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Characterization of protein tyrosine phosphatase E Melhado, Ian Granville 1999

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CHARACTERIZATION OF PROTEIN TYROSINE PHOSPHATASE e by Ian Granville Melhado B.Sc., Simon Fraser University, 1988  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Medicine)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 1999 © Ian Granville Melhado, 1999  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  fflKDlcZi t^-P-  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ii  Abstract  Protein tyrosine phosphatases have been shown to play an important role in immune cell regulation. This study focused upon identifying PTPases that were regulated during inflammation. Using PCR mediated differential hybridization PTPs was identified as a candidate PTPase mRNA upregulated by pro-inflammatory stimuli. Northern blotting analysis confirmed that the message for this PTPase was induced by a limited number of stimuli in cells of the monocyte/macrophage lineage. With the development of highly specific polyclonal antibodies, monocyte/macrophage derived PTPs was observed as a 72/74 kDa doublet. p72/74PTPs was found to possess PTPase activity in vitro, predominantly within the cytosol, and its expression was observed to be regulated during cellular differentiation. Further characterization of PTPs indicated that the PTPase was phosphorylated  and tyrosine phosphorylation could be induced in vivo. Tyrosine  phosphorylation was found to induce the association of the small adapter protein GRB2 with PTPs in pervanadate treated cells. These observations and others pointed to a novel isoform of the previously described transmembrane PTPs PTPase.  An in vitro analysis of recombinant PTPs was undertaken to characterize the PTPase activity of the enzyme. This analysis revealed that the PTPase had a pH optimum  Ill  of 5.6. PTPs exhibited some substrate specificity especially when compared to other recombinant PTPases such as CD45 and PTPa. From the preceding analysis and the fact that the closely related PTPa appears to show specificity for pp60 ' , the src family c src  kinases were the signaling proteins initially investigated as potential in vivo substrates of PTPs.  By utilizing cDNAs encoding both transmembrane and non-transmembrane isforms of PTPe, PTPases were introduced into mammalian cells. In the case of transmembrane PTPe, various mutants were characterized in vivo. These studies revealed that plOO/HOPTPs is a highly glycosylated membrane protein, found predominantly at cell-cell junctions. While plOO/1 lOPTPs was capable of associating with and modulating the phosphotyrosine content and kinase activity of the src family kinase members pp60 " c  src  and pp59*" p72/74PTPs was not.  This phenomenon may be explained by the  observation that the localization of p72/74PTPe was to the nucleus and not the plasma membrane where src family kinases are found.  Finally, the PTPs homologue was reported to contain a SH3 binding site, however, this site was not conserved in PTPs. A proline residue was introduced at position F l 12 and resulted in the reconstitution of an SH3 domain-binding site thus turning PTPe into a PTPa like molecule. PTPsF112P exhibited altered substrate specificity showing increased activity towards src family kinases and reduced activity towards the in vivo substrate Crkll.  Table of Contents  Title Page Abstract  ii  Table of Contents  iv  List of Figures and Tables  vii  Abbreviations  xiii  Acknowledgements  xv  Chapter 1 — Introduction  1  Chapter 2 - Materials and Methods  17  2.1  Materials  17  2.2  Buffers  17  2.3  Rapid mRNA purification  18  2.4  Northern Blot  19  2.5  Differential hybridization screening  19  2.6  Cloning of PTPs intracellular domains  20  2.7  Recombinant expression of PTPs, PTPa, and CD45  21  intracellular domains 2.8  PTPase purification  22  2.9  Production of PTP mutant cDNAs  22  2.10  Antibodies  23  2.11  Cell culture and transfection  24  2.12  Immunoprecipitation and immunoblotting  26  2.13  Immunoflourescence  27  2.14  Surface Biotinylation  27  2.15  Malachite green solution preparation  28  2.16  Inorganic phosphate standard curve  28  2.17  Phosphotyrosine containing peptides  28  2.18  Phosphatase Assays  29  2.19  Liquid Chromatography/Mass Spectroscopic analysis  30  2.20  Kinase assays  31  Chapter 3 - The Screen for PTPases Regulated by IFNy and PTPe  32  Expression in Cells of the Monocyte/Macrophage Lineage 3.1  Differential hybridization Screening System  32  3.2  Northern Blot Analysis of PTPe Expression.  39  3.3  Expression of PTPe protein in hemopoietic cells  43  3.4  p72/74 possesses PTPase activity  52  3.5  Liquid Chromatography/Mass Spectrometric analysis of p74  54  3.6  Expression of the cDNA encoding non-transmembrane PTPe  55  3.7  Phosphorylation of PTPe  58  3.8  Association of small adapter proteins with PTPe  61  3.9  Summary  63  Chapter 4 - Bacterial Expression and in vitro substrate specificity of  65  PTPe and Substrate specificity of native PTPe 4.1  Bacterial expression of PTPases  65  4.2  Bacterial expression of the cytosolic region of PTPe  67  4.3  Purification of PTPases  68  4.4  Synthetic substrate preparation  70  4.5  In vitro characterization and substrate specificity of PTPe  71  4.6  Comparison of substrate preference of recombinant  74  vs. native PTPe 4.7  Summary  77  vi  Chapter 5 - Over-expression of transmembrane and non-transmembrane  78  P T P s in mammalian cells and the role of a putative SH3 binding motif in P T P a / P T P s family of PTPases 5.1  Expression o f PTPs i n H E K 293 cells  5.2  Phosphorylation of  78  PTPs expressed i n H E K 293 cells  90  and total cell lysate phosphotyrosine profiles 5.3  Src family kinases  94  5.4  C r k l l phosphorylation in cells expressing transmembrane PTPs  99  5.5  Localization of  5.6  Significance of poly-proline sequences in  5.6.1  Structural basis o f F112P mutant  118  5.6.2  Characterization o f H E K 293 cells expressing F112P mutant  120  5.7  Summary  127  PTPs expressed in H E K 293 cells PTPs  Chapter 6 — General Summary 6.1  Upregulation o f PTPs in cells o f the monocyte/macrophage  107 113  129 130  Lineage 6.2  PTPs, PTPa and CD45  132  6.3  Expression o f transmembrane and non transmembrane PTPs  136  6.4  PTPs signal transduction  138  Chapter 7 - Bibliography  149  vii  List of Tables and Figures  Chapter 1  Figure 1.1  Illustration of the src family of tyrosine kinases  3  Figure 1.2  Illustration of the Protein tyrosine phosphatase family  4  Figure 1.3  Illustration of cytosolic PTPases  4  Figure 1.4  Illustration of transmembrane PTPases  5  Figure 1.5  Illustration of the catalytic mechanism of PTPases  8  Figure 3.1.1  Differential hybridization screening  34  Figure 3.1.2  Degenerate oligonucleotide selection  Chapter 3  35  Figure 3.1.3a Degenerate primer PCR products Figure 3.1.3b Cloning of PCR fragments into pSSBS  36  Figure 3.1.4  Autoradiograph of PTPase PCR fragments  37  Figure 3.2.1  PTPe message levels in response to LPS, LFNy, and  41  Phorbol esters Figure 3.2.2  Expression of PTPe mRNA in response to Vanadate  42  Figure 3.3.3  Expression of PTPe mRNA in response to Cytokine  43  viii  stimulation Figure 3.3.1  Regions of antibody peptide selection in PTPs  Figure 3.3.2 Testing of PTPs specific antibodies with recombinant  44 47  PTPe and PTPa Figure 3.3.3 Expression of PTPs in PMA treated HL-60 cells  48  Figure 3.3.4 Expression of PTPs  50  Figure 3.3.5 PTPs expression in hemopoietic cells  50  Figure 3.3.6 PTPs expression during Granulocytic differentiation  51  of HL-60 cells Figure 3.4.1 Phosphatase activity of immunoprecipitated p72/74  52  Figure 3.4.2 Kinetic analysis of p72/74PTPs  53  Table 3.5.1  Liquid Chromatography/Mass Spectorscopic analysis  54  of p74 tryptic peptides Figure 3.6.1 Expression of non-transmembrane isoform of PTPs  56  Figure 3.6.2 PTPs sub-cellular localization  57  Figure 3.7.1 Phosphorylation of PTPs in HL-60/mac cells  58  Figure 3.7.2 Effects of pervanadate on p72/74 phosphotyrosine  60  content in HL-60 cells Figure 3.8.1 Association of GRB2 with p72/74 in HL-60 cells  62  after pervanadate stimulation Figure 3.9.1 Nucleic acid sequence comparison of transmembrane and cytosolic PTPs  64  Chapter 4  Figure 4.1.1  Prokaryotic expresssion vector pGEX-2T  66  Figure 4.2.1 Construction of pGEX.2T-PTPs  67  Figure 4.3.1  SDS-PAGE analysis of recombinant PTPs  69  Figure 4.3.2  SDS-PAGE analysis of purified recombinant PTPases  69  Figure 4.5.1 pH Optimum of PTPs  72  Table 4.5.2  Kinetic analysis of PTPs, PTPa and CD45  73  Table 4.5.3  In vitro substrate analysis of recombinant PTPs  76  Figure 5.1.1  The pBCMGSneo-PTPs expression vector  80  Figure 5.1.2  Crystal structure of Human PTP IB  80  Figure 5.1.3  Overlap extension PCR mutagenesis of C334  82  Chapter 5  andC629ofPTPs Figure 5.1.4  Transient expression of PTPs in HEK 293 cells  Figure 5.1.5 Expression levels of wild-type and various mutants  83 85  in transfected HEK 293 cells Figure 5.1.6 Morphology of cells expressing PTPs Figure 5.1.7 Protein tyrosine phosphatase activity of ectopically  87 88  expressed PTPs and mutants Figure 5.1.8 De-glycosylation of PTPs expressed in HEK 293 cells  89  Figure 5.2.1 Phosphotyrosine profiles of HEK 293 cells  90  expressing PTPe and mutant forms Figure 5.2.2 Pervanadate treatment of HEK 293 cells expressing  92  PTPs cDNAs Figure 5.2.3 GRB2 association with PTPs  93  Figure 5.3.1 Phosphotyrosine content of pp60src in HEK 293  95  cells expressing wild-type PTPs or PTPs mutants Figure 5.3.2 Decrease in phosphotyrosine content of other src  96  family members in HEK 293 cells expressing PTPs Figure 5.3.3 Activation of pp60.src in HEK 293 cells expressing  97  wild-type PTPs and PTPs mutants Figure 5.3.4 Co-immunoprecipitation of pp60src with wild-type  98  PTPs and mutants in HEK 293 cells Figure 5.4.1 Modulation of Crkll phosphorylation in HEK 293 cells  100  expressing PTPs and PTPs mutants Figure 5.4.2a Over-expression of Crkll-myc with PTPs  103  Figure 5.4.2b Association of Crkll with PTPs  103  Figure 5.4.3 Transient expression of non-transmembrane PTPs  105  in Jurkat and BaF/3 cells Figure 5.4.4 Phosphotyrosine content of pp60src in HEK 293 cells  106  expressing non-transmembrane PTPs Figure 5.4.5 pp60s7r activity in HEK 293 cells expressing non-transmembrane PTPs  107  Figure 5.5.1 Surface biotinylation of HEK 293 cells expressing PTPs  108  Figure 5.5.2 Phosphotyrosine content of plOO/HOPTPs  109  Figure 5.5.3 Immunoflourescence staining of PTPs in HEK 293 cells  111  expressing PTPs in a stable manner  Figure 5.6.1 Alignments of membrane proximal regions of  116  transmembrane PTPases and SH3 binding sites of other signaling molecules Figure 5.6.2 Space filling model of the SH3 domain of the src  118  kinase family member Hck Figure 5.6.3 Crystal structure of PTPa  121  Figure 5.6.4 PTPase activity of PTPsFl 12P  122  Figure 5.6.5 Cellular morphology of HEK 293 cells  123  Figure 5.6.6 pp60src tyrosine phosphorylation in HEK 293 cells  124  expressing PTPsFl 12P mutant Figure 5.6.7 Activation of pp60src in HEK 293 cells expressing  125  PTPsFl 12P mutant Figure 5.6.8 Crkll phosphorylation in HEK293 cells expressing  126  PTPsFl12P Figure 5.7.1 Summary of in vivo observations with PTPs mutants  128  Chapter 6  Figure 6.1  Homology between C-terminal regions of src family  members  Abbreviations  AMLV  Abelson murine leukemia virus  ATP  Adenosine triphosphate  BPV  Bovine papilloma virus  CD  cluster differentiation  CMV  cytomegalovirus  CSF-1R  colony stimulating factor receptor  DNA  deoxyribonucleic acid  ECM  extracellular matrix  EGF-R  epidermal growth factor receptor  GAP  GTPase activating protein  GRB2  growth factor receptor bound protein 2  GST  Glutathione S Transferase  HEK  human embryonal kidney  ICAM-1  intracellular adhesion molecule 1  Ig  immunoglobulin  IL  interleukin  IPTG  isopropyl-1 -thio-P-D-galactopyranoside  LAR  leukocyte common antigen related protein  MAb  monoclonal antibody  MAP kinase  mitogen activated protein kinase  MMTV  mouse mammory tumour viruse  PBS  phosphate buffered salin  PCR  polymerase chain reaction  PDGF-R  platelet-derived growth factor  PH  pleckstrin homology  PI3K  phosphatidylinositol-3 -kinase  PKC  protein kinase C  PLCyl  phospholipase C gamma 1  PMA  phorbol myristate acetate  PTB  phosphoprotein binding  PTP  protein tyrosine phosphatase  PTPase  protein tyrosine phosphatase  PTK  protein tyrosine kinase  RNA  ribonucleic acid  SDS-PAGE  sodium dodecyl sulfate — polyacrylamide gel electrophoresis  SH2  src homology 2  SH3  src homolgy 3  SYP  SH2-containing tyrosine phosphatase  TCR  T cell receptor  TNF-a  tumour necrosis factor alpha  XV  Acknowledgements  I am indebted to a number of people who aided me during the course of this thesis. In particular I would like to thank Nicola Melhado (Mann) and the Mann family for all their support. I am forever grateful to Frank Jirik for encouragement and unwavering support during years prior to and during this course of study. A special thanks to Nicole Janzen a life long friend and co-worker, to Dr. Donald Semenuik, Gloria and Bruce Thomson, and my advisory committee. I would also like to acknowledge Ken Harder for collaborative efforts, discussions and assistance with the Fl 12P mutants, Scott Pownall for assistance in expression of the cytosolic isoform of PTPe and the remaining Jirik lab for continued support during the past 5 years. Finally, I would like acknowledge the solid support of my family, Vernal, Barbera, Mark and Joy with a special thank you to Caroline Proctor.  