitates were washed three times in lysis buffer. After the washes, reducing sample buffer was added to the immunoprecipitates before boiling and analysis by SDSP A G E (10%). The samples were transferred over to a P V D F membrane and analyzed by immunoblotting with antiserum against CD45 or CD45ap.  2.2.12  Binding assay of recombinant C D 4 5 to Lck in the presence or absence of  CD45ap  6-His-CD45, 6-His-CD45ap (del 157-197), GST-Lck, and GST-Grb2 were purified from E. coli. transformed with the appropriate expression vector using the respective method as outlined in Materials and Methods. 6-His proteins were eluted from the nickel beads and passed through a PD10 buffer exchange column (Amersham Pharmacia Biotech) and subsequently eluted from the PD10 column with binding buffer  48  (20 m M Tris-HCI p H 7.5, 150 m M NaCI, 0.05% TX-100, 0.2 m M PMSF, 1 pg/ml aprotinin, 1 pg/ml leupeptin, 1 pg/ml pepstatin). GST proteins were left bound to the G S H beads. 2 pg of GST-Lck (610 nM) or GST-Grb2 (910 nM) bound to G S H beads were washed three times and resuspended in 40 pi of binding buffer and incubated with 610 n M of 6-His-CD45 and 0, 610, or 3050 n M of 6-His-CD45ap with shaking at 4°C for two hours. Subsequently, the G S H beads with the bound proteins were washed with radioimmunoprecipitation assay (RIPA) buffer (20 m M Tris-HCI pH 7.5, 500 m M NaCI, 0.5% deoxycholic acid, 0.1% SDS, 1% Nonidet P-40), followed by a wash in a buffer containing 20 m M Tris pH 7.5, 500 m M NaCI, 0.5% TX-100, followed by a wash in RIPA buffer, followed by a wash in a buffer containing 20 m M Tris pH 7.5, 1 M NaCI, 0.5% TX-100, and finally followed by a wash in RIPA buffer.  After the washes,  reducing sample buffer was added to the immunoprecipitates before boiling and analysis by SDS-PAGE (10%). The samples were transferred over to a P V D F membrane and analyzed by immunoblotting with antiserum against 6-His.  2.2.13 S D S - P A G E Visualization of proteins was performed by separation by SDS-PAGE (5%, 10%, or 12.5% acrylamide). A l l samples were suspended in reducing sample buffer and heated to 100°C for five minutes prior to being loaded onto the gel.  2.2.14 Immunoblotting With the exception of anti-phosphotyrosine (4G10) blots, dried P V D F membranes were incubated for one hour with the primary antibody diluted in TTBS (20 m M Tris-  49  HC1 p H 7.5, 150 m M NaCl, and 0.1% Tween-20) and 5% skim milk powder. The membranes were then washed for three five minute intervals with TTBS. Following an incubation with the HRP conjugated secondary antibody diluted (1/5000) in TTBS with 5% skim milk for one hour, the membrane was washed for three five minute washes with TTBS. Visualization by chemiluminescence was carried out using E C L HRP substrate (Amersham Pharmacia Biotech) and exposure to Kodak BioMax film (Kodak). AntiM B P , anti-GST, anti-CD45ap, anti-6-His, R02.2, W W 1 , and WW2 antisera were used at 1/5000 dilutions. The mouse 9E10 monoclonal antibody and the anti-actin antiserum were used at 1/500 dilutions. Incubations with primary and secondary antibodies were at room temperature for one hour with shaking at 100 rpm. 4G10 (used at a 1/5000 dilution) anti-phosphotyrosine immunoblots were carried out as above except for the use of 0.5% B S A (Sigma) instead of skim milk powder diluted in TTBS as a blocking agent.  2.2.15 Densitometric Analysis: A l l densitometric analysis to determine the relative intensities and areas of ethidium bromide stained D N A bands on agarose gels or protein bands on SDS-PAGE gels or films were carried out on the Alpha Imager System (Alpha Innotech Corporation) or the VersaDoc Imaging System (Biorad).  50  3. Results  51  3.1 Investigation of CD45ap degradation  3.1.1 Generation and purification of degraded recombinant MBP-, GST-, and cmvcCD45ap Expression of recombinant eukaryotic proteins in bacterial systems can prove to be challenging.  Not all recombinant proteins are equal in their stability as certain  proteins may be more susceptible to proteolysis and/or may be toxic to the bacterial cells when expressed. In addition, the generation of fusion proteins may complicate matters further as different protein tag-fusion protein combinations can have different effects on the stability of the fusion protein. Thus, in an attempt to express recombinant CD45ap for use in in vitro binding assays, it was prudent to assess the expression of recombinant CD45ap in several different expression systems employing different protein tags. Constructs encoding for recombinant proteins containing the cytoplasmic domain of CD45ap fused to one of three different protein tags (MBP, cmyc, or GST) were constructed and transformed into bacteria. The extracellular and transmembrane domains of CD45ap were not included in the fusion proteins to decrease solubility problems of the recombinant protein that may be caused by the inclusion of the hydrophobic residues present in the transmembrane domain of CD45ap.  A schematic of the potential  recombinant proteins generated is provided in figure 3.1. Bacteria transformed with the different constructs were induced with IPTG to produce protein before lysis and the purification of each of the recombinant proteins was carried out as outlined in Materials and Methods.  The M B P , cmyc, and GST purified proteins were analyzed by  immunoblotting with anti-MBP, anti-cmyc, and anti-GST antibodies, respectively (figure  52  cmyc - CD45ap  MBP - CD45ap  GST - CD45ap 126 y  deleted  GST - CD45ap (del 52-126)  |  142  gzzzzzzzzzzzzzzzzzzz •  deleted  ,  157  GST - CD45ap (del 142-197) GST - CD45ap (del 157-197) GST - CD45ap (del 176-197)  186 deleted.  GST - CD45ap ( d e  | 186-197)  Figure 3.1. CD45ap recombinant proteins and mutants generated. Schematic illustrating the fusion proteins generated. cmyc-CD45ap, M B P - C D 4 5 a p , and G S T CD45ap fusion proteins contain amino acid residues 42-197 from CD45ap. Illustrations for G S T - C D 4 5 a p (del 52-126) (GST-CD45ap with a deletion o f amino acid residues 52126), G S T - C D 4 5 a p (del 142-197), G S T - C D 4 5 a p (del 157-197), G S T - C D 4 5 a p (del 176197), and G S T - C D 4 5 a p (del 186-197) are also provided.  53  3.2). Both M B P and GST fusion proteins were produced and migrated to their predicted molecular weights (77 and 56 kDa, respectively) but were highly degraded as evidenced by the presence of numerous lower molecular weight bands. No expression of the cmyc fusion protein could be detected by immunoblotting. Lack of expression may be due to the toxicity of the cmyc-CD45ap protein or high levels of fusion protein degradation. The use of the UT5600 strain of E.coli., which is deficient in an outer membrane protease, instead of the non-protease deficient DH5oc strain as an expression host, as well as a 2.5 hour versus overnight induction time with IPTG in attempts to decrease the amount of recombinant protein degradation, only increased the stability of the M B P and GST fusion proteins marginally (data not shown).  3.1.2 Identification of a degradation signal in the cytoplasmic domain of CD45ap by mutational analysis in E. coli. In vitro protein binding assays would be difficult to perform i f CD45ap is expressed as such a highly degraded recombinant protein. Thus, in order to gain insight into the factors controlling the degradation of CD45ap and to achieve a stably expressed recombinant form of CD45ap, deletion analysis of CD45ap was carried out in bacteria to identify the region responsible for its instability. Constructs encoding for three truncated forms of GST-CD45ap, GST-CD45ap (del 52-126) (removal of amino acids 52-126 from the cytoplasmic domain of CD45ap), GST-CD45ap (del 142-197), and GST-CD45ap (del 157-197) were constructed by restriction digests. A schematic representation of the deletions is provided in figure 3.1. The mutational analysis was performed with the GST-CD45ap construct as this protein  54  Figure 3.2. Recombinant CD45ap fusion proteins. D H 5 a E. coli. cells were transformed with constructs encoding for the cytoplasmic domain of CD45ap as an M B P (A), cmyc (B), or GST (C) fusion protein. Cells were grown and induced with IPTG overnight to produce the fusion proteins as outlined in Materials and Methods. Whole cell lysates were separated by SDS-PAGE (10%), transferred to a P V D F membrane, and the presence of the fusion proteins were detected by immunoblotting with the appropriate antiserum against M B P , cmyc, or GST. Whole cell lysates from non-transformed cells were included as a control. As additional controls, lysates from cells transformed with constructs encoding for M B P or GST alone were also included. Molecular mass standards are indicated on the left in kDa.  55  showed a slightly more favorable fusion protein to degradation product ratio than M B P CD45ap. Bacteria transformed with the different GST-CD45ap mutant constructs were induced with IPTG to produce protein and the purification of each GST recombinant protein was carried out as outlined in Materials and Methods. The recombinant proteins were then analyzed by SDS-PAGE (10%) and immunoblotting with an antibody against GST (figure 3.3). Since the GST-CD45ap (del 52-126) deletion construct removed one PEST sequence (the one with the higher PEST-FIND score), implicated in ubiquitin mediated degradation (reviewed in 94), from the cytoplasmic domain of CD45ap, it was anticipated that this deletion would yield a considerable increase in the stability of the fusion protein. Contrary to expectation, GST-CD45ap (del 52-126) showed no improvement in stability and was degraded to levels comparable to that of GST-CD45ap.  On the other hand, the  assessment of the stability of the GST-CD45ap (del 142-197) and GST-CD45ap (del 157197) fusion proteins produced intriguing results.  As seen in figure 3.3, both GST-  CD45ap (del 142-197) and GST-CD45ap (del 157-197) migrated as a single band. Very minor amounts of GST alone degradation products from GST-CD45ap (del 142-197) and GST-CD45ap (del 157-197) were detected.  