Chapter 1  Introduction  With the discovery by Sefton, Hunter and Beemon in 1979 of the cellular homologue of v-src, the kinase within the Rous sarcoma virus responsible for the tumorigenicity of the infectious particle, the family of signaling proteins now known as protein kinases was catapulted to the forefront of cancer research (figure 1.1). Further work by Collett et al, 1980 culminated in the report that the tumorigenic activity in the Rous sarcoma virus was a kinase specific for protein tyrosine residues. Despite the critical importance of tyrosine phosphorylation for differentiation and intracellular regulatory events associated with cell growth it was demonstrated to only constitute 0.1 % of the phosphate on proteins within a cell. In contrast, serine and threonine phosphorylations were found to comprise greater than 99% of the polypeptide associated phosphate within the cell. Based on these observations, it was assumed that serine and  1  threonine phosphorylation was the most important phosphorylation type with respect to physiological events occuring within cells. This may account for the fact that the serine and threonine kinases were initially the most widely studied of protein kinases. The work of Collett et al, 1980 however, indicated that although tyrosine phosphate was relatively rare, a single tyrosine kinase was capable of inducing single step transformation of susceptible cells. Thereafter,  numerous  groups reported that virally  transformed  homologues of cellular protein tyrosine kinases were the tumorigenie agents within the genomes of a number of different cancer inducing viruses (Rohrschneider et al, 1979, Yoshida et al, 1980). For example, the fyn, lyn, abl, fes, and erbB moleucles were all found to be tyrosine kinases present within the genomes of cancer causing retroviruses. In searching viral genomes for 'hijacked' signaling proteins, it rapidly became evident the viral counterparts of the cellular genes often led to truncated proteins or molecules containing amino acid substitutions. The sites of these mutations often yielded important biochemical information with respect to gene function since many of the mutations gave rise to abnormal protein products constituitive activation, abnormal dimerization or altered subcellular localization. Thus, tyrosine kinases play a crutial role in the regulation of intracellular signal transduction mechanisms and likely modulate key cellular processes.  2  M  SH3  N-term  SH2  C-terminal  Figure 1.1 Illustration of the src family of tyrosine kinases. M denotes meristylation of the N terminus. N-term is the unique N-terminal region of this family of kinases. SH3 is the src homology 3 domain, SH2 the src homology 2 domain and SHI the src homology 1 or kinase domain which contains the enzyme active site as well as the site for a stimulatory phosphotyrosine residue. The C-terminal tail 'Y' indicates the C-terminal regulatory phosphorylation site  Since the molecular cloning of the first PTPase, PTP1B, the number of members of the PTPase family has grown considerably with the nucleic acid sequences of more than 50 PTPases reported. This family of proteins can be divided into three broad groups (figure 1.2). The dual specificity PTPases, such as mkpl/CL100/3CH314, and CDC25 are predominantly nuclear enzymes having a single catalytic domain (Sun et al, 1993, Guan et al, 1995). These PTPases are characterized by their ability to catalyze the removal of phosphate groups from both threonine and tyrosine residues. The dual specificity PTPases also possess strict substrate specificity and are amongst the few PTPases whose substrates have been identified, these include the erk family of kinases (Guan et al, 1995).  3  Fibronectin III Repeals Ig-like Domains I  I  Alternately Spliced Exons SH2 Domains MAM Domain  n  % i  &  i  Carbonic Anhydrase-Iike Domain  Talin-Related Domain  0  PTPase Domain -240 aa. Cys-to-Asp substitution in PTP Domain  §& s O  >-  pc E > >  Figure 1.2 The Protein tyrosine phosphatase family. The PTPase family is broken into three broad groups. The transmembrane PTPases, the cytosolic PTPases and the nuclear PTPases. Transmembrane PTPases are characterized by diverse extracellular domains, a transmembrane domain and with the exception of PTPp tandem catalytic domains. Cytosolic PTPases often contain various signaling domains including SH2 domains and PEST domains. The majority of nuclear PTPases are dual specificity PTPases capable of catalyzing the dephosphorylation of threonine and tyrosine residues.  N-terminal region  PTPase domain  C-terminal tail  DFG Figure 1.3 Illustration of cytosolic PTPases  4  The cytosolic PTPases comprise a broad range of PTPases (figure 1.3). As with many other cytosolic molecules involved in cell signaling, this sub-group often has notable protein-protein interaction domains in addition to the single catalytic domain. The SH2 domain containing PTPases SHP-1  and SHP-2, for example, have been shown to  interact with a wide variety of activated growth factor receptors either potentiating (SHP2Xpr inhibiting (SHP-1) signal transduction cascades _emanating from these receptors (Rivard et al, 1995, Su et al, 1996, Tomic et al, 1995, Neel et al, 1997). Ezrin domain containing PTPases, such as PTP-MEG, associate with cytoskeletal proteins and are thought to regulate cellular architecture (Moller & Ullrich etal, 1994). The PDZ domain containing PTPase FAP-1 associates with the Fas receptor blocking the apoptotic signal delivered by FasL (Sato et al, 1995). The cysolic PTPase group also includes PTPases, such as PTP1B and T-cell PTPase that lack known protein interaction domains (Charbonneau et al, 1988, Cool et al, 1989). The activity of PTP1B can be regulated by limited proteolysis of the C-terminal tail (Liu et al, 1996). Its functions range from growth factor receptor de-phosphorylation (PTP1B) (Goldstein et al, 1998) to regulation of hematopoiesis (Tremblay et al 1998) to control of glucose homeostasis (Kennedy et al 1999).  5  Inter-catalytic domain Figure 1.4 Illustration of transmembrane PTPases  The transmembrane or receptor-like PTPases are probably the most diverse sub family of PTPases (figure 1.3). Differentially glycosylated extracellular domains of widely varying size, a single membrane-spanning region and, with the exception RPTPp, which has a single catalytic domain, tandem catalytic domains distinguishes this group of PTPases. The extra-cellular domains contain an array of common receptor motifs including immunoglobulin superfamily domains and fibronectin type-Ill repeats. The catalytic domains of this group of PTPases, in contrast, share high degrees of sequence identity (Levy etal, 1993). The crystal structure of PTPa 's first catalytic domain, as solved by Kurian and Hunter in 1997, indicated that PTPa may exist as inactive homodimers. The hypothesis argues that the membrane proximal region, located before the beginning of the catalytic  6  domain, forms a unique helical wedge structure. The wedge is capable of insertion into the first catalytic domain of a second PTPase, abrogating the interaction of the enzyme with its substrate through competitive inhibition.  Multiple mRNA splice variants is also a hallmark of this sub-family of PTPases. CD45, otherwise known as leukocyte common antigen (LCA), is an archetypal member of this family, apart from being one of the most widely studied and best understood transmembrane PTPase. With more than five different splice variants giving rise to a number of different isoforms, including T200 and B220, this PTPase exhibits cell lineage-specific isoform expression and function (Streuli et al,  1987, Thomas et  al,  1987, Ralph et al, 1987). The T200 isoform, for example, dephosphorylates and activates the membrane associated tyrosine kinase pp55  /ci  in mature T-cells during  TCR  aggregation, resulting in T lymphocyte activation (Pingel and Thomas 1989, Mustelin et al.,  1989, Ostergaard et al,  1989, Mustelin et al,  1990). Dephosphorylation and  activation of another src kinase family member, pp53/56 ", by the B220 isoform in /;;  mature B-cells during BCR aggregation, similarly results in B-cell activation (Katagiri et al,1995, Yanagiefa/., 1996).  As regulators of tyrosine phosphorylated PTKs and their substrates, both receptor and cytosolic protein-tryosine phosphatases have been identified as important regulatory components within signaling complexes (Charbonneau and Tonks, 1992; Stone and Dixon, 1994; Hunter, 1995). Furthermore, a given phosphatase may demonstrate either stimulatory, inhibitory or both effects within specific intracellular signaling pathways  7  (Feng and Pawson, 1994; Trowbridge and Thomas, 1994). While commonly referred to as "receptor" PTPases the putative ligands for this sub-family of PTPases are largely unknown. With the discovery of the ligand for RPTPp as the extracellular domain of another RPTPp molecule on an adjacent cell, many reports have subsequently implicated transmembrane PTPases in the regulation of cell-cell adhesion (Peles et al, 1998). CD45 also possesses an extracellular domain that resembles those of certain adhesion molecules. CD45 is also thought to have a ligand that is bound to the surface of thymic epithelial cells (Thomas personal communication).  The catalytic domains of all known PTPases contain a common active site motif, (Ff/V)C(X) R(ST), that is essential for the hydrolyis of substrates via a common 5  mechanism (figure 1.4). The first step of the reaction involves attack by the thiolate anion of the conserved cysteine residue within the above motif on the phosphate ester, with simultaneous departure of the tyrosyl residue aided by general acid-base catalysis involving an invariant aspartic acid residue. The resulting phosphoenzyme intermediate then undergoes a rate-limiting hydrolysis step to form inorganic phosphate and the free enzyme. The invariant arginine in the signature motif is crucial for binding of the substrate through electrostatic interaction with the phosphate moiety and also for transition state stabilization (Barford et al, 1998, Zhang 1998, Zhao etal, 1998, Denu et al,  1996, Denu et al.,1996). The catalytic domain has also recently been shown in  some cases to have a broad specificity for phosphorylated moieties. In the case of PTEN, for example, recent reports demonstrate that this PTPase is not only capable of catalyzing  the removal of phosphate groups from tyrosyl residues but also from the 3-position of phosphatidyl inositol (PI) group of phospholipids (Maehama & Dixon 1998).  Figure 1.5 Illustration of the catalytic mechanism of PTPases  The first member of the PTPase superfamily of signaling molecules to be characterized in detail was the cytosolic PTPase PTP1B (Charbonneau et al, 1989). Comprised of short N-terminal and C-terminal sequences flanking a single catalytic domain, PTP1B has been shown associate with the rough endoplasmic reticulum through its C-terminus. Its activity can be modulated by limited proteolytic cleavage of its tail (Liu et al, 1996). However, work by Lammers, Moller, and Ullrich in 1997, implicated PTP1B PTPase activity in insulin receptor signaling as well as the signal transduction pathways activated by various other growth factor receptors. It has recently been shown that the stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H 2 O 2 that is required for growth factor-induced protein  tyrosine phosphorylation (Sundaresan et al, 1995, Denu et al, 1998). PTP1B has been implicated in EGF-R signal transduction through a physical association of EGF-R in cells expressing a mutant PTP1B where the catalytic cysteine had been mutated to a serine residue. While the resulting mutant is capable of binding its substrate through phosphotyrosine independent interactions, it is incapable of forming a thio-phosphate ester intermediate. In this way, PTP1B mutant effectively traps the substrate, in this case the EGF-R (Milarski et al, 1993). The effect of H2O2 produced in response to epidermal growth factor (EGF) on the activity of protein-tyrosine phosphatase IB (PTP1B) has been investigated in A431 human epidermoid carcinoma cells. H2O2 was shown to inactivate recombinant PTP1B in vitro by oxidizing its catalytic site cysteine. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. When the oxidation-state of PTP1B was monitored, oxidized PTP1B reached maximal levels ten minutes after stimulation of cells with EGF and returned to baseline levels by forty minutes, suggesting that the oxidation of PTP1B is reversible in cells. These results elegantly indicated that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells and that concurrent inhibition of protein-tyrosine phosphatases by (Lee etal,  H2O2  might also be required  1998). This observation touches upon the novel methods of regulation that  may be discovered in signal transduction systems within the cell. However, it remains to be shown whether this is a common mechanism of PTP regulation in vivo.  10  Shortly after the initial molecular characterization of PTP1B, database searches revealed that CD45 exhibited sequence similarity. Using a variety of molecular cloning techniques, a number of receptor like PTPases have subsequently been described. One of the first in this family was LAR, a molecule that is characterized by tandem catalytic domains and a large extra-cellular domain composed of both fibronectin type-Ill repeats and Ig domains that are similar to those of the homophilic adhesion molecule NCAM. A role for LAR as well as PTPP has been reported in axon guidance (Serra-Pages et al, 1998, Holland et al., 1998) and mammary gland development (Serra-Pages et al., 1998). In cultured cells, LAR has been shown to bind to the intracellular, coiled coil LARinteracting protein at discrete ends of focal adhesions, implicating these proteins in the regulation of cell-matrix interactions (Serra-Pages et al., 1998). Seven LAR-interacting protein homologues have been described in humans and Caenorhabditis elegans that make up the liprin gene family (Serra-Pages et al., 1998). Based on sequence similarities and binding characteristics, liprins are subdivided into alpha-type and beta-type liprins. The C-terminal, non-coiled coil regions of alpha-liprins bind to the membrane-distal phosphatase domains of LAR family members, as well as to the C-terminal, non-coiled coil region of beta-liprins (Serra-Pages et al., 1998). Both alpha- and beta-liprins homodimerize via their N-terminal, coiled-coil regions. Liprins are thus multivalent proteins that potentially form complex structures. Co-expression studies indicate that liprin-a2 alters LAR cellular localization and induces clustering of this PTP which results in localization of LAR family tyrosine phosphatases at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates (Serra-Pages et al., 1998). Such membrane localization  11  may explain the possible role of LAR in insulin receptor signaling. In this regard, a functional and physical association between LAR and the insulin receptor within endosomes after stimulation has been described (Ahmad and Goldstein 1997). The receptor's activation was observed to influence the affinity of LAR for the receptor. Such results highlight the importance of cellular localization in defining the functions of receptor PTPases. Evidence suggests that individual receptor PTPases may be involved in different signaling pathways dependent upon their proximity to particular molecules within specific signaling complexes. In the case of LAR it is possible that interactions between the extracellular domain and signaling complexes involving cell-cell adhesion occur, in addition to its association with the activated insulin receptor. Thus, it is probable that insulin signals in a cell may be regulated by concurrent signals received by cell-cell or extracellular matrix interactions that are transduced through receptor PTPases.  The hemopoietic cell lineage gives rise to the wide-variety of cell types found in the immune system. Through differentiation, guided by coordination of a complex network of granulocytic and macrophage growth factors and cytokines, hemopoietic progenitors give rise to the cells of the reticuloendothelial system. The reticuloendothelial system includes microglia in the brain, dendtritic cells within the lymph nodes and monocytes which roam the vasculature. At sites of inflammation, it is the monocyte lineage, which responds in collaboration with granulocytes and neutrophils. Following a concentration gradient of chemotactic factors known as chemokines monocytes slow their progress through blood vessels and begin a phase of "rolling adhesion" as cellular  12  adhesion molecules are upregulated both on monocytes and endothelial cells. At sites of inflammation, monocyte migration is halted and the activated cells invade the tissue. Once activated, monocytes differentiate into macrophages and acquire various functions related to wound healing. Phagocytosis, production of reactive oxygen intermediates and secretion of pro-inflammatory cytokines, such as IL-1 and TNFa, are among the properties displayed be activated macrophages. These functions are also responsible for the symptoms of certain inflammatory autoimmune diseases such as arthritis, multiple sclerosis and lupus. Thus, a better understanding of macrophage signaling and differentiation may  yield novel strategies in the treatment of various autoimmune  diseases.  There is much evidence supporting the role of various PTKs in the proliferation, differentiation, and function of hemopoietic cells (Chan et al., 1994, Gold & DeFranco, 1994, Ihle, 1995). However, with the exception of molecules such as CD45, the SH2 domain-containing PTPases (Trowbridge & Thomas, 1994; Feng & Pawson, 1994), and the novel tumour suppressor PTPase PTEN, knowledge about the specific functional roles of many PTPases is lacking. With the importance of PTKs and PTPases in many leukocyte functions, we decided to examine which PTPases are upregulated by macrophages after differentiation, thus, providing some insight into the roles PTPases play in macrophage function.  An initial approach in elucidating the role of these molecules has been to establish the spectrum of expressed PTPases, both during cell differentiation as well as in  13  terminally differentiated cells. Efforts to determine the expression patterns of PTPases in monocytic cells have, for example, capitalized on systems allowing the external control of cellular differentiation. In this respect, the promyelocyte leukemia cell lines U937 and HL-60 have been useful models. In response to various stimuli, HL-60 cells undergo differentiation into various cell types (Collins et al, 1987). Following phorbol-ester treatment, HL-60 cells develop a number of monocyte-like characteristics. In contrast, DMSO treatment results in cells with granulocytic features (Collins et al., 1978; Collins et ah, 1987). The regulation of a number of PTPases has been studied during HL-60 differentiation. For example, Uchida et al., (1993), as well as Zhao et ah, (1994), described both induction of PTP1C phosphorylation and increases in transcript and protein levels within phorbol-ester stimulated HL-60 cells. In addition, the transcriptional regulation of a number of PTPases, including protein tyrosine phosphatases alpha (PTPa) and epsilon (PTPs) and PTP-MEG2 has been described in the U937 and HL-60 cell lines following phorbol-ester administration (Seimiya et al., 1995). macrophage cell line RAW  Using the murine  264.7 as well as primary macrophage cultures as model  systems the expression patterns of PTPases was studied further.  PTPs is a member of the family of receptor-like PTPases, which includes PTPa, LAR, and CD45 among others (Charbonneau and Tonks, 1992). PTPs is composed of a protein having two tandem intracellular catalytic domains, a transmembrane segment, and following signal peptide processing, a ~25-residue extracellular domain (Krueger et al, 1990). Within the group of receptor-like PTPases, PTPs bears closest similarity to the widely expressed phosphatase, PTPa (Matthews et al., 1990; Sap et al, 1990; Kaplan et  14  al, 1990; Jirik et al, 1990). The similarity between these two PTPases is striking when the amino acid sequences of the intracellular domains are compared (-85% identity) (figure 3.3.1). In contrast, the extracellular regions of the two molecules are dissimilar, both in length and amino acid sequence (Krueger et al, 1990). Moreover, the expression of PTPe appears to be more restricted in its distribution than that of PTPa (Hendriks et a/.,...1994; Elson and Leder,__1995a, 1995b). PTPe_ transcripts were. detected in the interleukin (IL)-3-dependent myeloid leukemia cell line DA-3 (Yi et al, 1991), mouse embryonic stem cells (Hendriks et al, 1994), and at low levels in NIH-3T3 cells (Yi et al,  1991). PTPs transcripts were also seen in MEL  erythroblastoid leukemia cells,  following induction with dimethyl sulfoxide (Kume et al, 1994). PTPe expression was recently discovered in mammary tumor cell lines derived from transgenic mice expressing MMTV-v-Ha-ras and MMTV-c-«ew (Elson and Leder, 1995a).  In a study aimed at characterizing PTPases expressed in cells of hemopoietic origin and in response to pro-inflammatory stimuli, PTPe was identified as a phosphatase upregulated by treatment of adherent cells obtained from murine peritoneal macrophages with LPS or IFNy. This thesis focuses upon a characterization of PTPe. In Chapter 3, "The Screen for PTPases Regulated by IFNy and PTPe Expression in Cells of  the  Monocyte/Macrophage Lineage", PTPe expression during monocytic cell differentiation is described. Here, the expression pattern and activity of PTPe was analyzed in phorbol ester-differentiated HL-60 cells. During this study a novel, primarily nuclear and cytosolic fraction-associated, p72/74 isoform of PTPe was identified, the only detectable form of this PTPase expressed in HL-60, Jurkat, and murine peritoneal-exudate cells.  15  Chapter  4,  "Bacterial Expression  PTPs and Substrate  Specificity  of  and  in  Native  vitro  Substrate  PTPs",  describes  Specificity  of  expression of  recombinant PTPs in bacteria. This section details the characterization of in vitro substrate specificity of PTPs using synthetic phosphopeptide substrates. The final chapter entitled "Over-expression of Transmembrane and Non-transmembrane PTPs Mammalian Cells and the Role of a Putative SH3 Binding Motif in  in  PTPa/PTPs  Family of PTPases" describes the characterization of PTPs isoforms by over-expression in mammalian cells. These studies compare the PTPs isoforms and makes use of the in vitro biochemical enzyme analysis in Chapter 4 to direct the search for in vivo substrates.  16  Chapter 2  Materials and Methods  2.1 Materials. Phorbol 12-myristate 13-acetate (PMA) was obtained from Gibco/BRL. Hydrogen peroxide, sodium orthovanadate and other chemicals were obtained from Fisher Scientific.  2.2 Buffers.  Buffer A: 25 mM  Tris-HCl pH 7.5, 150 mM  NaCl, 10 mM  2-  mercaptoethanol, 1.0% Triton X-100, soybean trypsin inhibitor 10 ug/ml, leupeptin 1.0 ug/ml, 1 mM phenylmethylsulfonylflouride, and pepstatin 1.0 pg/ml. Buffer B: 50 mM Tris-HCl pH 8.0, 150 mM  NaCl, 2.5 mM  CaCl , 10 mM 2  2-mercaptoethanol.  Northern/Southern Hybridization buffer: 25% Formamide, 1.0% BSA, 7.0% SDS, 0.5 M NaHP0 pH 7.2, 10 mM EDTA. GT buffer: 4M guanidine isothiocyanate, 50 mM MOPS 4  17  pH 7.0, 1.0% 2-mercaptoethanol, 1.0% Sarcosyl. 2x Bind buffer: 1M NaCl, 50 mM MOPS pH 7.0, 10 mM EDTA pH 8.0. Ix Wash buffer 150 mM NaCl, 25 mM MOPS pH 7.0, 2 mM EDTA, 0.1% Sarcosyl. TX100 lysis buffer: 100 mM NaCl, 25 mM Tris-HCl pH 7.5, 2 mM EDTA, 1 mM DTT, 1.0% Triton X-100, 10 ug/ml aprotinin, 10 ug/ml bestatin, 10 fig/ml leupeptin, 5 ug/ml pepstatin, 10 ug/ml soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3V04, 50 mM NaF. RIPA buffer: 100 mM NaCl, 25 mM Tris-HCl pH 7.5, 2 mM EDTA, 1 mM DTT, 1.0% (v/v) Triton X-100, NaF, 0.5% (w/v) Deoxycholate, 0.1 % (w/v) SDS, 10 ug/ml aprotinin, 10 ug/ml bestatin, 10 ug/ml leupeptin, 5 ug/ml pepstatin, 10 ug/ml soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na V0 , 50 mM NaF. 3  4  2.3 Rapid mRNA purification. Cells or tissue were homogenized in 1-5 ml of GT buffer using a 22G needle and syringe or tissue homogenizer. Equal volumes of 2x Bind buffer was added and DNA/protein precipitate was pelleted at 10,000g for 10 min. The supernatant was transferred to afreshtube containing 150 ul of a 50% slurry of poly-U Sepharose and incubated at room temperature on a rotating wheel for 30 min. The beads were collected by centrifugation at 250g for 2 min. The beads were washed 3 times with lx Wash buffer. Messenger RNA was liberatedfromthe beads by the addition of 50 ul of distilled water and incubation at 70°C for 10 min followed by removal of the beads by centrifugation at 10,000g for 2 min. If subsequent enzymatic reactions were to be performed on the mRNA sample Sarcosyl was omittedfromthe Wash buffer.  