The fact that these two fusion proteins  exhibited excellent stability and minimal sensitivity to degradation suggests that the removal of the 40 C-terminal amino acid residues (amino acid residues 157-197) from CD45ap is sufficient to prevent the degradation of the GST-CD45ap fusion protein and may indicate the presence of a destabilizing region within the cytoplasmic domain of CD45ap.  56  Figure  3.3.  Mutational analysis of  CD45ap  in E. coli. DH5a E. coli. cells were  transformed with constructs encoding for GST alone, GST-CD45ap, GST-CD45ap (del 52-126) (GST-CD45ap with the removal of amino acid residues 52-126 from the cytoplasmic domain of CD45ap), GST-CD45ap (del 142-197), or GST-CD45ap (del 157197) and induced with IPTG overnight to produce the fusion proteins as outlined in Materials and Methods. Whole cell lysates were separated by SDS-PAGE (10%), transferred to a PVDF membrane, and the presence of the fusion proteins were detected by immunoblotting with antisera against GST. Molecular mass standards are indicated on the left in kDa.  57  Attempts to further localize the destabilizing signal were not as successful. Two additional mutants, GST-CD45ap (del 176-197) and GST-CD45ap (del 186-197), were constructed by P C R and subsequent cloning. A schematic of these fusion proteins is provided in figure 3.1. The residues to be deleted from the GST-CD45ap (del 176-197) and GST-CD45ap (del 186-197) proteins were selected upon examination of the sequence alignment between mouse CD45ap and human L P A P C-terminal sequences (figure 3.4). This alignment illustrates three conserved regions between mouse and human CD45ap beyond amino acid residue 157 that are interrupted by one or two nonconserved residues.  Since important degradation motifs would most likely be  evolutionarily conserved, deletions from amino acid residues 176-197 and residues 186197 were chosen to allow for the assessment of the contributions of each of these three regions to the instability of GST-CD45ap. Nevertheless, GST-CD45ap (del 176-197) and GST-CD45ap (del 186-197) exhibited much greater resistance to degradation than GSTCD45ap but was more degraded than GST-CD45ap (del 142-197) and GST-CD45ap (del 157-197) (figure 3.5).  3.1.3 No significant difference between the half life of CD45ap and CD45ap (del 157-197) in L cells Several proteins involved in cell signaling have been found to be rapidly degraded after phosphorylation in a ubiquitin dependent manner (96 and reviewed in 94). This is not unexpected as proteins involved in signaling may often be short lived in order to achieve a highly regulated response.  Since CD45ap has been implicated in T C R  signaling by potentially playing an adaptor like role, its turnover rate may be critical to  58  CD45ap and LPAP C-terminal Sequence Alignment (del 142-197)  (del 157-197)  (del 176-197)  (del 186-197)  begins here  begins here  begins here  begins here  \  1  1  1  Murine CD45ap DPRDTDSD—GGLGLSSQGPVGSGSSAEALLSDLHAFSGSAAWDDSAGGAGGQGLRVTAL-STOP Human LPAP AEEARDSDTEGDLVLGSPGPASAGGSAEALLSDLHAFAGSAAWDDSARAAGGQGLHVTAL-STOP  t  t  \  /  Conserved  Conserved  Conserved  region 1  region 2  region 3  Figure 3.4. Sequence alignment of the C-terminus of C D 4 5 a p and LPAP. Indicates the presence of three conserved regions beyond amino acid residue 157 of CD45ap, interrupted by one or two non-conserved residues. The locations of the residues where the deletions were performed to construct the GST-CD45ap (del 142-197), GST-CD45ap (del 157-197), GST-CD45ap (del 176-197), and GST-CD45ap (del 186-197) recombinant protein mutants are indicated.  59  O) ^™  1  ,^ CO CN  cn T—  *7 CN  m a> •a a  CD h-  (£> 00 f-  aj •a a  "aJ •o  O  *r Q o  O  O  o  o  CD  O  in  "53 a  re W Q  1— CO  ro m  ro m Q  1— CO  1—  Q.  ro m Q  1— CO  62 —  Figure 3.5. Further mutational analysis of C D 4 5 a p in E. coli. D H 5 a E. coli. cells were transformed with constructs encoding for GST-CD45ap (del 176-186) and GSTCD45ap (del 186-197) and induced with IPTG overnight to produce the fusion proteins as outlined in Materials and Methods. Whole cell lysates were separated by SDS-PAGE (10%), transferred to a P V D F membrane, and presence of the fusion proteins were detected by immunoblotting with antisera against GST. For comparison, cell lysates from cells producing the stable GST-CD45ap (del 157-197) and the unstable GST-CD45ap (del 52-126) fusion proteins were also included. Molecular mass standards are indicated on the left in kDa.  60  the modulation of the T cell response.  In addition, it has been shown that CD45 is  required for CD45ap expression - that is, in the absence of CD45, CD45ap is rapidly degraded (63, 66) and N . Gill and P. Johnson, unpublished data). This suggests that the interaction between CD45ap and CD45 is necessary to prevent its degradation.  It is  possible that the association of CD45ap with CD45 may mask a degradation signal sequence located within the cytoplasmic tail of CD45. Since the expression of various cytoplasmic deletion mutants of CD45ap in bacteria revealed a region in the molecule that was responsible for its degradation in bacteria, it was of interest to determine i f the deletion of this 40 amino acid sequence conferred CD45ap with a longer half-life in eukaryotic cells. To address this, L cells were transfected with CD45ap and CD45ap (del 157-197), the latter of which is a form of CD45ap containing a 40 amino acid C-terminal deletion. The L cell system was chosen as CD45ap is not normally expressed in L cells and this system would allow for the comparison of the half lives of CD45ap and CD45ap (del 157-197) in the absence of endogenously expressed CD45ap, a scenario which would not be possible in normal T cells which express CD45ap. The L cells expressing CD45ap or CD45ap  (del 157-197) were metabolically labeled by  14  C-labeled amino acid  incorporation and cell lysates were collected at after 0, 2, 4, 6, and 8 hour incubations in 14  C label free media. CD45ap was then immunoprecipitated from the lysates and the  samples were analyzed by SDS-PAGE (12.5%) and autoradiography. Surprisingly, both CD45ap and CD45ap (del 157-197) were observed to have similar half lives, 7.6±3.4 and 7.3±2.8 hours, respectively  (figure  3.6).  It is interesting  to note that the  immunoprecipitated CD45ap and CD45ap (del 157-197) ran as two distinct bands and  61  CD45ap Time (hrs):  0  2  4  CD45ap del 157-197 6  8  Time (hrs):  32.5—  0  2  4  6  8  32.5—  Figure 3.6. Half lives of CD45ap and CD45ap (del 157-197) in L cells. (A) L cells were transfected with constructs encoding for CD45ap or CD45ap with a C-terminal 40 amino acid deletion (CD45ap (del 157-197)). CD45ap was subsequently immunoprecipitated from C metabolically labeled lysates (two hour labeling time) from 5 x 10 L cells expressing CD45ap or CD45ap (del 157-197) chased with unlabeled media for 0, 2, 4, 6, or 8 hours. Immunoprecipitates were separated by SDS-PAGE (10%), transferred to a P V D F membrane, and exposed to film at -70°C. Molecular mass standards are indicated on the left in kDa. (B) B y densitometric analysis, CD45ap and CD45ap (del 157-197) half lives were calculated from eight and nine experiments, respectively. Half lives of CD45ap and CD45ap (del 157-197) were found to be 7.6±3.4 and 7.3±2.8 hours, respectively. Graphical analysis of this is provided here. 1 4  6  62  that over time, a decrease in the levels of the upper bands was observed while the lower band levels remained fairly stable.  3.2 Generation of antisera against CD45ap In order to generate antisera against CD45ap for use in the detection of recombinant and cellular CD45ap as well as the immunoprecipitation of CD45ap from cellular lysates, rabbits were subjected to an immunization program using a purified GST fusion protein containing the W W domain of CD45ap (GST-WW). Antisera collected after four injections with GST-WW demonstrated that only GST specific antibodies were produced (data not shown). Thus, in subsequent immunizations of the same rabbits, attempts to produce CD45ap specific antibodies required the use of a CD45ap recombinant protein with a smaller and less immunodominant epitope tag.  A  recombinant 6-His fusion protein containing the cytoplasmic domain of CD45ap with a deletion of 40 C-terminal amino acid residues was constructed and purified as outlined in Materials and Methods. The 6-His-CD45ap recombinant fusion protein was constructed with a 40 amino acid C-terminal deletion to allow the expression of a non-degraded recombinant protein and similar to GST-CD45ap (del 157-197), 6-His-CD45ap (del 157197) could be expressed as a non-degraded recombinant protein (data not shown). The immunization of the rabbits with this protein resulted in the successful production of CD45ap specific antibodies. In figure 3.7, an immunoblot with the prepared antiserum shows the detection of CD45ap in lysates from a murine cell line expressing CD45 (BW 5147+), but not in lysates from cells deficient in CD45 expression (BW 5147-) in which  63  CD45ap  Figure 3.7.  Testing of antiserum generated against CD45ap. Lysates from 1 x 10  6  murine B W 5174 T cells expressing CD45 (BW+) or deficient in CD45 (BW-) were prepared and separated by SDS-PAGE (10%), transferred to a P V D F membrane and immunoblotted with a 1/5000 dilution of antiserum from rabbit WW1 prepared as outlined in Materials and Methods. CD45ap expression was detected in BW+ cells but not in B W - cells where CD45ap expression levels remain below detectable levels due to the absence of CD45. WW2 antiserum performed similarly (data not shown). Molecular mass standards are indicated on the left in kDa.  64  CD45ap expression levels remain below detectable levels due to its rapid degradation in the absence of CD45.  