18  2.4 Northern Blot.  Purified mRNA was prepared as described above and subjected to  electrophoresis through 0.7% agarose gels containing 7.5% formaldehyde, lx MOPS buffer for 4 hours at 150V in lx MOPS running buffer. After electrophoresis gels were washed for 15 min in distilled water then mRNA transferred to GeneScreen nylon membranes by capillary action with 2x SSC. The transferred mRNA was then immobilized to the membranes by a single 150 kJ dose of UV-C  irradiation and efficient  transfer visualized by UV light. Blots were pre-hybridized for 20 min at 65 °C with Hybridization buffer then hybridized with the appropriate probes overnight at 65 °C in a rotating hybridization oven. Then washed until background had decreased to acceptable levels with O.lx SSC / 0.1% SDS at the appropriated temperature (65°C).  2.5 Differential hybridization screening. Randomly primed cDNA's were prepared by reverse transcription of purified mRNA samples using Superscript Reverse Transcriptase (GIBCO/BRL) in the following reaction. Five hundred nanograms of mRNA was added to 5 pi of 5x Superscript buffer, 500 uM dNTPs, 25 pmol pdN , 5 U RNasin, 10 U 6  Superscript reverse transcriptase and the final volume adjusted to 25 pi with distilled water. The reaction was then allowed to proceed at 45 °C for 30 min then at 55°C for 30 min before termination by incubation at 70°C for 15 min. PCR fragments of conserved PTPase catalytic domains from PTPases upregulated in response to IFNy were generated by  touch-down PCR using  degenerate oligonucleotides  Deg #1 (DFW)  [5'-  CTCAGTCGACCTT T/C TGG C/A G I ATG G/A T I TGG GA A/G C], GXGXXG [5'CTCAGTCGACCC I A C/T I CC I GC A/G CT G/A CAGTG], and Deg #2 (QYWP)  19  [5'-CTCAGTCGACCAA A/G TG T/C G C/A I CA A/G TA T/C TGGCC] using cDNAs generated from mRNAs purified from RAW264.7 cells stimulated with 500 U/ml murine IFNy. PCRfragmentswere agarose gel purified and sub-clbried blunt-ended into EcoRV digested pSSBS. Positive clones were selected on the basis of blue/white selection using X-gal (100 ug/ml) and IPTG (2 mM) within the bacterial culture plates. Positive clones were confirmed with a Sal T restriction digest liberating the 350 bp insert. DNA from positive clones was prepared and equal amounts immobilized on Hybond-N nylon membranes by slot blotting. A PCR probe was prepared from cDNAs generated from unstimulated mRNA samples using identical conditions as described previously with the addition of 75 u.Ci of [a P] dCTP (3000 Ci/mmole). Slot blots were hybridized with the 32  PCR probe overnight at 65°C in Southern hybridization buffer. Slot blots were then washed at 65°C in O.lx SSC / 0.1%SDS for 30 min and exposed to X-ray film at -80°C. Upregulated PTPase messages were identified on the basis of the absence of a hybridization signal due to under representation of the PTPase mRNA in the PCR probe prepared from the unstimulated sample. Clones representing candidates for upregulation were sequenced manually using the chain termination method (Sanger et al, 1977).  2.6 Cloning of  PTPe  intracellular domains.  The cDNA encoding the intracellular  domains of PTPe were generated by PCR using 5' and 3' oligonucleotide primers: [5'CTCGGATCCCCCATGAGGAAGCAGAGGAAAGCTGTGGTC]  and  GAGGGATCCAATTGCGGCCGCTCATTTGAAATTAGCATAATCAGA]  [5'as  described in the published sequence (Krueger et al., 1990) synthesized on a Applied  20  Biosystems DNA Synthesizer (model 391). Messenger RNA was prepared from HL-60 cells as described earlier. Complementary DNA  strands were generated by reverse  transcription by the addition of the reaction mixture (5 pi 5x Superscript buffer, 500uM dNTPs, 25 pmol pdN6, 5 U RNAsin, 10 U Superscript Reverse transcriptase final volume 25 pi) to 1.0 pg of purified mRNA in distilled water. Reactions were carried out at 45°C for 30 min followed by incubation at 55°C for 30 min. Five microlitres of the reverse transcriptase reactions were then subjected to touch-down PCR amplification using oligonucleotides described earlier under the following reaction conditions (10 pi lOx Taq buffer, 200 uM dNTPs, 1.5 mM MgCl , 1 uM 5' primer, 1 uM 3' primer, 1 U Taq DNA 2  polymerase final volume adjusted to 100 pi). Full-length cDNAs were purified by agarose gel electrophoresis then digested with BamHI and Muni restriction enzymes restriction sites for these enzymes were included within the 5' primer, and 3' primer, respectively. Digested DNA fragments were gel purified and directionally sub-cloned into the BamHI, EcoRI digested pGEX-2T expression vector utilizing the fact that Muni and EcoRI share compatible ends.  2.7 Recombinant expression of PTPs, PTPa, and CD45 intracellular domains.  pGEX-2T-PTPe, pGET-2T-tag-PTPa, pGEX-2T-tag-CD45  prokaryotic  The  expression  vectors were transformed into competent XL-1 blue bacteria. 50 ml overnight cultures were grown in 100 pg/ml ampicillin/Luria broth and added to 1 litre of the same culture medium and grown to an ODeoo of 0.6 at 37°C in the absence of IPTG. Recombinant PTPe, PTPa, and CD45 were induced with the addition of 0.1 mM  IPTG and cultures  21  grown in the presence of IPTG for 6 hours at 23°C. Bacteria were pelleted by centrifugation at 5,000g for 15 min at 4°C. The bacterial pellets were resuspended in 10 ml of Buffer A and lysed by sonication with 3x 30 second pulses on ice. The bacterial extracts were then subjected to ultracentrifugation at 100,000g for 30 min at 4°C and the supernatants stored at -80°C prior to further purification.  2.8 PTPase purification.  The bacterial extracts were added to 1.0 ml of a 50%  glutathione Sepharose slurry and incubated at 4°C for 1 hour. The beads were washed 4 times with Buffer A and once with Buffer B before cleavage in 1.0 ml of Buffer B containing 50 pi of thrombin (400 ug/ml). Glycerol was added to 50% (v/v) final concentration before storage at -80°C. Protein concentration was determined by SDSPAGE and coomassie staining in the presence of BSA standards. Protein concentrations were adjusted to 50 pg/ml before storage.  2.9 Production of FTP mutant cDNA's. Mutant PTPe cDNA's were generated by overlap extension PCR (Ho et al, 1989) using oligonucleotide pairs Ml3 reverse primer +  mCl>Aa  primer amplify  [5 '-ACCCACGCCCGCGCTAGCGTGAACC ACAATGGG],  + mCl>As PTPeC334A  M13(-20)  [5'-CCCATTGTGGTTCACGCTAGCGCGGGCGTGGGT] to fragments;  M13  reverse  primer  +  mC2>Aa [5'-  CCCTGCTCCCGCCGTAGCGTGCACGGTGATGGG], M13(-20) primer + mC2>As [5-CCCATCACCGTGACAGCTAGCGCGGGAGCAGGG] to amplify PTPeC629A  22  fragments;  M13  reverse  primer  +  CACGGGC ATGGGGGGGA ACTTCTTGGG], M13(-20) primer  mPPIPa +  [5'-  mPPIPs [5'-  CCCAAGAAGTTCCCCCCCATCCCCGTG] to amplify PTPeFl 12P fragmentsfromthe full-length cDNA in the pBluescript derivative pSSBS (REF). Amplification was carried out under the following reaction conditions (5 ul lOx Vent Buffer, 200 uM dNTP's and 4 U of Vent DNA polymerase the final volume adjusted to 50 u.1) using touch-down PCR (2 cycles of 96°C for 45 sec, annealing at 65 °C for 1 min, 73 °C for 1 min, 2 cycles annealing at 64°C, 2 cycles annealing at 63°C, 2 cycles annealing at 62°C, 2 cycles annealing at 61°C, 2 cycles annealing at 60°C, 2 cycles annealing at 59°C, 2 cycles annealing at 57°C) and PCRfragmentspurified by gel electrophoresis in a 1.0% agarose gel and resuspended in 20 ul of distilled water. Full-length cDNA's carrying mutations were generated from the PCR fragments by extension of the overlap contained within the synthetic oligonucleotides with a second round of touch-down PCR using 1.0 ul of each agarose gel purified PCRfragmentsand M13 reverse + M13(-20) primers. Full-length cDNA's were gel-purified and digested with Not I then sub-cloned into pSSBS for sequence analysis. Mutated PTPs cDNAs were then shuttled into the Not I site of the CMV  promoter based episomal expression vector pBCMGSneo (Karasuyama and  Melchers, 1988).  2.10 Antibodies.  The anti-GRB2 monoclonal antibody was from Transduction  Laboratories. The anti-GRB2 polyclonal antibody and the SRC2 antiserum specific the C-terminal peptide sequence 509-533 of Src and conserved sequences of Fyn and Yes  23  was from Santa Cruz Biotechnology. The anti-phosphotyrosine monoclonal antibody (4G10) as well as the anti-src monoclonal (GD11) were obtained from Upstate Biotechnology Inc. The FITC labeled goat anti-rabbit IgG F(ab')2 antibody, and TRITC labeled Goat anti-Mouse IgG F(ab')2 antibody were obtained from Caltag. The rabbit polyclonal antibody PTPel5ic was raised against a PTPe-unique 12-residue peptide derived  from  the  intercatalytic  (TMHGTTTHFDKI), and corresponding  to  region  PTPsl6d2 a  second  was  sequence  of  similarly  raised  PTPs-unique  human  PTPs 413-424  against a peptide  sequence  555-587  (IKNDTLSEAISIRDFLVTLNQPQARQEEQVRW) within the C-terminal catalytic domain (Krueger et al, 1990). Both peptides were coupled with N-terminal cysteinelinked keyhole limpet hemocyanin and anti-sera generated by repeated immunization of New  Zealand white rabbits. Anti-sera were purified by peptide affinity column  chromatography. Columns prepared by conjugation of synthetic peptide to thiol Sepharose and anti-sera eluted with 0.1 M glycine pH 2.5.  2.11 Cell culture and transfection. HL-60, 293, and Jurkat cell lines were obtained from the American Type Culture Collection. RAMOS, WEHI 231, and 2PK3 B-cell lines were provided by M. Gold (University of British Columbia). AKR1.G.1.26 , BWT200+ and T28 murine T-cell lines were provided by P. Johnson (University of British Columbia).  HL-60 cells were maintained  in RPMI (StemCell Technologies)  supplemented with 10% fetal bovine serum (Hyclone), 2 mM  L-glutamine, 2 mM  pyruvate, 50 U/ml penicillin G, 50 mg/ml streptomycin. PMA  sodium  was added to cells at a  24  final concentration of 16 nM for various times as indicated. Culture medium containing undifferentiated and dead cells was discarded after 48 h. Adherent, PMA-differentiated HL-60 cells (designated here as HL-60/mac cells) were washed and allowed to recover for 24 h prior to further treatment. For pervanadate stimulation, cells were exposed to a freshly-prepared mixture of hydrogen peroxide and vanadate with final concentrations being 1 mM  and 0.1 mM, respectively. Murine peritoneal exudate cells, obtained by  peritoneal lavage of Balb/c mice 3 d after intra-peritoneal thioglycollate administration, were purified by adherence to plastic tissue culture dishes for 2 h at 37 °C. A cDNA encoding the transmembrane form of PTPe (Krueger et al., 1990), generously provided by A. Schmidt (Merck, West Point, PA), was inserted into a human cytomegalovirus immediate-early gene promoter-based eukaryotic  expression vector for transient  expression in 293 cells. Lipofectamine (GEBCO/BRL) was used for transient as well as stable transfections. For transfections 6-well plates were coated with 20 mg/ml bovine fibronectin in PBS overnight at 4 °C then washed twice with PBS prior to the cells being plated. Cells were seeded at 2.0 x 10 cells/well on to thefibronectin-treatedplates the 5  day before transfection. The following day, 2.5 ug of DNA in 100 ul of serum free media (SFM) (Optimem, GEBCO/BRL) was combined with 15 u.1 of lipofectamine in 100 ul SFM. The DNA/lipofectamine transfection cocktail was incubated for 30 min. HEK293 cells were carefully washed once with SFM to remove any serum then the transfection cocktail with the addition of 800 ul SFM was layered onto the cells. After 5-7 hours at 37 °C the transfection was terminated by the addition of 2 ml of 10% serum-containing media. The transfected cells were allowed to recover 24 hours prior to lysis and analysis. Stable transfectants were obtained by dilution cloning. After gentle typsinization and  25  dilution to 40 ml of serum containing media, supplemented with 600 pg/ml G418. Cells were then seeded onto four 96-well dishes at 100 ul/well and selected for a period of 10 days with the addition of 100 pi of G418 containing complete media after 5 days. The final concentration of G418 used was 600 pg/ml. In previous experiments this concentration was determined to kill G418 sensitive cells within 7 days.  2.12 Immunoprecipitation and immunoblotting.  Cells were lysed in TX100 lysis  buffer or RIPA buffer and incubated on ice for 10 min. Insoluble material was pelleted by centrifugation 10,000 x g for 10 min and lysates were adjusted to a protein concentration of 1.0 mg/ml using the BCA Test kit (Pierce), then pre-cleared with Gammabind G Sepharose (Pharmacia). Lysates were then incubated for 1 h on ice with 1.0 pg PTPel5ic anti-sera.  