3.3 CD45ap and its effect on CD45 expression, half-life, and transport  3.3.1 Expression of CD45ap results in increased total but not cell surface levels of CD45 in L cells The fact that CD45 is required for stable CD45ap expression (63, 66) and that most CD45 molecules in lymphocytes are found constitutively associated with CD45ap at a 1:1 stoichiometric relationship (64-66), suggests an intimate relationship between CD45 and CD45ap.  The biosynthesis of CD45 is complex with multiple CD45 isoforms  expressed due to alternative exon splicing (reviewed in (153-158).  Significant post-  translational modifications of CD45 that include both N - and O- linked glycosylation are also known to occur (159 and reviewed in 153). Since CD45 associates with CD45ap within minutes of biosynthesis (151), it is possible that CD45ap may act as a chaperone to aid CD45 transport through the E R and Golgi and to the cell surface. However, the fact that CD45ap remains associated with CD45 at the cell surface suggests that CD45ap does not act solely as a chaperone for CD45. As cell surface CD45 levels were seen to be decreased in two out of three CD45ap mouse knock out models (73, 74), it was interesting to examine i f CD45ap could moderate CD45 transport to the cell surface and/or total CD45 levels. To examine the effects CD45ap may have on CD45 expression, L cells previously transfected with CD45 were transfected with CD45ap and CD45ap (del 157-197). The L cell system was chosen  65  as CD45 and CD45ap are not normally expressed in L cells and this system would allow for the examination of CD45 expression and transport in the presence or absence of CD45ap, a scenario which would not be possible in normal T cells which express both of these molecules. As seen in figure 3.8, both the transfected CD45ap and CD45ap (del 157-197) migrated as two distinct bands when analyzed by SDS-PAGE (12.5%) and immunoblotting with antibodies against CD45ap. As a caveat to CD45 and CD45ap not being endogenously expressed in L cells, it was first important to demonstrate the association of these two molecules in L cells. A CD45-CD45ap association in L cells was demonstrated through the coimmunoprecipitation of CD45ap with CD45 from L cells transfected with both of these molecules (figure 3.9). Similarly, CD45ap (del 157197) could be coimmunoprecipitated with CD45 from L cells transfected with these two molecules (figure 3.9). The L cells transfected with CD45 alone, both CD45 and CD45ap, and both CD45 and CD45ap (del 157-197) were analyzed by flow cytometry to examine the cell surface levels of CD45 in each of these cell lines.  F A C S analysis of five clones  transfected with CD45ap and CD45 and six clones transfected with CD45ap (del 157197) and CD45 demonstrated that the cell surface levels of CD45 was not significantly different when CD45ap or CD45ap (del 157-197) was cotransfected with CD45 (figure 3.10). To look at potential differences in the total CD45 levels in these three L cell lines, cell lysates were taken and analyzed by SDS-PAGE (10%) and immunoblotting with antibodies against CD45. Here, it was shown that the total CD45 levels in the cell  66  cn  T—  m aS  3,  a (0  a  ro m  m  •<*  Q  Q  O  O  oa in  m  •<t Q  Q  o  O  O  +  +  o  a> o  «  w H  _l  -J  CD45  175 —  47.5—  CD45ap 32.5-  ^  CD45ap del 157-197  Figure 3.8. Transfection of L cells expressing CD45 with CD45ap and CD45ap (del 157-197). L cells previously transfected with CD45 were transfected with constructs encoding for CD45ap or CD45ap with a 40 amino acid C-terminal deletion (CD45ap (del 157-197)) using Gene Porter2 as outlined in Materials and Methods. Lysates from 1 x 10 cells were prepared and separated by SDS-PAGE (10%), transferred to a P V D F membrane and immunoblotted with antisera against CD45 and CD45ap to check for the expression of both proteins. Non-transfected L cells were included as a control. Molecular mass standards are indicated on the left in kDa. 6  67  CN CO  CN CO  m CO  o  Q O  •  a  Q O  C  0  CO CN CO  o  CD45  CD45ap CD45ap "(del 157-197)  Figure 3.9. Demonstration of an association between CD45 and CD45ap in L cells. CD45 was immunoprecipitated from lysates (prepared in lysis buffer containing 1% Brij97) from 5 x 10 L cells transfected with (a) CD45 and CD45ap or (b) CD45 and CD45ap (del 157-197) as outlined in Materials and Methods. Analysis of the immunoprecipitates by SDS-PAGE (10%), transferring to a PVDF membrane, and immunoblotting with antisera against CD45 and CD45ap demonstrated the coimmunoprecipitation of CD45ap or CD45ap (del 157-197) with CD45. Molecular mass standards are indicated on the left in kDa. 6  68  c.6 c.5 c.4 CD45 + CD45ap (del 157-197) c.3 c.2 c.1 c.5 c.4 c.3 CD45 + CD45ap c.2 c.1 CD45 L cells  "Tirliii II  »•»  B  Cell Surface C D 4 5 Levels in L Cell Transfectants 1.4 1.2  w  1.0  o  0.8 0.6  O  0 4  Su vel  U ra t  D45  CD  5 o >  O  •rt  ro <u  Ct  -j  CD45  CD45 + CD45ap CD45 + CD45ap del 1! 7-197  0.2 0.0  Figure 3.10. Cell surface CD45 levels in L cells transfected with CD45, CD45 and CD45ap, and CD45 and CD45ap (del 157-197). (A) 2 x 10 L cells transfected with 5  CD45 alone, CD45 and CD45ap, or CD45 and CD45ap (del 157-197) were incubated with antibodies against CD45 and subsequently with a FITC conjugated secondary antibody as outlined in Materials and Methods. The cells were then analyzed by flow cytometry to examine the levels of cell surface CD45 in the different cell lines. Five different clones of the cells transfected with CD45 and CD45ap were analyzed while six different clones of the cells transfected with CD45 and CD45ap (del 157-197) were analyzed. (B) Graphical analysis of the data shown in (A). The relative amounts of cell surface CD45 in the cell lines transfected with CD45 and CD45ap or CD45 and CD45ap (del 157-197) were averaged between the clones. The relative amounts of cell surface CD45 in the CD45 and CD45ap or CD45 and CD45ap (del 157-197) co-transfectants compared to that of the CD45 alone transfectant were 1.0±0.2 and 0.8±0.4 fold, respectively. 69  O)  a c ma  T t  Q  O  as ro m  If) T t  Q  o  + T t  Q O  T~  d  —  0)  2, m +  T t  T t  O  O  Q  Q  in  T -  C\| d  t--  d  CN d CD45  175"  47.5-  Actin 32.5"  B  Total CD45 Levels in L Cell Transfectants  Figure 3.11. Total CD45 levels in L cells transfected with CD45, CD45 and CD45ap,  and CD45 and CD45ap (del 157-197). (A) Lysates from 1 x 10 L cells transfected with CD45 alone, CD45 and CD45ap, or CD45 and CD45ap (del 157-197) were prepared and separated by SDS-PAGE (10%), transferred to a PVDF membrane, and immunoblotted with antisera against CD45 and actin. Two clones of L cells transfected with CD45 and CD45ap as well as two clones of CD45 and CD45ap (del 157-197) transfected L cells were analyzed. Molecular mass standards are indicated on the left in kDa. (B) Graphical analysis of the data in (A) and three other experiments. The data in (A) was analyzed by densitometric analysis and the amounts of total CD45 in the cell lines transfected with CD45 and CD45ap or CD45 and CD45ap (del 157-197) were expressed as amounts relative to CD45 levels in cells transfected with CD45 alone. Cells cotransfected with CD45 and CD45ap or CD45 and CD45ap (del 157-197) exhibited total CD45 levels of 2.6±0.8 and 2.3±0.7 times the levels of CD45 in cells transfected with CD45 alone, respectively. 6  70  increased with the cotransfection of CD45ap (2.6±0.8 (n=4) fold increase) or CD45ap (del 157-197) (2.3±0.7 (n=4) fold increase) (figure 3.11).  3.3.2 No significant difference in the half life of CD45 in the presence or absence of CD45ap in L cells To address whether the increase in the total levels of CD45 observed with the cotransfection of CD45ap was due to the ability of CD45ap to moderate the turnover of CD45, half life studies of CD45 in the presence or absence of CD45ap were performed. Since CD45ap associates with CD45 on the cell surface, it was foreseeable that a potential role of CD45ap may be to stabilize cell surface CD45 and prevent its degradation.  L cells transfected with CD45 or both CD45 and CD45ap were  metabolically labeled by S-labeled amino acid incorporation and cell lysates were 35  collected after 0, 24, 48, 72, and 96 hour incubations in S label free media. CD45 was 35  then immunoprecipitated from each of the samples and the immunoprecipitates were analyzed by SDS-PAGE (5%) and autoradiography. Nonetheless, these half life studies indicated that the half life of CD45 in the presence or absence of CD45ap was not significantly different (75±2 (n=4) and 77±6 (n=4) hours, respectively) (figure 3.12).  3.3.3 No significant difference in the transport time (T\n to Endoglycosidase H resistance) of CD45 in the presence or absence of CD45ap in L cells Chaperone proteins are critical to ensure the proper folding and assembly of various proteins before they exit from the ER. It was shown previously that not only does CD45ap associate with Endoglycosidase H (Endo H) sensitive forms of CD45,  71  A  +CD45ap  -CD45ap Time (hrs):  0  24 48  72  96  Time (hrs):  0  24 48  72  96  212 —  212 —  Figure 3.12. Half life of CD45 in the presence or absence of CD45ap in L cells. (A) CD45 was immunoprecipitated from S metabolically labeled lysates from 5 x 10 L cells expressing CD45 alone or CD45 and CD45ap chased with unlabeled media for 0, 24, 48, 72, or 96 hours. Immunoprecipitates were separated by SDS-PAGE (5%), transferred to a P V D F membrane, and exposed to film at -70°C. Molecular mass standards are indicated on the left in kDa. (B) By densitometric analysis, CD45 half lives were calculated from four experiments. The half life of CD45 in the absence and presence of CD45ap was calculated to be 75±2 and 77±6 hours, respectively. Graphical analysis of this is provided here. 35  6  72  suggesting that CD45ap associates with CD45 in the ER, but also that this association occurs within minutes of CD45 biosynthesis (151).  