Immune complexes were collected with 10 pL of  Gammabind G sepharose for 1 h at 4 °C On a rotating wheel, and washed 5 times with lysis buffer. Immunoprecipitates were boiled for 10 min in sample buffer and separated using 10% SDS-PAGE before transfer to Duralose (Stratagene) or Immobilon-P membranes (Millipore). Membranes were blocked with 5% (w/v) bovine serum albumin in TBST (20 mM  Tris-HCl pH 7.5, 150 mM  NaCl, 0.05% Tween-20) at room  temperature for 1 h, and incubated with 0.2 pg/ml of polyclonal antiserum (in TBST and 5% bovine serum albumin) for 1 h. Following incubation with the secondary antibody, blots were visualized via an Enhanced Chemiluminescence (ECL) system (Amersham). Cells were also scraped into hypotonic lysis buffer (25 mM Tris-HCl pH 7.5, 2 mM EDTA, 1 mM EGTA, 1 mM Na3V04, 250 mM  sucrose, with protease inhibitors),  26  followed by cell lysis on ice with 20 strokes of a glass dounce homogenizer. Nuclei and large debris were removed by centrifugation at 800 x g for 15 min. The particulate material was obtained by centrifugation at 100,000 x g for 1 h; the supernatant was the cytosolic fraction.  2.13 Immunoflourescence. Cells were grown on coverslips coated overnight with bovine fibronectin at 20 ug/ml. After washing with PBS the cells were fixed with 3.0% paraformaldehyde in PBS for 10 min. at room temperature. Coverslips were washed with PBS then cells permeablized with 0.2% Triton X-100 in PBS. After washing, the cells were blocked with 2.5% BSA in PBS and then stained with PTPel5ic polyclonal antibody at 0.5 ug/ml for 1 hour. After extensive washing the cells were stained with FITC labeled goat anti-rabbit IgG F(ab')2 at 1/250. Coverslips were mounted and photographed with a CCD camera, mounted to a Carl Zeiss  immunoflourescence  microscope, using a standard FITC filter set.  2.14 Surface Biotinylation. HEK 293 cells maintained in log phase growth were cultured to -80% confluency on T175 culture flasks. Cells were transferred to ice and washed 3 times with ice cold HBS (Hepes Buffered Saline) [20 mM Hepes pH 7.4, 150 mM NaCl]. Ten millilitres of Biotinylation buffer [HBS + 0.2 mM NHS-LC-Biotin] was incubated with the washed cells for 1 hour on ice. Biotinylation buffer was removed and cells washed 2x with HBS and remaining active NHS-LC-biotin was neutralized by incubation  27  with three changes of TBS (Tris Buffered Saline) [20 mM  Tris-NH pH 7.4, 150 2  mM  NaCl] 15 minutes each. Cells were then lysed with the addition of TX100 lysis buffer and scraped. Cellular debris was removed by centrifugation and cleared lysates stored at —80 °C.  2.15 Malachite green solution preparation.  One volume of 4.2% (w/v) ammonium  molybdate in 4N HC1 was added to 3 volumes of 0.045% (w/v) malachite green. This solution was stirred for a minimum of 30 min before filtering through a 0.22 um filter. The filtered solution was stored for up to 6 months at 4°C. Tween-20 (0.01% v/v) was added to an aliquot offilteredmalachite green solution prior to use.  2.16 Inorganic phosphate standard curve. Standards were prepared from KH2PO4 that had been desiccated at 80°C for 5 hours. Appropriately diluted inorganic phosphate standards in a volume of 25 ul were delivered to half volume wells of a 96 well microtitre plate, followed by the addition of 50 ul of the malachite green working solution. After a 15 min incubation at room temperature the optical density of each well was measured at 620 nm.  2.17 Phosphotyrosine containing peptides.  Phosphotyrosine containing peptides were  synthesized using solid-phase methods with Y-butyloxyl carbonyl (t-boc) and alpha-  28  protected amino acids, with the appropriated side chain protection, as described (ClarkLewis et al., 1991). Phosphopeptides were synthesized with t-boc Tyr PO4 (Bzl^OH. After chain assembly, the peptides were deprotected via triflouromethane sulfonic acid (TFMSA). Each 100 mg of peptide resin was stripped with 100 pi thioanisole, 50 pi ethandithiol and 1 ml of triflouroacetic acid (TFA). 100 pi of TFMSA was added and allowed to react for 2 hours at room temperature. The peptide was precipitated with diethyl ether, washed, and then dissolved in 6 M guanidine HCl, pH 8.5. For purification this mixture was loaded directly onto reverse phase HPLC (Clark-Lewis et al. 1991). The mass of each peptide was confirmed using ion-spray mass spectroscopy on a model API III triple quadrupole mass spectrometer (SCIEX, Thronhill, Ont.,) with a liquid delivery interface. The amino acid composition was comfirmed by amino acid analysis.  2.18 Phosphatase Assays.  PTPe was immunoprecipitated from varying quantities of  cell lysate protein using 1 pg of polyclonal antibody, as described above but with omission of PTPase inhibitors, followed by two washes with PTP buffer (50 mM NaCl, 50 mM KOAc pH 6.0, 0.2 mM dithiothreitol). Otherwise recombinant PTPe was used. In certain instances during determination of pH optima 50 mM Mes pH6.5-8.0 and 50 mM Tris-HCl pH8.0-9.5 were used. PTPase reactions were carried out (Harder et al., 1994) using the following phosphopeptide substrates, synthesized as previously described (Dechert et al, 1994): s r c PDGF-R.Y1021 STAT91  Y701  Y 5 2 7  (TSTEPQpYQPGENL),  (NEGDNDpYIIPLPD),  hck  EFNy-RaY440  Y  m  (TATESQpYQQQP),  (APTSFGpYDKPHVL),  (GPKGTGpYDCTELI). Reactions were initiated by the addition of 30 pL  29  of PTP buffer containing 200 uM of synthetic phosphopeptide. Inorganic phosphate liberated was quantitated by the addition of 60 ul of malachite green solution then incubated for 15 min at room temperature before optical density measurement at 620 nm.  2.19 Liquid Chromatography/Mass Spectroscopic analysis.  Instrumentation used and  LC-ESI-MS analyses was a modification of those described elsewhere (Hess et al, 1993). Following SDS-PAGE purification and Coomassie blue staining/destaining, protein bands were excised and partially dried. Following complete pulverization of the gel, proteins were digested in  situ  at 37 °C, 16 h in 50 mM ammonium bicarbonate pH  8.3 with either 12.5 ug/ul trypsin (Promega) or chymotrypsin (Sigma). Sample volume was reduced to 10-20 uL in a Speedvac concentrator before loading onto a 320 um (internal diameter) x 15 cm long C18 reverse phase HPLC column (Micro-Tech Scientific). The column was connected via a Rheodyne sample injection valve to the gradient pump outlet of a Michrom Ultrafast Microprotein Analyzer HPLC system (BioResources Inc.). The downstream end of the HPLC column was connected to the ESI interface of a PE-Sciex API-Ill triple quadrupole mass spectrometer (MS) by a 50 cm long x 50 um internal diameter x 150 um outside diameter fused silica capillary (Polymicro Technologies). The chromatography mobile phases were (phase A): 0.05% TFA and 2% acetonitrile, and (phase B): 0.045% TFA, 80% acetonitrile. Tuning and calibration of the MS was performed either with polypropylene glycol or myoglobin. The quadrupole was scannedfromm/z 300 to 2000 at 2.9 s/scan. All mass spectra data were 'background subtracted'.  30  2.20 Kinase assays.  Src was immunoprecipitated from 500 pg of cell lysate with 2 pg  of anti-src monoclonal antibody GD11 as described in section 2.12. Immunoprecipitates were washed 5 times with RIPA buffer followed by a single wash with Kinase buffer (20 mM  PIPES [piperazine-JV, JV-bis 2-ethanesulfonic acid] pH 7.2, 10 mM  MgCl , 5 2  mM  MnCb); Kinase assays were initiated with the addition of 10 pCi [y P] ATP (3000 Ci/mmole) and terminated after 10 minutes at 25 °C by the addition of 1/5 vol. of 5x SDS-PAGE sample loading buffer. Reactions were boiled for 5 minutes then proteins separated via SDS-PAGE. After electrophoresis gels were dried X-ray films exposed.  . 31  Chapter 3  The Screen for PTPases Regulated by IFNyand PTPe Expression in Cells of the Monocyte/Macrophage Lineage  3.1 Differential hybridization Screening System  To investigate PTPase expression in macrophage cells under pro-inflammatory conditions, the mouse macrophage cell line RAW  264.7 was used as a source of  messenger RNA (mRNA) for a differential hybridization PCR screening system. The RAW  264.7 cell line was originally produced from thioglycolate elicited Balb/c  peritoneal macrophages, after transformation by infection with the Ableson Murine Maloney leukemia virus (Raschke et al., 1978). Originally derived from near fully differentiated macrophages, this line exhibits many of the functions associated with true monocyte/macrophage cells while retaining the ability to divide, a cellular function lost in terminally differentiated macrophages. Macrophage functions, including adherence to  32  the substratum, active phagocytosis, p r o d u c t i o n o f T N F a a n d IL-1 i n response to L P S a n d I F N y a n d a l l o f the w e l l k n o w n m a c r o p h a g e c e l l surface markers i n c l u d i n g C D 14, CD16  (FcyRIII),  CD32  (FcyRII), C D 6 4  (FcyRI), a n d the C D l l b / C D 1 8  complex  p r e v i o u s l y k n o w n as Mac-1 o r Complement-3 receptor ( C R 3 )  are a l l expressed b y  RAW264.7  i n response  cells. T h e s e  inflammatory  features i m p l y  that  gene regulation,  cytokines, w i l l c l o s e l y m i m i c that o b s e r v e d  i n ex vivo  to pro-  primary  cells,  m a k i n g R A W 264.7 cells an ideal m o d e l c e l l type f o r s t u d y i n g P T P a s e gene r e g u l a t i o n i n cells o f the monocyte/macrophage lineage.  T h e a p p r o a c h taken was to prepare m R N A f r o m R A W 264.7 cells w i t h or without exposure to m u r i n e I F N y f o r a p e r i o d o f 24 hours (figure 3.1.1). M e s s e n g e r R N A s stimulated R A W  264.7 cells were u s e d as templates to prepare r a n d o m p r i m e d  from  cDNAs.  R a n d o m p r i m i n g was u s e d instead o f p o l y - A p r i m i n g so as not to bias c D N A p r o d u c t i o n towards the 3 p r i m e e n d i n the event that m R N A q u a l i t y v a r i e d from b a t c h to batch. T h e c D N A reactions were then u s e d as substrates f o r the P C R o f P T P a s e catalytic d o m a i n s u s i n g the degenerate oligonucleotides d e g 1 a n d G X G specific f o r h i g h l y c o n s e r v e d n u c l e o t i d e sequences w i t h i n the P T P a s e catalytic d o m a i n (figure 3.1.2) (as d e s c r i b e d i n M a t e r i a l s a n d Methods). T h e o l i g o n u c l e o t i d e sequences were c h o s e n b y c o m p i l a t i o n o f a l l k n o w n P T P a s e catalytic d o m a i n s at the time, i n c l u d i n g b o t h m e m b r a n e p r o x i m a l a n d distal catalytic d o m a i n s i n the case o f dual d o m a i n P T P s . F i g u r e 3.1.2 shows the resulting PTP more  catalytic d o m a i n alignment. A t n u c l e o t i d e sequence sites w h e r e the o c c u r r e n c e o f than  2 nucleotides w a s probable, the n u c l e o s i d e a n a l o g  Inosine ( I ) w a s  33  incorporated because of its ability to hydrogen bond in a stable manner with both purines and pyrimidines. The ability to base pair freely with both types of nucleotides  Differential Hybridization Method of Cloning Novel, Inducible PTPases  murine RAW 264.1 monocyte cell line or normal Human Monocytes  500U/ml ylFN  1-20 hrs.  isolate pA RNA random hexamers reverse transcriptase cDNA degenerate oligos  Taq polymerase  PCR product agarose gel isolate  Slot blot approx. 50ng (1ul) mini prep DNA prepare PCR probe from nonstimulated cells  hybridize O/N wash  pSSBS EcoRV cut  clone PCR product and blue/white selectfor +ve clones  isolate differentially hybridized clones geneclean remainder of DNA prep  confirm white clones with Sail restriction digest  Figure 3.1.1  partially sequence and analyze  Differential hybridization screening  34  Figure 3.1.2 Degenerate oligonucleotide selection  effectively decreases the degeneracy of the oligonucleotide while allowing the maximum number of permutations for annealing.  The expected size of the PCR product was -350 base pairs upon agarose gel electrophoresis (figure 3.1.3a). Amplification fragments this size were purified using the Pharmacia Sephaglass band prep kit and cloned into the EcoRV site of the pUC based plasmid pSSBS (figure 3.1.3b). The plasmid pSSBS is identical to the pBluescript II KS vector with the addition of a second Sail site at the expense of a Xbal site. The Sail sites in pSSBS flank the EcoRV site to allow efficient excision of PCR fragments.  35  1  2  F i g u r e 3.1.3a Degenerate p r i m e r P C R p r o d u c t s . Photograph of ethidium bromide stained 5% agarose gels. Lane 1 PCR products were derived from deg#l, deg#2 degenerate oligonucleotides. Arrow A indicates the 250 bp PCR fragment for subcloning. Lane 2 PCR products were derived from deg#l, deg#3 degenerate oligo-nucleotides. Arrow B indicates the 387 bp PCR fragment for subcloning into the pBluescript derived plasmid, pSSBS.  c D  F i g u r e 3.1.3b C l o n i n g o f P C R fragments i n t o pSSBS. Photograph of ethidium bromide stained 1 % agarose gels containing Sail digests of D N A preperations from pSSBS, PCR fragment ligations. Arrow C indicates the linearized parent plasmid. Arrow D indicates the 287 bp PCR fragment liberated from the plasmid.  