In addition, in two out of three  CD45ap knock out mouse models, ablation of CD45ap results in decreased cell surface levels of CD45 (73, 74). It is thus foreseeable that CD45ap may act as a chaperone to aid in the transport of CD45 through the ER and Golgi and to the cell surface. To address whether the increase in total CD45 levels observed in the presence of CD45ap in L cells was due to the ability of CD45ap to facilitate CD45 exit from the ER and transport to the cell surface, i.e., the accumulation of Endo H resistant forms of CD45, L cells transfected with CD45 alone or both CD45 and CD45ap were metabolically labeled by S-labeled amino acid incorporation and cell lysates were 35  collected after 0, 15, 30, 60, and 120 minute incubations in S label free media. Due to a three minute delay required for the removal of the metabolic label and a requisite wash step prior to cell lysis, there was a 3 minute delay before the lysis of the cells. This is reflected in the labeling of the timepoints in figure 3.13.  CD45 was then  immunoprecipitated from the samples and half of each sample was subsequently subjected to an overnight Endo H digestion. The samples were then analyzed by SDSP A G E (5%) and autoradiography. However, in the end, there was no observed difference in the T1/2 to Endo H resistance of CD45 in the absence or presence of CD45ap (17±6 (n=4) and 18±4 (n=5) minutes, respectively) (figure 3.13).  3.4 C D 4 5 a p  affects T cell signaling  Controversy amongst CD45ap mouse knock out models exists with one model demonstrating that CD45ap-null mice have reduced T cell proliferation upon stimulation  73  A -CD45ap  - "do H (min): 212  —  175—  3 18 33 63 123 3  «*•* w j g * »  m *>»  +CD45ap  +Endo H  E  -Endo H  18 33 63 123  Time (min): 3 18 33 63 123 3 18 33 63 123  212^  * * * * *  175  >  ***  +Endo H  #»•«  —  II  M fl H  j  1 1  Figure 3.13. CD45 transport time in the absence or presence of CD45ap in L cells. (A) CD45 was immunoprecipitated from S metabolically labeled lysates from 5 x 10 L cells expressing CD45 alone or CD45 and CD45ap chased with unlabeled media for 3, 18, 33, 63, or 123 minutes. The immunoprecipitates were then divided in two, of which one half was treated with Endoglycosidase H (Endo H) and the other was not. Subsequently, the immunoprecipitates were separated by SDS-PAGE (5%), transferred to a P V D F membrane, and exposed to film at -70°C. Molecular mass standards are indicated on the left in kDa. (B) B y densitometric analysis, the time for half of the CD45 molecules to become Endo H resistant (T to Endo H resistance) was calculated from four experiments for cells expressing CD45 alone and from five experiments for cells expressing both CD45 and CD45ap. The T to Endo H resistance for CD45 in the absence and presence of CD45ap was calculated to be 17±6 and 18±4 minutes, respectively. Graphical analysis of this is provided here. 35  6  ]/2  1/2  74  and decreased T cell mediated cytolysis of infected cells (72) while two other models demonstrate that the T cell response to foreign antigen remains normal in mice where CD45ap  has  been  knocked  out  (73,  74).  Regulated  phosphorylation and  dephosphorylation of tyrosine residues within various molecules involved in T cell signaling are critical to the activation of a T cell. With the ability of CD45ap to associate with various T C R components such as CD3-<^, CD4, and CD8, the tyrosine phosphatase CD45, the tyrosine kinase Lck, and to a lesser extent, the tyrosine kinase ZAP-70, it was foreseeable that CD45ap might play an adaptor protein like role and modulate some T cell signaling phosphorylation events by modulating the interactions between signaling molecules (76, 77). In order to gain insight into the role of CD45ap in modulating the T cell signal, differences in the tyrosine phosphorylation profiles between T cells expressing endogenous levels of CD45ap and those overexpressing CD45ap in response to CD3 stimulation were examined.  CD3 stimulation using antibodies against CD3  molecules results in the generation of stimulatory signals due to the CD3 antibody mediated clustering of the T C R complex, which is thought to mimic T C R contact with an MHC-peptide complex on an A P C . B W 5147 murine T cells that endogenously express both CD45 and CD45ap were transfected with a construct encoding for cmyc-CD45ap. F A C S analysis and CD45ap immunoblots of cell lysates confirmed that these transfected B W cells expressed increased amounts of CD45ap (figure 3.14).  Equal numbers of cells from both the  parental B W 5147 cells and those overexpressing CD45ap were incubated with an antiCD3 antibody as outlined in Materials and Methods. Cells lysates were collected for lysis at 0, 10, 30, 60, and 120 minute timepoints and analysed by SDS-PAGE (10%).  75  Mir  CD45ap  CD3 stimulation (min):  0  cmyc-CD45ap CD45ap  10  30  60  120  Figure 3.14. Effects of CD45ap overexpression in B W 5174 murine T cells. B W 5147 CD45 positive (BW+) T cells were transfected with a construct encoding for cmycCD45ap expression. (A) 2 x 10 B W 5147 cells (BW+) or B W 5147 cells expressing cmyc-CD45ap (CD45ap) were incubated with antibodies against cmyc and subsequently with a FITC conjugated secondary antibody followed by analysis by flow cytometry as outlined in Materials and Methods. (B) Lysates from 1 x 10 B W 5147 cells (BW+) or B W 5147 cells expressing cmyc-CD45ap (CD45ap) were prepared and separated by SDSP A G E (10%), transferred to a P V D F membrane and immunoblotted with antiserum against CD45ap. (C) 2 x 10 cells expressing endogenous (BW+) or overexpressed (CD45ap) levels of CD45ap were incubated with purified anti-CD3 antibody as outlined in Materials and Methods for 0, 10, 30, 60, and 120 minutes. Lysates were prepared and separated by SDS-PAGE (10%), transferred to a P V D F membrane, and immunoblotted with an anti-phosphotyrosine antibody (4G10). Molecular mass standards are indicated on the left in kDa. 5  6  6  76  Clear differences in the tyrosine phosphorylation profiles between B W cells expressing endogenous levels of CD45ap and those overexpressing CD45ap upon stimulation were observed in the anti-phosphotyrosine immunoblot (figure 3.14). Of particular interest were bands at 120-130, 70, 59, 56 and 40 kDa which became hyperphosphorylated and remained phosphorylated longer in cells overexpressing CD45ap compared to cells expressing endogenous levels of CD45ap. It is also noteworthy to mention that the basal phosphorylation level of proteins in the CD45ap overexpressing B W cells was observed to be slightly higher than B W cells expressing endogenous levels of CD45ap.  3.5 The WW domain of CD45ap interacts with a 50 kDa tyrosine phosphorylated protein Since W W domains have been implicated in binding proline rich sequences (88), or phosphorylated serine or threonine residues (88), the W W domain may provide CD45ap with a site for interactions with other proteins and may allow for its functioning as an adaptor molecule. Further, the fact that CD45ap contains this putative proteinprotein interaction domain coupled with its ability to associate with CD45 and other T C R signaling molecules suggests that it might play an adaptor protein role. To determine if CD45ap participates in any novel interactions with other T cell proteins, a GST recombinant protein containing the W W domain of CD45ap was constructed to test for novel interactions with T cell proteins from T cell lysates.  Purified GST-WW was  incubated with lysates from pervanadate treated T cells and the samples were subsequently analyzed by SDS-PAGE (10%) and an anti-phosphotyrosine (4G10) immunoblot. T cells were treated with pervanadate, which is a potent inhibitor of protein  77  50 kDa interacting protein  Figure 3.15. G S T - W W pull down assay from T cell lysates. Recombinant GST fusion protein containing the W W domain of CD45ap (GST-WW) was incubated with lysates (prepared in lysis buffer containing 1% Tx-100) from 1 x 10 B W 5147 T cells treated with pervanadate as outlined in Materials and Methods. After incubation with the lysates, the GST-WW protein, along with any other interacting proteins was analysed by SDSP A G E (10%), transferred to a P V D F membrane, and immunoblotted with an antiphosphotyrosine antibody (4G10). Proteins interacting with GST alone were included as a control. Molecular mass standards are indicated on the left in kDa. 7  78  tyrosine phosphatases including CD45 (160), in order to facilitate the identification any interacting tyrosine phosphorylated proteins.  This pull down assay demonstrated the  interaction of GST-WW with a 50 kDa tyrosine phosphorylated protein (figure 3.15). Attempts to identify this interacting protein have been unsuccessful.  Immunoblotting  with Lck, Csk, Fyn, WASP, Nek, V A S P , Dok-2 and She antibodies were performed but did not yield conclusive results.  3.6 CD45ap modulates the CD45-Lck interaction The ability of CD45 to associate with and activate Lck is critical to T cell activation (13-16, 161). Since CD45ap has been shown to bind Lck directly (76, 77), it was meaningful to examine the ability of CD45ap to enhance the CD45-Lck interaction in vitro as it has been proposed that CD45ap may aid in "presenting" Lck to CD45 for activation. Having achieved a stably expressed form of recombinant CD45ap, CD45ap (del 157-197), it was possible to examine the in vitro effects CD45ap could have on the CD45-Lck interaction. Using soluble, purified, stably expressed 6-His-CD45ap (del 157197) fusion protein, an in vitro binding assay was performed in collaboration with D. Lefebvre to determine if CD45ap could modulate the interaction between 6-His-CD45 and GST-Lck immobilized on G S H sepharose beads (figure 3.16). In the absence of CD45ap, CD45 bound to Lck as expected.  However, when CD45ap was added at  equimolar amounts as a competitor for Lck binding, CD45ap was shown to decrease the binding of CD45 to Lck by 65±17% (n=3) compared to CD45 binding to Lck in the absence of CD45ap. When five molar amounts of CD45ap was added, CD45ap was able to decrease the binding of CD45 to Lck by 81±11% (n=3) compared to CD45 binding to  79  Q O 0  Q O o  1  i  CN  Lck  Grb2 0  1  1  10  N 1 0 1 5 1  mol GST-fusion  111  01  5 molGstSon  17583-  4— CD45  32.5<— CD45ap  25-  83 • 6247.5-  +— GST - Lck +— GST - Grb2 Coomassie blue  32.525-  B  Effect of CD45ap on the CD45-Lck Interaction 120  1  2  3  4  Molar ratio (CD45ap:CD45)  5  Figure 3.16. Effect of CD45ap on the CD45-Lck interaction. (A) Equimolar amounts of 6-His-CD45 and GST-Lck immobilized on GSH beads were incubated with shaking at 4°C as outlined in Materials and Methods with equimolar or five molar amounts of 6-HisCD45ap (del 157-197). In addition, equimolar amounts of 6-His-CD45 and GST-Lck as well as equimolar amounts of 6-His-CD45ap (del 157-197) and GST-Lck were incubated with shaking at 4°C. The G S H beads with the associated proteins were then washed and separated by SDS-PAGE (10%), transferred to a P V D F membrane, and immunoblotted with an anti-6-His antibody. As a negative control, the experiment was also preformed as above, except with GST-Grb2 used in place of GST-Lck. Molecular mass standards are indicated on the left in kDa. (B) By densitometric analysis of the data from three replicate experiments, CD45ap was shown to decrease the binding of CD45 to Lck by 65±17% compared to CD45 binding to Lck in the absence of CD45ap when added at equimolar amounts to CD45. When 5 molar amounts of CD45ap were added, the binding of CD45 to Lck was decreased by 81±11% compared to the binding of CD45 to Lck in the absence of CD45ap. Graphical representation of this is provided here. 80  Lck in the absence of CD45ap. As a negative control, it was shown that Grb2 had no effect on the CD45-Lck interaction. As CD45ap was found to bind Lck in vivo (63), it was expected that CD45ap would be found to bind Lck in vitro.  Additional in vitro studies performed by D.  Lefebvre using the 6-His-CD45ap (del 157-197) fusion protein have demonstrated that the CD45ap-Lck interaction occurs via the kinase domain of Lck and is independent of Lck phosphorylation state (D. Lefebvre, unpublished results; data not shown).  Also,  Lefebvre has shown that in the presence of CD45ap, the rate of dephosphorylation of the F505 Lck mutant (believed to be constitutively active) by CD45 was significantly decreased while the dephosphorylation of the F394 Lck mutant (believed to be constitutively inactive) by CD45 was only slightly decreased (D. Lefebvre, unpublished results; data not shown).  81  4. Discussion  82  4.1 CD45ap degradation  4.1.1 Achieving stable expression of CD45ap as a recombinant fusion protein in bacteria  In order to study the role of CD45ap in T cell signaling, a protein expression system in bacteria was chosen to express CD45ap as a fusion protein in order to allow for the purification of large amounts of usable protein for use in in vitro assays. Expression of the cytoplasmic domain of CD45ap as a recombinant fusion protein was attempted to allow for the examination of the in vitro interaction between Lck and CD45ap as well as the effect of CD45ap on the CD45-Lck interaction. Attempts to express CD45ap with several different protein tags (MBP, cmyc, and GST) were made as recombinant protein stability and yield can vary greatly between different epitope tag-fusion protein combinations. Certain fusion proteins may be toxic to the host cells or highly susceptible to degradation. As expected, the stability of the fusion proteins, MBP-CD45ap, cmyc-CD45ap, and GST-CD45ap varied. No expression of the cmyc fusion protein was detected while MBP-CD45ap and GST-CD45ap were expressed as highly degraded protein products. Neither the use of the UT5600 strain of outer membrane protease deficient E.coli. nor shorter protein production periods seemed to significantly decrease the amount of fusion protein degradation. Using truncation mutants of GST-CD45ap, it was determined that the removal 40 amino acid residues from the C-terminus of CD45ap (amino acids residues 157-197) was sufficient to stabilize the GST-CD45ap fusion protein that was previously significantly  83  degraded. This suggested that a particular motif present within the region of amino acid residues 157-197 of the cytoplasmic domain of CD45ap may be acting to destabilize CD45ap and promote its rapid degradation in bacteria.  Alternatively, perhaps the full  length CD45ap fusion proteins folded in a manner different to that of the GST-CD45ap (del 157-197) protein, such that it was subject to higher levels of degradation. The fact that the GST-CD45ap (del 52-126) protein remained highly degraded suggests that it was not the primary sequence length of the protein that promoted its degradation as this protein contained less amino acid residues than the stable GSTCD45ap (del 157-197), but possibly the presence of a destabilizing sequence within the last 40 residues of CD45ap. In addition, the fact that a 6-His-CD45ap (del 157-197) fusion protein could be expressed as a stable fusion protein further suggests that a destabilizing motif exists in the last 40 amino acid residues of CD45ap and that the stability of GST-CD45ap (del 157-197) was not specific only to GST CD45ap fusion proteins carrying a C-terminal 40 amino acid residue deletion. Attempts to further localize the destabilizing sequence to the last 21 or 11 amino acid residues of CD45ap were not as successful.  Although being degraded far less  compared to GST-CD45ap, GST-CD45ap (del 176-197) and GST-CD45ap (del 186-197) were shown to be more degraded than GST-CD45ap (del 157-197). Perhaps the partial presence of the putative destabilizing sequence within the last 40 amino acid residues of CD45ap present in the GST-CD45ap (del 176-197) and GST-CD45ap (del 186-197) fusion proteins resulted in the decreased but still active effects of the destabilizing sequence.  84  It would be interesting to attach this 40 amino acid potentially destabilizing motif to a stably expressed protein in bacteria and assess whether its stability is affected.  A  motif that targets proteins for degradation may have practical applications. For example, the fusion of this degradation motif to vaccine proteins may enhance antigen presentation by M H C molecules on APCs and potentially improve the effectiveness of the vaccine.  4.1.2  Assessment of the stability of CD45ap with a C-terminal 40 amino acid  deletion in eukaryotic cells It is not surprising that many proteins involved in signaling are often short lived in order to achieve a regulated response. As mentioned, CD45ap is known to associate with CD45 (62-64), a major participant in T cell signaling, as well as other molecules critical to an effective T cell signal such as Lck and ZAP-70 (76, 77). If CD45ap does indeed play a role in T cell signaling, it may not be surprising that it may contain a destabilizing sequence in order to modulate its activity. As previously mentioned, CD45ap was found to be rapidly degraded in T cells lacking CD45 (63, 66 and N . Gill and P. Johnson, unpublished data), suggesting that the interaction of CD45ap with CD45 may be necessary to prevent its degradation, possibly via CD45 masking a degradation signal sequence located within the cytoplasmic tail of CD45ap. Perhaps, the highly stable GSTCD45ap (del 157-197) recombinant protein has this degradation sequence removed, resulting in the prevention of its degradation in bacteria. As some proteolytic systems are conserved between prokaryotes and eukaryotes, it is not unforeseeable that the potential degradation sequence within amino acid positions 157-197 of CD45ap may be recognized by both prokaryotic and eukaryotic degradation systems.  85  The presence of one or two PEST sequences and a W W domain in the cytoplasmic domain of CD45ap leads to speculation that CD45ap is specifically targeted for degradation, perhaps though the ubiquitin-proteasome pathway. It is important to note though that to date, there has been no post translational tagging mechanism described in prokaryotes that is equivalent to the eukaryotic ubiquitin system. Thus, i f CD45ap is indeed degraded through the ubiquitin-proteasome pathway in eukaryotes, speculation that the mechanism through which CD45ap is degraded is conserved between prokaryotes and eukaryotes may be difficult. However, one tagging mechanism to effect the elimination of protein fragments synthesized from damaged m R N A has been discovered in E. coli. (162) and one of the genes of the Microbin C7 bacterial microcin antibiotic system found in some Enterobacteriaceae strains, MccB, has been shown to have homology to the eukaryotic ubiquitin activating enzyme U B A 1 (163-166).  In  addition, even though CD45ap contains one or two PEST sequences and a W W domain, this does not guarantee that it is degraded through a specific protein targeting degradation system and not through a pathway that is conserved between eukaryotes and prokaryotes. A short summary of the evidence for similarities between prokaryotic and eukaryotic proteases follows. Generally, in prokaryotes, substrate selection by proteases is handled by approximately six energy dependent proteases, each of which is a large oligomeric assembly whose proteolytic site is hidden in a chamber. Entry to this site is believed to be mediated by ATPases that are also responsible for substrate specificity (reviewed in 167). Nevertheless, many specific proteases found in E. coli. are well conserved in both prokaryotes and eukaryotes. For example, the energy-dependent protease Lon is found in  86  E. coli. and in the mitochondria of eukaryotic cells (168-170), the A A A class of prokaryotic ATPases contains some subunits of the 26S proteasome (171), and the HslV prokaryotic energy-dependent protease has homology to the proteasome subunit of the eukaryotic ATP-dependent proteasome (172, 173). Also, many misfolded, mutant, and truncated proteins are often rapidly degraded in similar fashions in both prokaryotes and eukaryotes (174-176). Finally, bacteria energy dependent proteases such as ClpAP may have the ability to recognize and degrade certain N-terminal motifs (130, 177) and the recognition and degradation of proteins that contain specific C-terminal motifs, such as the P22 Arc repressor protein, have also been examined (178-180). As the GST-CD45ap (del 157-197) protein was determined to be the most stable mutant form of CD45ap fusion protein examined in bacteria, L cells were transfected with both CD45ap and CD45ap (del 157-197) to compare the stability of the two proteins in an eukaryotic system.  