One hundred nanograms of each clone was immobilized onto nylon membranes and subjected to hybridization with a [a P]-dCTP labeled PCR probe prepared from cDNA reverse transcribed from an unstimulated mRNA sample. After extensive high stringency washing candidate upregulated PTPase cDNAs were chosen by an under represented signal on autoradiography (figure 3.1.4).  36  t  I  M l  I  I  I I I I  I  M  i  I I  l  l  H l i i i l l  II  I  1 1 1 I I f  M U M  I I I ! 11  M J |  Figure 3.1.4 Autoradiograph of PTPase PCR fragments. Autoradiograph of  slot blotted plasmids containing PTPase PCR fragments from stimulated R A W 264.7 cells. Blots were probed with P a d C T P labeled PCR reactions using m R N A purified from non-stimulated cells. After hybridization with P labeled probes blots were washed with high stringency. Dashes indicate m R N A species corresponding to this plasmid is not present in non-stimulated R A W 264.7 cells. Dots indicate the possibility of low abundance messages. 32  3 2  Candidate clones were manually sequenced using T3 and T7 sequencing primers. Approximately 250 base pairs of nucleic acid sequences were obtained from each clone. Sequence analysis of the candidate PTPases was carried out on a Sparc Unix workstation using Blast or Fasta sequence similarity analysis programs with global matching, a ktup of 4, and no gaps. Sequence searches were carried out against the rodent nucleotide database within Genbank.  Screening in this manner, using IFNy stimulation varying from 6 hours to 24 hours, of 173 clones isolated 12 clones were designated as putatively regulated PTPases. Sequence analysis of the 12 candidate clones provided only 4 distinct PTPases cDNAs, 3  37  transmembrane PTPases and one cytosolic. PTPa, CD45, PTP1B and PTPe were identified as candidate mRNAs for induction in mouse macrophages in response to IFNy and highlighted for further investigation.  Each of these PTPs identified in our search had been previously cloned and characterized. PTPa is a widely expressed transmembrane PTPase possessing two tandem catalytic domains and is characterized by a small heavily glycosylated extracellular domain. Recent reports implicate PTPa in regulation of pp60  csrc  activity and in  control of neurite out-growth and neuronal differentiation (den Hertog et al, 1993). CD45, also known as leukocyte common antigen (LCA), is a well-known leukocyte cell surface marker (Thomas 1989). Characterized by highly regulated exon splicing to produce several variants with distinct extra-cellular domains, CD45 has also been shown to play a role in the regulation of src family kinases (Mustelin et al, 1989, 1990, 1992, Trowbridge et al, 1991, Ostergaard et al, 1990). The PTPase activity of CD45 has been shown essential in the activation of p561ck during antigen receptor stimulation in T lymphocytes and p53/561yn in the case of B-lymphocytes (Weiss 1993, Ales-Martinez et al, 1991). Unlike the two previous candidates, PTP1B is a non-transmembrane PTPase (Tonks et al, 1989). This PTPase appears to be activated by proteolytic cleavage at its Cterminal tail, which anchors the PTPase to the endoplasmic reticulum (Frangioni et al, 1993). While its function remains unclear, PTP1B has been implicated in insulin receptor signaling, EGF receptor signalling as well as intra-ER tyrosine kinase dephosphorylation and regulation (Lammers et al, 1997, Milarski et al, 1993). PTPe, the last of the four PTPases identified, shares very high sequence homology with PTPa (-85%) and is also  38  characterized b y a small extra-cellular domain however PTPs's extra-cellular domain shows no sequence similarity with that o f PTPa. Although little is known o f the function o f P T P s , recent reports have implicated PTPs i n cellular adhesion and/or insulin receptor signaling (Lammers et al,  1997). Due to the paucity o f information regarding the  function o f PTPs, i n addition to its homology with P T P a which may afford re-use o f certain existing reagents, PTPs was chosen for further analysis as an upregulated PTPase in the macrophage cell line.  3.2 Northern Blot Analysis of PTPe Expression.  For further analysis o f PTPs expression, the human monocyte cell line H L - 6 0 was included as a representative human cell line with macrophage/monocyte characteristics. Developed i n 1979 i n the laboratory o f R . C . Gallo at N I H , H L - 6 0 was derived from a patient with leukemia (Collins et al, 1978). A growth factor independent cell line with myeloid characteristics, H L - 6 0 is capable o f differentiation i n response to a number o f agents. A s a result, H L - 6 0 cells have been induced to differentiate towards a variety o f hemopoietic  cell  lineages.  DMSO,  and  retinoic  acid  cause  granulocyte-like  differentiation. Vitamin D 3 and J-FNy cause monocyte differentiation, while treatment with phorbol-esters for 48 hours causes adherence to plastic, cessation o f proliferation, and an increase i n the following: complement receptors, F c receptors, lysozyme activity, phagocytosis, microbicidal activity, src family kinase expression. These characteristics and others identify phorbol ester-differentiated H L - 6 0 cells as macrophage-like cells,  39  possibly making these a human counterpart for RAW 264.7 cells. In addition, HL-60 affords  the  opportunity to  study  PTPe  expression  during the  process  of  macrophage/monocyte differentiation. To observe PTPe mRNA expression in the two representative cell lines, oligonucleotides corresponding to the 5 prime membrane proximal area of human PTPe (ptpel5) and to the 3 prime region of human PTPe (ptpe25) were used to PCR amplify the cytosolic portion of human PTPe. Complementary DNA reverse transcribedfromHL60 mRNA was used as a substrate in the PCR reactions and purified DNA fragments were used as DNA probes as well as subcloned into the prokaryotic expression vector pGEX-2T.  Messenger RNA purified from RAW 264.7 cells and HL-60 cells after treatment with various stimuli was subjected to Northern blot analysis with human PTPe probes. Figure 3.2.1 reveals that PTPe mRNA level is induced in HL-60 cells only after stimulation with a few key pro-inflammatory stimuli. Lipopolysaccaride (LPS) (figure 3.2.1a) and IFNy (figure 3.2.1b) both induce -6.5 kb, -2.1 kb and -1.8 kb PTPe messages in HL-60. The presence of three hybridizing transcripts was unexpected since only a single -8 kilobase message had been previously reported for PTPe (Krueger et al. 1990). However, similar transcript heterogeneity was observed in MEL cells treated with various stimuli (Kume et al, 1994). In addition, evidence of as many as four different transcripts was obtained from analysis of various mouse mammary tumor lines (Elson and Leder, 1995a). Since high stringency conditions were used during hybridization and subsequent washings, the possibility of a cross-reacting PTPase message, while not eliminated, is  40  unlikely. The most probable explanation for the number o f messages observed is alternative splicing o f a single primary transcript. The presence o f alternative transcripts is a common feature o f transmembrane PTPases.  Figure 3.2.1 shows a representative  northern blot  from  four  independent  experiments. The induction o f PTPe message followed similar kinetics regardless o f whether L P S or IFNy was used as the stimulus. W h i l e PTPe m R N A was detectable in control unstimulated cells, an increase in levels was not detectable until at least 3 hours had elapsed (figure 3.2.1). Induction o f PTPe message generally reached maximal levels after approximately 20 hours o f stimulation (figure  <  PTPe B PTPs  <4  1  2  3  4  5  [3-actin  1  2  iH  3  4  5  IFNy <  <  PTPe  p-actin  PMA PTPe  Figure 3.2.1. PTPe message levels in response to, L P S (a) and IFNy (b) Phorbol esters (c). PTPe message levels observed by Northern blot analysis in response to 1.0 ug/ml LPS (A), 100 U/ml IFNy (B), or 16 n M P M A (C). Lane 1, unstimulated control; lane 2, 1 hour exposure; lane 3, 3 hour exposure; lane 4, 6 hour exposure; lane 5, 20 hour exposure. Lower panel represents human p-actin re-probe of the blot above.  41  3.2.1) with densitometric analysis revealing a 16-fold (+/- 0.4) increase in message levels (figure 3.2.1). While the mechanism responsible for PTPe induction in response to LPS and IFNy remains to be elucidated, figure 3.2.2 shows a similar induction of PTPe message in response to ortho-Vanadate, a powerful PTPase inhibitor.  To assess PTPe expression at the message level in response to other proinflammatory stimuli a panel of hemopoietic growth factors, as well as chemotactic cytokines (chemokines) were used to treat HL-60 cells in a time course from 1 hour to 20 hours. Figure 3.2.3 shows the PTPe mRNA expression profiles from these experiments, using GM-CSF, IL-1, MCP-1 as the pro-inflammatory stimuli. Interestingly,  1  2  3  4  5 PTPE  Figure 3.2.2 Expression of P T P e mRNA in response to Vanadate.  MRNA isolated from HL-60 cells in response to 50 | j M Vanadate. (Lane 1) control unstimulated cells. (Lane 2) cells stimulated for 1 hour, (Lane 3) 3 hour stimulation, (Lane 4) 6 hour stimulation, (Lane 5) 20 hour stimulation.  of the stimuli tested only MCP-1 elicited a modest induction of PTPe messenger RNA. The mRNA expression levels appear to show a gradual increase in response to LPS and IFNy, as well as phorbol esters.  <  B  *  PTPE  GM-CSF  PTPE  IL-1  PTPE  MCP-1  Figure 3.2.3. Expression of P T P E m R N A in response to Cytokine stimulation. M R N A isolated from control unstimulated HL-60 cells (Lane 1). (Lane 2) cells stimulated for 1 hour, (Lane 3) 3 hour stimulation, (Lane 4) 6 hour stimulation, (Lane 5) 20 hour stimulation. With 200 ng/ml G M - C S F (panel A), 40 U/ml IL-1 a (panel B), 200 ng/ml MCP-1 (panel Q .  3.3 Expression of PTPs protein in hemopoietic cells  To study the product of the PTPs gene, three rabbit polyclonal antibodies were generated against synthetic peptides corresponding to regions within PTPs unique to the PTPase. PTPsEC polyclonal antisera was generated against the synthetic peptide sequence PTPs 21-44 (RGNETTADSNETTTSGPPDPGASQ). This sequence  43  | 1 MEPFCPLLLASFSLSLARAG aGNDTTPTESNWTSTTAGPPDPGASQ PLLTWLLLPLTiTiTiT, MEPLCPLLLVGFSLPLARAL RGNETTADSNE-TTTTSGPPDPGASQ ?LLAWLLLPLLLLL ** ** * * * * * * * * * * * * k** * * * * * * * * * * *** ***** *******  1  F--LLAAYFFRFRKQRKAWSSNDKKMPNGILE^ LVULiLAAYFFRFRKQEKAWSTSDKKMPNGIU:^  ******************  ***************************** *******  IJ25EIRVRSADIX?KRFREEFNSLPSGHIQGTFEI^ T .P.RRTRTR.qAnnrKQFRKRFMg!T .PSflHTQCrTFRT AMKKKMRRKNRVPWTT .PWDHSRVTT .S **************************************************** ***** QVTCIPCSIWINASYIDGYKEKNKFIAAQGPKQFVIWDFWR^ QLIX5IPCSDYINASYIIX3YKFJQQKFIAACX5PKQETV^  * ******************************************** *************  KEFJCCYQYWrXXOnYGNIRVCVEIX^A/LVDYTIR^ KEEKCHQYWIXXSCVmrGNIRVCV^^ *****  IHPQLPDSCKAPRLVSQLHFT IQPQLPDGCKAPRLVSQLHFT ********************************** ***** *************  SWPDFGVPFTPIGMLKFLKKVKTLNPSHAGPIVVHCSAGVGRTGTFIV SWPDFGWFTPIGMLKFTjKKvTCTLOTVHAGPIVVHCSAGVGRTGTF^ ************************** **************************  * * **  KVIWFEFVSRIRNQRPQMVQTDVQYTFIYQAT ,T iKYYLYGDTELDv'SSLERHLQ TLHSTAT KVDVFEFVSRIRNQRPQMVQTEMQYTFIYQALLEYYLYGDTELDVSSLEKHLQ rMHGTTT  ********************** ************************** ****Mr  *  *  *  HFDKI 3T,F,KF,FRKLTOMmiKE3^TGNLPANMKKARVIQIIPYDFN^ HFDKI 3LEEEFRKLTNVRIMKENMRTG^PANMKKARVIQI IPYDFNRVILSMKRGQEYT  ********************************************************** *  DYINASFIIX3YRQKDYFMATC<3PLAHTVEDFWRMVWEWK^  DYINASFIKSYRQKDYFIATCXSPLAHTVEDFWRM^ ***************** **************** ************************* WPTEGSVTHGDITIE rKSDTLSEAISVRDFLVTFKQPLARQEEQVRMV XQFHFHGWPEVG WPTEGSVTHGEITIE rKNDTLSEAISIRDFLVTLNQPQARQEEQVRVV S.QFHFHGWPEIG ********** ****** ******** ****** ** ******** *********** * IPAEGKGMIDL:Ti^VQKQC4X2TG^PI IPAEGKGMIDLIAAVQKC<XOTGNHPIT^  ************************************************************ QAVKSLRLQRPHMVQTLF^YFJCTKVVQDFIDIFSDYANFK QAVKSLRLQRPHMVQTLEQYEFCYKWQDFIDIFSDYANFK ***************************************** Figure 3.3.1  Regions of antibody peptide selection in PTPe The polypeptide sequences of murine  PTPe above and human PTPe below. Identical amino acids are indicated with a star, homologous substitutions with a dot. The regions highlighted in gray indicate the putative signal peptide and tranmembrane domian. The first catalytic domain and second tandem catalytic domain are indicated by horizontal line. Boxed regions denote areas used as templates for synthesis of immunogens for polv clonal antibodies.  44  PTPsl5ic was ( T M H G T T T H F D K I )  generated against the amino acid sequence PTPe 413-424 which is found with the intervening region between the tandem  catalytic domains (figure 3.3.1). PTPsl6d2 was raised against the oligo-peptide PTPs 555-587  ( I K N D T L S E A I S I P v D F L V T L N Q P Q A R Q E E Q V R V V )  and is found within the C-  terminal catalytic domain of PTPs (figure 3.3.1). The sequence PTPs 555-587 along with those peptides used for the previously described antibodies also exhibit little sequence homology with PTPa.  