Unfortunately, there was no significant difference observed  between the half lives of the full length CD45ap protein and CD45ap (del 157-197). It is noteworthy to mention that the half life study of these two proteins were performed in the L cell line which is a fibroblast cell line and not a lymphocyte cell line where CD45ap has been previously found to be expressed at lower levels and have a shorter half life in the absence of CD45 (63, 66 and N . Gill and P. Johnson, unpublished data). CD45ap and CD45 are not normally expressed by L cells.  Also,  It is possible that the  mechanisms or proteins responsible for the rapid degradation of CD45ap in lymphocytes are not present in fibroblasts. It would be useful to compare the half lives of full length CD45ap in the presence and absence of CD45 in L cells to demonstrate whether the  87  degradation rate of CD45ap is affected by the presence of CD45 in fibroblasts as it is in lymphocytes. Currently, it is unclear as to whether the potential degradation signal in the last 40 C-terminal amino acids of CD45ap is specific to bacteria or if it is also functional in eukaryotes. Clearly, various proteolytic systems are conserved between prokaryotes and eukaryotes and thus it is foreseeable that a degradation signal functional in prokaryotes would still be functional in eukaryotes. It is also possible that the mechanism which is responsible for CD45ap degradation is not conserved between prokaryotes and eukaryotes and that the full length recombinant CD45ap protein expressed in bacteria folds in such a way that lends itself to be susceptible to proteolysis whereas the way in which the CD45ap (del 157-197) mutant protein folds leads it to be resistant to degradation by the same protein(s). However, the fact that 6-His-CD45ap (del 157-197), which would most likely fold in a different manner than GST-CD45ap (del 157-197), is also stably expressed, suggests that it is the removal of a destabilizing motif rather than a conformational change arising from the 40 amino acid C-terminal deletion that leads to the acquired stability of CD45ap fusion proteins with a 40 amino acid C-terminal deletion. The increase in half life that a 40 amino acid C-terminal deletion from CD45ap confers the molecule in lymphocytes remains to be examined. The rapid degradation of CD45ap in the absence of CD45 has begged for answers to questions asking which features of CD45ap cause it to be short lived, if this degradation can be moderated, and which proteolytic system is responsible for its degradation. Attempts to isolate a potentially destabilizing sequence in CD45ap should continue with the assessment of the stability of a CD45ap mutant with the potentially  88  phosphorylated "stronger" PEST sequence (residues 94-127) removed. In addition, it may also be helpful to isolate the mechanism by which CD45ap is degraded utilizing specific proteolytic inhibitors such as NH4CI and chloroquine for the lysosome or peptide ketones or lactacystin for the 26S proteasome.  In addition, the use of inhibitors of  specific proteases, for example, leupeptin for serine and cysteine proteases, calpain inhibitors, and assessment of the ubiquitination status of CD45ap may also aid in isolating the mechanism responsible for CD45ap degradation. It  is  interesting  to  note  that  CD45ap  and CD45ap  (del  157-197)  immunoprecipitated from L cells during the half life studies showed both CD45ap and CD45ap (del 157-197) migrating as two distinct bands with an approximate 1-2 kDa difference in their apparent molecular weights when analyzed by SDS-PAGE. In the half life studies, it was noticed that during the eight hour chase period with unlabeled amino acid supplemented media, the levels of the higher apparent molecular weight radiolabeled bands decreased while the lower band levels remained approximately the same.  This  suggests that the turnover rates of the higher apparent molecular weight forms of CD45ap and CD45ap (del 157-197) are higher than that of their respective lower apparent molecular weight forms. The migration of CD45ap when analyzed by SDS-PAGE has always been anomalous with its observed apparent molecular weight of around 32 kDa when its predicted molecular weight is 19 kDa. Treatment of CD45ap with phosphatases does not alter its migration (63) and it is not known to be glycosylated. It has also been observed that CD45ap and L P A P can migrate as more than one distinct form when subjected to SDS-PAGE. CD45ap has been observed to migrate at the following apparent molecular  89  weights, probably due to differential phosphorylation of the different forms: 29 kDa and 30 kDa (181); 29 kDa, 30 kDa, 31 kDa and 32 kDa (62, 63); and 32 kDa-33 kDa (104). Interestingly, in a human T cell line, L P A P was observed to alternate between 29 kDa and 32 kDa forms in resting cells and 30 kDa and 31 kDa forms in stimulated cells (62, 63).  In this case, it was shown that the treatment of all four forms of CD45ap with  alkaline phosphatase, resulted in the migration of CD45ap as a single 32 kDa band when analysed by SDS-PAGE, suggesting that the 29 kDa, 30 kDa, and 31 kDa CD45ap forms were the result of differential phosphorylation of L P A P (63). The cytoplasmic domain of CD45ap contains two potential PEST sequences, with the more N-terminal PEST sequence having a much higher potential to be a true PEST sequence than the other one as scored by PESTfmd (94).  Since at least one PEST  sequence bearing potential phosphorylation sites is present on both CD45ap and CD45ap (del 157-197) as well as other potential phosphorylation sites present in these two proteins, perhaps the two different forms of CD45ap and CD45ap (del 157-197) observed in the half life studies are due to differential phosphorylation of the two forms, one of which is more sensitive to degradation. Since both CD45ap and CD45ap (del 157-197) appear to be potentially phosphorylated in the same manner as evidenced by the appearance of both CD45ap and CD45ap (del 157-197) as two distinct bands 1-2 kDa apart when analyzed by SDS-PAGE, this suggests that phosphorylation of CD45ap in eukaryotic cells may only occur N-terminal to residue 157 in CD45ap. In the CD45ap (del 157-197) protein transfected into L cells, the second PEST sequence is mostly ablated, but the first "stronger" PEST sequence remains as it does in the full length CD45ap protein. Thus, both CD45ap and CD45ap (del 157-197) still  90  contain at least one PEST sequence allowing CD45ap degradation via PEST sequence phosphorylation and ubiquitination i f in fact that is how CD45ap is degraded in L cells. In bacteria, it is likely that the more N-terminal "stronger" PEST sequence does not play a role in the rapid degradation of CD45ap as the GST-CD45ap (del 52-126) protein, which does not contain this PEST sequence, was subject to rapid degradation in bacteria.  4.2 Potential roles for CD45ap in T cell signaling  4.2.1 CD45ap overexpression studies Due to the controversial nature of the CD45ap knock out models with one group finding that CD45ap is critical for efficient T cell response to stimulation (72) while two other groups found that CD45ap is not necessary for an efficient T cell response (73, 74), the potential role of CD45ap in T cell signaling has became a greater mystery. Yet the exclusive expression of CD45ap on lymphocytes (66, 70), its tight association with CD45 (62-66), and its interactions with key signaling molecules such as Lck and ZAP-70 (76, 77) have teased researchers to explore its raison d'etre. The overexpression of CD45ap in B W 5147 murine T cells has offered some insight into the role of CD45ap in the T cell response. Prolonged phosphorylation and hyperphosphorylation of T cell proteins were observed in lymphocytes overexpressing CD45ap compared to those expressing endogenous levels CD45ap. Proteins of particular interest that were hyperphosphorylated and phosphorylated for extended periods were seen at apparent molecular weights of 120130 kDa, 70 kDa, 59 kDa, 56 kDa, and 40 kDa, which may represent the pl20P  130 7pl25 Ca  Fak  /pll5  Pyk2  complex, p70  ZAp  - , p59 , p56 , and p40 70  91  fyn  lck  LAT  , respectively.  The Cas/Fak/Pyk2 complex of phosphorylated proteins together with the phosphatase PTP-PEST has been implicated in focal adhesion formation and the regulation of cell migration and spreading (182, 183). Cas is an adaptor protein involved in focal adhesion formation while Fak and Pyk2 are protein tyrosine kinases that colocalize with integrins in cellular focal adhesions. However, the physiological relevance of the Cas/Fak/Pyk2 complex to the T cell response is unclear as focal adhesions do not exist in immune cells. The other hyperphosphorylated proteins critical to T cell signaling, ZAP-70, Fyn, Lck, and L A T , have been previously discussed.  When overexpressed in lymphocytes,  CD45ap enhances T cell signaling as evidenced by the prolonged phosphorylation and hyperphosphorylation of various proteins involved in signaling.  This suggests that  CD45ap plays a role in promoting efficient T cell signaling. Perhaps by moderating the interactions between various proteins involved in T cell signaling, CD45ap may be involved directly or indirectly in the facilitation of the phosphorylation of these proteins as well as in the maintenance of their phosphorylated state. Since CD45ap has been previously shown to associate with ZAP-70 (76, 77), coupled with the fact that a 70 kDa band was seen to be hyperphosphorylated and phosphorylated for extended periods in lymphocytes overexpressing CD45ap, it would be interesting to assess if CD45ap overexpression results in an enhancement of ZAP-70 association with CD3<^ and/or enhancement of ZAP-70 phosphorylation and activation. Increased CD45ap association with ZAP-70 upon stimulation has been previously observed (76) suggesting that CD45ap-ZAP-70 interactions may be important in T cell activation.  Likewise, examinations of the CD45ap-Lck association, states of Lck  92  activation, and the CD45-Lck interaction in cells overexpressing CD45ap would be insightful.  4.2.2 CD45ap and its effects on the CD45-Lck interaction Utilizing the stably expressed CD45ap (del 157-197) recombinant protein, it was shown in collaboration with D . Lefebvre that CD45ap could indeed act as an adaptor molecule in vitro and modulate the CD45-mediated dephosphorylation of Lck. It was first demonstrated that CD45ap and Lck could interact in vitro and that the kinase domain of Lck was necessary and sufficient for this interaction to occur. This confirmed the in cell data by Veillette et. al. (77) and Motoya et. al. (76), which demonstrated that an association between CD45ap and Lck could occur.  However, Veillette et. al. also  demonstrated in the cell that the F505 Lck mutant (believed to be constitutively active) showed an increased association with CD45ap compared to the F394 Lck mutant (believed to be constitutively inactive), while D. Lefebvre showed no difference between the in vitro direct binding of CD45ap to F505 or F394 Lck. This may suggest that other molecules in the cell may be necessary to effect the increased binding of activated Lck to CD45ap. Interestingly, it was also demonstrated that CD45 and CD45ap competed for binding to Lck in vitro and that CD45ap was able to outcompete CD45 for association with Lck. In an in vitro competition binding assay, at 1:1 and 5:1 molar ratios of CD45ap to CD45, CD45 binding to Lck was decreased by 65±17% and 81±11%, respectively, when compared to CD45 binding to Lck in the absence of CD45ap. It was also shown in vitro by D. Lefebvre that CD45ap could significantly decrease the rate at which CD45  93  could dephosphorylate the F505 Lck mutant (believed to be constitutively active) but could only slightly decrease the rate at which CD45 could dephosphorylate the F394 Lck mutant (believed to be constitutively inactive) (D. Lefebvre, unpublished data). Given that CD45 mediated dephosphorylation of inhibitory tyrosine residues is necessary for Src-family P T K activation and subsequent signal transduction (6, 13, 14, 154) and that CD45 is also capable of down regulating T cell signaling events by counteracting Srcfamily signaling activity by dephosphorylating Src-family activating tyrosine residues as well as Src-family substrates such as ZAP-70 and the CD3-<^ chain (184, 185), a regulation of CD45 activity is essential to achieve a strong yet controlled signal. Taken together, the results of the in vitro binding assays involving recombinant CD45ap (del 157-197), Lck, and CD45 suggest that not only does CD45ap serve as an effective modulator of CD45 access to Lck by being a stronger competitor for Lck than CD45, but also serves as a promoter of T cell activation by its ability to promote the CD45 -mediated dephosphorylation (and potential subsequent activation) of inactive Lck rather than the dephosphorylation (and potential subsequent inactivation) of active Lck.  This model  correlates nicely with the CD45ap overexpression studies in T cells that suggest a positive role for CD45ap in effecting the T cell signal. Some controversy does exist, however, in the role of CD45ap in the moderation of the CD45-Lck interaction between the findings described above and the findings by other groups. One CD45ap mouse knock out model demonstrated decreased CD45-Lck association upon stimulation (72) while two other models showed that this interaction was not affected in the absence of CD45ap (73, 74). In addition, studies in the human Jurkat T cell line, demonstrated that the absence of L P A P did not alter the CD45-Lck  94  interaction or Lck enzymatic activity, suggesting that L P A P is not important in moderating the interaction between CD45 and Lck (75). It must be noted that the in vitro binding assays described above using the CD45ap (del 157-197) recombinant protein were performed using a form of CD45ap that does not contain an extracellular or transmembrane domain, and carried a C-terminal deletion of 40 amino acid residues in order to stabilize its expression. This truncated recombinant CD45ap protein may behave differently from full length CD45ap, but it was used as it was the best tool available for in vitro studies involving CD45ap. Efforts are now ongoing to determine i f the full length CD45ap recombinant protein, although expressed as a somewhat degraded protein, behaves similarly to the CD45ap (del 157-197) protein in in vitro binding assays involving CD45 and Lck. Methods to minimize the degradation of the full length CD45ap recombinant protein such as using protease deficient bacterial strains, adding protease inhibitor cocktails, using shorter protein production times, and utilizing lower temperatures during protein production will be applied.  4.2.3 Novel interactions of CD45ap with other T cell proteins With the presence of a W W domain, a putative protein-protein interaction domain, within the cytoplasmic domain of CD45ap, speculation that CD45ap interacts with other T cell proteins is tempting. Using a GST fusion protein containing the W W domain of CD45ap, the W W domain of CD45ap was observed to interact with a tyrosine phosphorylated T cell protein with an apparent molecular weight of 50 kDa. Speculations on the identity of this interacting protein based on its apparent molecular weight, the fact that it was tyrosine phosphorylated, and the fact that it might be involved  95  in T cell signaling lead to immunoblotting with various antibodies in an attempt to identify the interacting band. Immunoblotting with antibodies against with Lck, Fyn, W A S P , Nek, V A S P , Dok-2, and Csk did not yield conclusive results. Future attempts at identifying this interacting protein using a yeast 2-hybrid screen employing the W W domain of CD45ap as bait will be attempted. It may also be interesting to overexpress the W W domain of CD45ap in T cells to produce a CD45ap dominant negative phenotype in order to give further insight into the role of CD45ap and this unidentified 50 kDa protein in T cell signaling.  This overexpressed W W domain protein would  potentially serve to bind up and sequester most of the available unidentified 50 kDa interacting protein molecules (and possibly other CD45ap interacting molecules) away from interactions with other proteins, thus leading to a CD45ap dominant negative phenotype.  4.3 CD45ap and its effect on CD45 expression, transport, and turnover  4.3.1 CD45ap and its effect on cell surface and total CD45 levels The intimate relationship between CD45 and CD45ap can be evidenced by the fact that almost all CD45 molecules in lymphocytes are found associated with CD45ap (64-66) and that CD45 is required for stable CD45ap expression. The CD45ap-CD45 association is also known to occur within minutes of CD45 biosynthesis (151). These facts, along with the complex nature of CD45 biosynthesis, with CD45 expressed as multiple isoforms (reviewed in 153-158) and modified by post-translational glycosylation (159 and reviewed in 153), led to speculation that CD45ap may serve as a CD45  96  chaperone, prompting examinations of the effect of CD45ap on CD45 expression, transport, and half life. L cells were transfected with CD45, CD45 and CD45ap, or CD45 and CD45ap (del 157-197) and chosen as a system for these studies as the absence of endogenously expressed CD45 and CD45ap in L cells would allow for the examination of CD45 expression characteristics in the absence or presence of CD45ap in the transfected cell lines. It was then established that CD45 and CD45ap could associate in these transfected L cell lines as they do in lymphocytes. Subsequent analysis of CD45 expression by flow cytometry demonstrated that CD45 cell surface expression was not affected by the presence or absence of CD45ap or CD45ap (del 157-197). However, it was observed that the cotransfection of CD45 expressing L cells with CD45ap or CD45ap (del 157-197) resulted in a 2.6±0.8 or 2.3±0.7 fold increase in the levels of total CD45 present in the cells, respectively. Decreased total levels of CD45 in the absence of CD45ap have been previously observed (63, 72, 73, 75, 105, 151).  4.3.2 CD45ap and its effect on CD45 transport Due to the close relationship between CD45ap and CD45, it is possible that CD45ap may act as a chaperone to aid CD45 transport through the E R and Golgi and to the cell surface. However, the fact that CD45ap remains associated with CD45 at the cell surface suggests that if CD45ap were to serve as a CD45 chaperone, this would not be its sole function. The hypothesis that CD45ap functions as a CD45 chaperone was tested by evaluating the Endo H sensitivity of radiolabeled CD45 molecules in presence or absence of CD45ap over a period of time.  As it was previously found that CD45ap could  associate with Endo H sensitive forms of CD45 (151), that CD45 cell surface levels were  97  decreased in two CD45ap mouse knock out models (64, 73), and that the presence of CD45ap resulted in increased total levels of CD45 in L cells, speculation that CD45ap could aid CD45 exit from the ER and transport to the cell surface was not unfounded. Nevertheless, the T1/2 of CD45 to become Endo H resistant was found to be not significantly different in the absence or presence of CD45ap (T1/2 to Endo H resistance of 17±6 and 18±4 minutes, respectively).  