Crude antiserum was purified by column chromatography using the immunizing peptide linked to thiol-Sepharose beads as the affinity purification resin. The resulting polyclonal antibodies were tested for specificity and affinity towards PTPs and PTPa by immunoblotting samples of the recombinant proteins (expression of rPTPs and rPTPa is described  elsewhere). The  antibodies were also  tested  for their ability to  immunoprecipitate PTPe and PTPa by immunoprecipitation from crude bacterial lysates expressing recombinant PTPs and PTPa G S T fusion proteins. Figure 3.3.2 summarizes the results of the antibody characterization. Lanes 1,3,5 and 7 in the upper panel indicate that PTPsl5ic, PTPsl6ic and anti-LRP (the PTPa specific antisera) are all able to immunoblot for PTPs. Indeed, PTPsl5ic appears to be the best antibody for immunoblotting with PTPel6ic exhibiting a somewhat lower signal. Lanes 2, 4, 6, and 8 provide internal controls since these lanes contain the ~60kDa GST-PTPeD2 fusion protein which migrates at a lower molecular weight than the larger GST-PTPe fusion protein. The lower panel of figure 3.3.2 indicates that all antisera tested except for antiL R P 150 (a PTPa specific antibody) are capable of immunoprecipitating GST-PTPe 45  fusion protein from bacterial cells overexpressing the fusion protein. Thus, for subsequent boosts, purifications and all further studies PTPel5ic and PTPel6d2 were used  for  protein  characterization  since,  both  PTPel5ic  and  PTPsl6d2  immunoprecipitated and immunoblotted recombinant PTPe but not PTPa. This implied that the reagents displayed high reacitivity towards PTPe and therefore it is fair to say specific for PTPe and unlikely to react with other PTPs. These results confirm that experiments performed with PTPel5ic and PTPel6d2 should not produce ambiguous results due to cross-reactivity with PTPa.  As previously reported, treatment of HL-60 cells with P M A resulted in cells having a monocyte-like appearance (Collins et al., 1987). Thus, within ~24 h of P M A exposure significant morphological changes were seen including vacuole formation and adhesion to tissue culture plastic of cells with a monocytoid appearance. After 2 to 3 days, maximum attachment of cells was observed and was accompanied by minimal cell death. Continuous exposure was not required for phorbol ester-mediated differentiation of HL-60 cells. Thus, after 2 days of stimulation, adherent cells were washed and cultivated in PMA-free media for a final 24 h to obtain HL-60/mac cells.  46  1  2  3  4  5  6  7  8 rPTPe  1  2  3  4  5  6  7  rPTPe  Figure 3.3.2 Testing of PTPe specific antibodies with recombinant PTPe. Upper Panel Lysates prepared from bacteria expressing GST-PTPe (lanes 1, 3, 5, 7) GST-PTPeD2 (lanes 2, 4, 6, 8) were western blotted with the following polyclonal antisera PTPel5ic (lanes 1 and 2), PTPel6ic (lanes 3 and 4), PTPel7ic (lanes 5 and 6), anti-LRP (anti-PTPa) (lanes 7 and 8). Lower Panel Lysates prepared from bacteria expressing  GST-PTPe  were  subject to  immunoprecipitation with 2.5 ng of the following antisera PTPel4ic (lane 1), PTPel5ic (lane 2), PTPel6ic (lane 3), PTPel7ic (lane 4), anti-LRP150 (lane 5), anti-LRP (lane 6), anti-LRPD2 (lane 7). Immunopreciptitates were then western blotted with PTPel5ic anti - P T P e polyclonal antibody.  Figure 3.3.3 illustrates the relative levels of PTPel5ic antibody-reactive proteins in PMA-stimulated HL-60 total cell lysates harvested at various time points as shown. The antibody detected a single major protein, migrating as a doublet, of approximately 72/74 kDa and a third cross-reacting protein migrating at ~85 kD.  The signal  corresponding to the p72/74 protein was present at low levels until approximately 20 hours post-PMA exposure of HL-60 cells. Once present, the level of p72/74 in the HL60 cells appeared to remain stable for up to 72 hours in culture (data not shown). The expression of p72/74 appears to correlate well with the mRNA expression patterns observed in the phorbol ester stimulation, figure 3.2.2. However, p72/74 expression does not correlate well with mRNA expression patterns in the LPS and IFNy stimulation (figure 3.2.1). This discrepancy in mRNA expression patterns was addressed in section 3.2. However, it appears the hypothesis for mRNA expression may be extended to  47  describe protein expression especially in the face observations that induction of message by LPS and IFNy doesn't result in protein translation in HL-60 cells as described in figure 3.3.4. These results further suggest that while they are able to rapidly induce mRNA expression and translation, LPS and IFNy are only capable of delivering a partial signal, which results in increased message but a block at the translation level. It is interesting that while PMA induction of PTPE results in mRNA expression at 3 hours, expression of PTPe protein (p72/74) is not observed until 20 hours of exposure. This time point coincides with morphological changes in HL-60 cells that are commensurate with adherence to the substratum. It might be speculated that mRNA upregulation and a second signal, such as adherence to the substratum is required for p72/74 expression.  97.4 kDa 69kDa  p72r74  46kDa Figure 3.3.3 Expression of PTPe in P M A treated HL-60 cells Immunoblot of HL-60 cell lysates, 10 (ag per lane, with PTPel5ic polyclonal antibody (0.2 ug/ml) following P M A treatment for the indicated times.  To compare the SDS-PAGE migration characteristics of the oberved p72/74 with the previously cloned cDNA for PTPs the pBCMGSneo eukaryotic expression vector  48  was u s e d to transfect H E K 2 9 3 cells w i t h a P T P e c D N A e n c o d i n g a p r e d i c t e d - 8 0 k D a transmembrane  form  of  this  phosphatase  (Krueger  et  al, 1990).  The  human  c y t o m e g a l o v i r u s i m m e d i a t e - e a r l y gene p r o m o t e r w a s u s e d to d r i v e e x p r e s s i o n o f a - 1 0 0 k D a p r o t e i n , that was r e c o g n i z e d b y the P T P s l 6 d 2 antibodies b y i m m u n o p r e c i p i t a t i o n (figure 3.3.4) as w e l l as i m m u n o b l o t t i n g ( F i g 3.3.4, lanes 1 t h r o u g h 8). T h i s p o l y p e p t i d e species  correlated  transmembrane  well  with  the  molecular  weight  of  the  putative  full-length  i s o f o r m o f P T P s . R e c o g n i t i o n o f b o t h p 7 2 / 7 4 a n d p 100/110 b y b o t h  P T P e l 5 i c p o l y c l o n a l a n t i b o d y (figure 3.3.4, lanes 3 a n d 4) and a n t i b o d y P T P e l 6 d 2 (figure 3.3.4, lanes 7 a n d 8) c o n f i r m e d the s p e c i f i c i t y o f the a n t i b o d y reagents for P T P s p o l y p e p t i d e s . T h e s e observations also i n d i c a t e d that b o t h P T P e s p e c i f i c epitopes  are  c o n t a i n e d w i t h i n the n o v e l p 7 2 / 7 4 species suggesting this p o l y p e p t i d e is a n i s o f o r m o f transmembrane P T P e . T h e P T P e l 6 d 2 antibodies appeared to be o f l o w e r a f f i n i t y than the P T P e l 5 i c antibodies o w i n g to their w e a k e r a b i l i t y to i m m u n o p r e c i p i t a t e p 7 2 / 7 4 a n d p l 0 0 / 1 1 0 (figure 3.3.4 lanes 7 and 8). T h e difference b e t w e e n the p r e d i c t e d (~80 k D a ) a n d o b s e r v e d (100 k D a ) m o l e c u l a r w e i g h t s for the transmembrane f o r m o f P T P e m a y h a v e b e e n due to g l y c o s y l a t i o n ( E l s o n and L e d e r , 1995a) as a diffuse b a n d , i n d i c a t i v e o f heterogeneous g l y c o s y l a t i o n , m i g r a t i n g at a p p r o x i m a t e l y 110 k D a w a s also o b s e r v e d .  49  1 2  3  4  5  6  7  8 pi (KM 110  97.4 kDa  p72/74  69 kDa  lysate PTPe 15c IP PTPe16d2IP  Figure 3.3.4, Expression of PTPe. Lysates obtained from control 293 cells (lane 1 and 5), and from 293 cells transfected with a c D N A encoding the transmembrane form of PTPe (lane 2 and 6). Lysates obtained from HL-60/mac cells (lanes 4 and 8) and from 293 cells transfected with the transmembrane form of PTPe (lanes 3 and 7 ) were immunoprecipitated with PTPel5ic antibodies (lanes 3 and 4 ) or with PTPel6d2 antibodies (lanes 7 and 8) and immunoblotted with PTPel6d2 antibodies. Lanes 1 through 4 were blotted with PTPel5ic. Lanes 5 through 8 were blotted with PTPel6d2.  An increase in the level of p72/74 was also seen in immunoprecipitations with PTPsl5ic antibodies from detergent-soluble lysates of PMA-treated, as compared to untreated, HL60 cells (figure 3.3.5). By densitometry this corresponded to a ~16-fold increase in p72/74-specific signal on the film. In contrast to the results obtained using PMA, differentiation of HL-60 cells with DMSO resulted in only slight upregulation of PTPe (figure 3.3.6). As can also be seen in figure 3.3.5, p72/74 was also detected in the Jurkat T-lymphocyte cell line, and in thioglycollate-elicited peritoneal-exudate derived cells.  1  2  3 p72/74  69 kDa  IgH 46 kDa  I  Figure  3.3.5,  ,.JL  JL.  PTPe  I I I expression  in  hemopoietic  cells.  PTPe was  immunoprecipitated from HL-60/mac cells (lanes 1 and 2) with PTPel5ic (2.0 ug) antibodies or normal rabbit serum control (lane 3), Jurkat cells (lane 4) or mouse thioglycollate-elicited  peritoneal-exudate  adherent cells  (lane  5).  PTPe  was  immunoblotted with PTPel5ic antibodies (0.2 ug/ml).  50  Detection of p72/74 in peritoneal macrophages as well as the Jurkat T cell line indicated that PTPs expression was not limited to hemopoietic cell lines, or to macrophages. However, in DMSO-mediated differentiation of HL-60 cells, which resulted in granulocytic differentiation, only a weak upregulation of p72/74 was observed indicating that while p72/74PTPe is expressed in a variety of hemopoietic cells and cell lines, its expression may not be ubiquitous in hemopoietic cells.  97.4 kDa 69kDa 46kDa  p72r74 IgH  Figure 3.3.6, PTPe expression during Granulocytic differentiation of HL-60 cells. PTPel5ic immunoprecipitates from untreated (lane 1), dimethyl sulfoxidetreated (Me2SO) (lanes 2 and 3), or PMA-treated Triton X-100 solublized HL-60 cells (lane 4), were immunoblotted with the PTPel5ic antibody.  51  3.4p72/74possesses PTPase activity  time (min)  Fig. 3.4.1 Phosphatase activity of immunoprecipitated p72/74. Inorganic phosphate release, quantitated by the Malachite green method, was plotted as a function of time in PTPase assays carried out using 200 synthetic phosphopeptides with p72/74 protein obtained by immunoprecipitation of HL-60/mac lysates with PTPsl5ic antibodies. A l l measurements were background subtracted using immunoprecipitation PTPase reactions in the absence of PTPel5ic. (o) I F N y - R a STAT91  Y 7 0 1  , (•) P D G F - R  Y 1 0 2 1  .  Y 4 4 0  ,  (A) hck  ,  Y501  (*) src  ,  Y527  Inset shows calibration of phosphate release from the P D G F - R  (0) Y 1 0 2 1  phosphopeptide using p72/74 immunoprecipitated from HL-60/mac cells using 1.0 ug of PTPsl5ic and increasing amounts of cell lysate.  52  V  km PDGFR-Y  1 0 2 1  Src-Y™ Hck-Y>  m  STAT-Y'"' IFNyR-Y-440  Vmax/kfn  197 +/- 9 288 +/- 15 293 +/- 4 882 +/- 34  265 m +/-12 ax 589 +/-30 455 +/-16 77 +/-13  0.09  -  -  -  v  1.3 2 1.6  Figure 3:4.2 Kinetic analysis of p72/74PTPe. PTPs was immunoprecipitated from HL-60/mac cells lysates using PTPel5ic polyclonal antibody. Immunoprecipitates were subjected to PTP assays as described in sections 2.12 and 2.18 (pg 25, pg 28). Km and Vmax values obtained are comparable to those observed with recombinant PTPe.  As can be seen in figure 3.4.1, PTPel5ic immunoprecipitated material contains an active PTPase. The inset of figure 3.4.1 shows the increasing activity immunoprecipitated with PTPsl5ic antibody using increasing amount of HL-60 crude protein lysate. From these results it was concluded that p72/74 was a PTPase. Experiments outlined in section 3.3 indicated that p72/74 was detectable by two different antibodies both specific for PTPe  provided convincing evidence that p72/74 was likely the only PTPase  immunoprecipitated using PTPel 5 ic" antibodies and was indeed a PTPe isoform. Further experiments in section 3.5 however provide confirmation of this statement.  