With respect to transmembrane protein  complexes that associate through their respective transmembrane domains, these complexes are generally formed either to allow chaperones to assist in the transport of the complex through the E R and to the cell surface or to serve as a cooperative signaling complex at the cell surface. For example, each M H C class I molecules is complexed with and retained in the E R by the chaperone calnexin via a transmembrane interaction until the M H C molecule associates with a peptide and P2-microglobulin. Only then is the complex is allowed to exit to the cell surface (186-188). M H C class I molecules and calnexin are never found associated at the cell surface. In comparison, the T C R chains and various CD3 components assemble via their transmembrane domains in the E R and migrate to the cell surface.  Any missing antigen receptor subunit causes the entire  complex to be retained in the ER (189-192). Since CD45ap associates early with CD45 in the E R but does not seem to assist in CD45 exit from the E R as evidenced by the similar times for CD45 to achieve Endo H resistance in the presence or absence of CD45ap, this suggests that CD45ap is complexed with CD45 not to assist in its transport to the cell surface but to serve in effecting the efficient transduction of the T cell signal in conjunction with CD45 at the cell surface. In addition, the fact that CD45 and CD45ap associate early in the E R while CD45 transport to the cell surface seems to be unaffected  98  by the absence of CD45ap suggests that it may be CD45ap that is retained in the E R and degraded in the absence of CD45, resulting in reduced CD45ap levels in CD45 deficient T cells.  4.3.3 CD45ap and its effect on CD45 half life As described above, it was found that when L cells transfected with CD45 were cotransfected with CD45ap, this resulted in an increase in the total amount of CD45 present in these cells.  Considering this data and the fact that most CD45 molecules  associate with CD45ap shortly after biosynthesis (151) and continue to be associated with CD45ap at the cell surface (64-66), it is not unreasonable to suggest that CD45ap may serve to stabilize CD45 expression and decrease its turnover rate. In spite of this, in L cells, the half life of CD45 was found to be not significantly different in the absence or presence of CD45ap at 75±2 and 77±6 hours, respectively. Thus, CD45ap does not seem to affect the turnover rate of CD45. It is interesting to note that the half life of CD45 (-76 hours) expressed alone is much greater than that of CD45ap (~7.5 hours) expressed alone in L cells. This suggests that surface CD45 molecules may dissociate from attached CD45ap molecules and reassociate with newly synthesized CD45ap molecules several times before being turned over. Alternatively, perhaps the expression of both CD45 and CD45ap in L cells may increase the half life of CD45ap such that both CD45 and CD45ap are turned over at similar rates. Tests are underway to examine which, if any, of these two scenarios is likely to be true.  99  Despite similarities in CD45 half life and transport time in the presence or absence of CD45ap, the finding of increased total levels of CD45 with the cotransfection of CD45ap remains intriguing. It has been demonstrated before that subcellular pools of CD45 exist and that these CD45 pools may not be brought to the cell surface to participate in cell signaling until the cells are stimulated. For example, CD45 expression at the cell surface of cytotoxic T cells was found to increase immediately upon phagocytic events, suggesting that a pool of subcellular CD45 that can be exposed upon demand at the cell surface exists (193). In addition, it was observed that cell surface CD45 levels in neutrophils could be increased under stimulatory conditions (194). It is possible that CD45ap aids in the maintenance of subcellular CD45 pools that are exposed at the cell surface at the initiation of T cell signaling. Perhaps in the L cell model, in the presence of CD45ap, the subcellular pools of CD45, but not cell surface CD45 levels are increased, leading to the observed increase in total but not cell surface CD45 levels in the presence of CD45ap. It is interesting to revisit the CD45ap overexpressing B W T cells at this point. In these cells overexpressing CD45ap, perhaps the presence of more CD45ap molecules allows the maintenance of larger subcellular CD45 pools.  These larger  cytoplasmic CD45 pools may lead to more efficient and stronger T cell signaling (evidenced by increased and prolonged tyrosine phosphorylation of signaling proteins) observed in these CD45ap overexpressing cells due to more CD45 molecules being available to dephosphorylate and activate key signaling proteins.  As an alternate  explanation, since not all CD45 molecules (approximately 30%) are associated with CD45ap in T cells expressing endogenous levels of both molecules (65), perhaps CD45ap overexpression in T cells allows for the formation of more CD45-CD45ap complexes to  100  function in signaling. Finally, the results of the effects of CD45ap on C D 4 5 half life and transport in L cells suggest that the hyperphosphorylation and prolonged phosphorylation of T cell proteins observed in the BW cells overexpressing CD45ap are probably not due to any effects CD45ap may have had on C D 4 5 half life or transport to the cell surface.  101  5. Conclusion  102  Although the current role of CD45ap in T cell signaling remains unknown, the lymphocyte exclusivity of CD45ap expression coupled with its intimate affiliation with CD45 and its ability to associate with Lck and ZAP-70 underscore its importance. The work presented in this thesis has continued to fuel interest into the elucidation of the function of CD45ap by not only outlining interesting characteristics regarding its degradation but also further implicating it in T cell signaling. By mutational analysis, it was shown that a 40 amino acid deletion from the Cterminus of CD45ap was sufficient to stabilize the expression of CD45ap in bacteria, which as an undeleted recombinant fusion protein was highly degraded. As CD45ap was found to be rapidly degraded in T cells deficient in CD45, possibly due to the exposure of a degradation signal in the absence of CD45, it was thought that a potential degradation signal in CD45ap defined by mutational analysis in bacteria may also be responsible for mediating CD45ap degradation in eukaryotic cells. Although many proteolytic systems are conserved between prokaryotes and eukaryotes, the deletion of 40 amino acid residues from the C-terminus of CD45ap did not affect the half life of the molecule in L fibroblast cells. Differences between prokaryotic and eukaryotic degradation systems as well as potential differences between the pathways and proteins responsible for CD45ap degradation in fibroblasts and lymphocytes may be possible explanations as to why the potentially active degradation signal in bacteria was found to be not functional in L cells. Studies are ongoing to determine if the 40 amino acid C-terminal deletion from CD45ap confers the molecule with a longer half life in lymphocytes, the pathways through which CD45ap is degraded, and if other regions of CD45ap may be responsible for its rapid degradation in the absence of CD45.  103  With the expression of a stable recombinant form of CD45ap, it was shown in vitro that CD45ap could outcompete CD45 for binding to Lck as well as significantly decrease the rate at which CD45 could dephosphorylate the F505 Lck mutant (believed to be constitutively active) but only slightly decrease the rate at which CD45 could dephosphorylate the F394 Lck mutant (believed to be constitutively inactive). Together, this suggests that not only can CD45ap serve as an effective modulator of the interaction between CD45 and Lck by being a stronger competitor for Lck binding than CD45, but also serve as a promoter of T cell activation by encouraging the CD45-mediated activation rather than deactivation of Lck. To further support the role of CD45ap in promoting T cell activation, B W 5147 T cells overexpressing CD45ap displayed prolonged phosphorylation and hyperphosphorylation of various T cell proteins upon stimulation compared to cells expressing endogenous levels of CD45ap.  Another  possible route through which CD45ap may promote T cell activation may be through the interaction of the W W domain of CD45ap with a currently unidentified 50 kDa tyrosine phosphorylated protein. Efforts to identify this interacting protein are ongoing. The fact that CD45 associates with CD45ap within minutes of biosynthesis and that CD45 levels were seen to be decreased in two CD45ap mouse knock out models prompted the investigation of the possibility that CD45ap may act as a chaperone and assist CD45 transport to the cell surface. However, in L cells, the transport rate (T1/2 to Endo H resistance) of CD45 was found to be not significantly different in the presence or absence of CD45ap.  As members of transmembrane protein complexes generally  associate via their transmembrane domains either to allow chaperones to assist in the transport of the complex to the cell surface or to function as a cooperative signaling  104  complex at the cell surface, the fact that CD45ap does not affect the transport rate of CD45 further supports its role as a functional member of a signaling complex together with CD45 rather than a CD45 chaperone. After the transfection of L cells with CD45 and CD45ap, it was noticed that total but not cell surface CD45 levels were increased in the presence of CD45ap, giving rise to speculation that CD45ap may allow for the maintenance of larger subcellular pools of CD45, leading to more efficient T cell signaling. The increased total levels of CD45 in the presence of CD45ap also led to the examination of the effect that CD45ap may have on the half life of CD45, but nonetheless, the half life of CD45 in the presence or absence of CD45ap was found to be not significantly different in L cells.  However, the  significant difference between the half lives of CD45 and CD45ap in L cells suggests that CD45 molecules may dissociate from attached CD45ap molecules and reattach to newly synthesized CD45ap molecules several times before being turned over.  Studies are  underway to investigate this scenario. 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