A survey of the following synthetic peptide substrates: IFNy-Ra src \ Y52  STAT91  Y701  , and PDGF-R  Y1021  , indicated that PDGF-R  substrate, in vitro, of the substrates tested, with STAT91  Y701  Y1021  , hck  ,  was the best  being the least favoured  substrate. Figure 3.4.2 shows the kinetic parameters for the time course presented in figure 3.4.1, obtained from three independent experiments. The results of these analyses  53  indicate that p72/74 also exhibits some degree of substrate specificity, a finding that potentially sheds some light on p72/74 function. It appeared that p72/74 displayed similar kinetic parameters to that observed with recombinant PTPs. The synthetic substrates appeared similar in their preference by PTPe. In general, though, the k values obtained m  with p72/74 were higher than those observed with recombinant PTPe. This may be due to the method of preparation of the immunoprecipitated material. In chapter 4 this data will be incorporated into a more in-depth analysis of the enzyme characteristics of PTPe utilizing recombinant PTPe in conjunction with immunoprecipitated material.  3.5 Liquid Chromatography/Mass Spectrometric analysis of p74  Peptide  Location  Molecular Predicted  Mass Observed  VDVFEFVSR  361-369  1097.56  1097.0  VIQIIPYDFNR  457-467  1377.75  1376.5  NDTLSEAISIR  557-567  1218.63  1218.0  DFLVTLNQPQAR  568-579  1401.75  1402.5  TGTFIALSNILER  637-649  1434.80  1433.5  AEGLLDVFQAVK  652-663  1289.71  1289.5  Table 3.5.1, Liquid Chromatography/Mass Spectroscopic analysis of p74 tryptic peptides. The sequences and molecular masses of p74-derived tryptic peptides are shown. Peptides whose fragmentation patterns revealed identifying sequence information are shown in bold lettering. Those not in bold displayed corresponding peptide masses in PTPe. Sequence coordinates for the peptides were according to Krueger et al. (1990).  54  As the molecular weight of PTPe, predicted from the cDNA (Krueger et al.,  1990)  in the absence of glycosylation was -80 kDa, it was critical to characterize the p72/74 protein further before it was considered to be a PTPe isoform. Thus, following  PMA  treatment, detergent-soluble lysates of large-scale HL-60/mac cell preparations were subjected to immunoprecipitation with PTPel5ic. After SDS-PAGE, Coomassie staining demonstrated the 72/74 kDa was a protein doublet (data not shown). To determine the nature of the p74 species, LC/MS analysis was performed on the more abundant -74 kDa gel band. LC-ESI-MS analysis revealed several peptide fragments in both the trypsin and chymotrypsin digested samples. The measured masses of several of the peptides matched predicted fragment masses derived from the PTPe sequence (Table 3.5.1). In addition, the mass spectra of three peptides generatedfragmentationpatterns corresponding to the predicted internal amino acid sequence of PTPe (Table 3.5.1). Peptides corresponding to the predicted extracellular domain of the mature PTPe protein (Krueger et al., 1990), however, were not identified.  3.6 Expression of the cDNA encoding non-transmembrane PTPe  With the cloning and transmembrane and  sequencing of a PTPe cDNA which lacked the  extracellular domain (Elson and  Leder  1995a) from the  monocyte/macrophage cell line HL-60, many questions were answered as to the origin and nature of the smaller PTPe isoform. However, the question arises as to how the 72/74 kDa doublet is generated. Post-translatiorial modifications, such as phosphorylation or proteolysis, may account for the doublet, however, one of the four messages observed by  55  Northern blotting may give rise to one of the polypeptides, in which case the cDNA cloned by Elson and Leder likely corresponds to one of the species in the 72/74 doublet. To determine the origin of the p72/74 doublet, the PTPe cytosolic cDNA under the control of the EF-la promoter was transiently transfected using the eukaryotic expression vector pcDEF3, into HEK 293 cells by lipofection. Expression of PTPe was observed by immunoprecipitation and immunoblotting with anti-PTPe polyclonal antibodies. As shown in figure 3.6.1, using PTPe immunoprecipitated from HL-60 lysates as a control (lane 1) as well as immunoprecipitates (lane 3) only the 74 kDa band of the p72/74 doublet was observed upon transfection with the cytosolic PTPe cDNA (lane 2). This was true for both hemopoietic and non-hemopoietic cell lines as the same results were obtained when non-transmembrane PTPe was transfected in to BaF/3 as well as Jurkat  1  23 72r74  69kDa  P  12  69 kDa  3 4  HE! 12  34  69 kDa Figure  3.6.1,  p74  p74  Expression of non-transmembrane isoform of PTPe. (Top panel) PTPel5ic HL-60 cell lysate (lane 1), PTPsl5ic  polyclonal antibody immunoblots of PMA-treated  immunoprecipitates from 293 cells transiently transfected with non-transmembrane PTPe (lane 2), PTPel5ic immunoprecipitates from PMA-treated  HL-60  cells (lane 3). (Middle panel)  PTPel5ic immunoblots of Jurkat cells transiently transfected with; control vector, 10 ug of protein lysate (lane 1), non-transmembrane PTPe cDNA, 10 ug of protein lysate (lane 2), control vector, PTPe immunoprecipitated with PTPel5ic antibody (lane 3), non-transmembrane PTPe cDNA, PTPe immunoprecipitated with PTPel5ic polyclonal antibody (lane 4). (Bottom panel) PTPel5ic immunoblots of BaF/3 cells transiently transfected with; control vector, 10 ug of protein lysate (lane 1), non-transmembrane PTPe cDNA, 10 pg of protein lysate (lane 2), control vector, PTPe immunoprecipitated with PTPel5ic antibody (lane 3), non-transmembrane PTPe c D N A , PTPe immunoprecipitated with PTPel5ic polyclonal antibody (lane 4).  56  cells lines (figure 3.6.1).  ^  'Jt  ^  ^  #  97.4 kDa 69 kDa  • p7274  • M&jm t^tm.,. -mmm*  46kDa  •^^^^^  "^IjPlippppBft  1  —f  igH  Figure 3.6.2, PTPs sub-cellular localization. PTPel5ic immune-precipitates  from Triton X-100 solublized PMA-treated HL-60 cells (lane 1), crude cytosol (lane 2) or crude  particulate (lane 3) protein immunoprecipitates were immunoblotted with PTPel5ic antibodies.  To determine the sub-cellular localization of the p72/74 doublet, membrane and cytosolic fractions were prepared from HL-60/mac cells. Anti-PTPe immunoprecipitates using l.Opg of PTPel5ic antibody were then compared from 1.0 mg of detergent soluble lysate, 500 pg of cytosolic fraction and 500 pg of the crude membrane fraction. As shown in figure 3.6.2 the majority of p72/74 was found in the cytosolic fraction of the HL-60/mac cells (lane 2). Densitometric analysis revealed that ~94% of the p72/74 signal was within the cytosolic fraction, with only -6% detected within the particulate fraction. Figure 3.6.2 , lane 3 also shows that of the two isoforms observed, p72/74, only the p74  57  isoform could be detected within the particulate fraction. This suggests the p72/74 isoforms can be differentially localized.  3.7 Phosphorylation of PTPe  Examples o f enzyme regulation by post-translational modifications are common. Protease cleavage, glycosylation and phosphorylation are just a few methods employed by biological systems to regulate enzymes. A number o f PTPases have been reported to become phosphorylated on serine, threonine, and tyrosine residue and to be regulated by these modifications (Flint et al, 1993, Bouchard et al, 1994, Stein-Garlach et al, 1995). Indeed, P T P e ' s closest relative, P T P a , has been shown to be phosphorylated on tyrosine as well as serine and threonine residues, the latter reported to be P K C dependent (Su et al, 1994, Hunter et al, 1995). In light o f these observations, phosphorylation o f P T P s was examined in resting H L - 6 0 cells as well as i n response to pervanadate stimulation. 1 2  3  p72/74  Figure 3.7.1 Phosphorylation of PTPe in HL-60/mac 69 kDa. cells. PTPe was immunoprecipitated, using PTPel5ic polyclonal antibody, from P M A differentiated HL-60 cells after labeling with P for 4 hours. (Top Panel) 18 46 kDa hour exposure. Unstimulated cells (lane 1), cells stimulated with pervanadate for 1 minute (lane 2), cells stimulated with pervanadate for 15 minutes (lane 3). (Bottom Panel) Blot in top panel immunoblotted with anti-phosphotyrosine monoclonal antibody. 3 2  1 2  3 p95  69 kDa 46kDa-  p72/74 p55 p46  58  3.7.1 Pervanadate induced tyrosine phosphorylation of PTPs  As illustrated by PTPa and the SH2 domain containing PTPases, PTPases may themselves be regulated by tyrosine phosphorylation events (Sun and Tonks, 1994). To induce the tyrosine phosphorylation of p72/74, the PTPase-inhibiting/PTK-activating agent pervanadate was employed (Heffetz et al., 1990; Secrist et al., 1993; Posner et al., 1994). P labelling of HL-60/mac cells revealed that p72/74 was phosphorylated at low 3 2  levels in resting cells but upon pervanadate stimulation the amount of  P labelled p72/74  increased ~8 fold. The anti-phosphotyrosine specific monoclonal antibody 4G10 was used for immunoblotting of gel-separated proteins. Triton X-100 solublized cell lysates showed a dramatic increase in phosphotyrosine in response to pervanadate stimulation (Figure 3.7.1 (lower panel), lanes 1 and 2). Phosphotyrosine was not detectable within p72/74 immunoprecipitates obtained from resting HL-60/mac cells (Figure 3.7.1 (lower panel), lane 1). However, following 15 min exposure to O.lmM pervanadate, a number of phosphotyrosine containing proteins were observed, including p72/74 (Fig. 3.7.1 (lower panel), lane 3). The p72/74-associated proteins were of approximate molecular weights 46 kDa, 55 kDa, 80 kDa and 95 kDa.  59  To determine the nature o f the p72/74 associating proteins in pervanadatestimulated HL-60/mac cells, immunoprecipitates were examined for the presence o f a number o f different molecules known to be commonly involved in signal transduction processes. Thus, based on estimated molecular weights o f phosphoproteins observed to be associated with p72/74 immunoprecipitated from pervanadate-stimulated cells (figure 3.7.2), antibodies against the following proteins were evaluated: csk, hck, lyn, She, S H P T P 2 , S T A T 9 1 , syk, vav. However none o f the tested proeins appeared to co-  1  2  3  4  5 6  Figure 3.7.2, Effects of pervanadate on p72/74 phosphotyrosine content in HL-60/mac cells. Anti-phosphotyrosine monoclonal antibody immunoblots of unstimulated (lane 1) and pervanadate stimulated (lane 2) HL-60/mac cell lysates. As well as p72/74 (PTPel5ic) immunoprecipitates from unstimulated and pervanadate stimulated HL-60/mac cells, or normal rabbit serum immunoprecipitates from unstimulated (lane 3) and pervanadate stimulated (lane 4) HL-60/mac cells. PTPel5ic immunoprecipitate from unstimulated H L 50/mac cells (lane 5) and pervanadate stimulated HL-60/mac cells (lane 6)  immunoprecipitate with p72/74  The results o f the pervanadate stimulation o f HL-60/mac cells led to efforts aimed at identifying the phosphotyrosine-containing proteins observed within p72/74-containing  60  immune complexes. Proteins having molecular weights of approximately 45 kDa, 55 kDa, 80 kDa, 95 kDa and 150 kDa were found associated with p72/74frompervanadatestimulated HL-60/mac cells. The identity of these proteins is unknown. It is important to note that pervanadate stimulation, is known to have reciprocal effects on PTPases and PTKs, and can potentially lead to protein-tyrosine phosphorylation events within diverse intracellular signaling molecules (Heffetz et al., 1990; Secrist et al., 1993; Posner et al., 1994).  3.8 Association of small adapter proteins with PTPe  As GRB2 was reported to associate with tyrosine phosphorylated PTPa (Su et al., 1994; denHertog et al., 1994), anti-GRB2 antibodies were used to assess whether the small SH2 and SH3 domain containing adaptor protein was present in a complex with PTPe. Use of the various antibodies revealed that GRB2 was present, although at low levels, and primarily within the p72/74 immunoprecipitates obtained from pervanadate stimulated HL-60/mac cells indicating an inducible association with PTPe (Figure 3.8.1). A trace signal corresponding to GRJ32 was detectable in p72/74 immunoprecipitates from unstimulated HL-60/mac cells. This indicated that PTPe was may be phosphorylated on tyrosine 676 of the DYANFK GRB2 binding motif previously identified in PTPa. Additionally, this observation revealed that PTPe is not constitutively associated with GRB2 and is thus not constitutively phosphorylated at this site.  61  1  2  3 4  5 6 GRB2  Figure 3.8.1 Association of GRB2 with p72/74 in HL-60 cells after pervanadate stimulation. Anti-GRB2 immunoblots of p72/74 immunoprecipitates from unstimulated or O.lmM pervanadate stimulated HL-60/mac cells for 10 minutes. Lane 1 and 2 contained 15 ng of the Triton X-100 soluble protein fraction from unstimulated (lane 1) and stimulated (lane 2) HL-60/mac cells. Normal rabbit serum immunoprecipitates of unstimulated (lane 3) and stimulated (lane 4) HL-60/mac cell lysates as well as PTPel5ic immunoprecipitates from unstimulated (lane 5) and stimulated (lane 6) HL-60/mac cell lysates.  GRB2 was identified as an associating protein in p72/74-containing complexes following pervanadate stimulation of the HL-60 cells. This finding was consistent with the previous observations showing that GRJ32 associated with the PTPs-like molecule, PTPa (Su et al, 1994), via the phosphorylation of PTPa residue Y789 (den Hertog et al, 1994). Sos, however, was not detected in the PTPa-GRJ32 complexes, raising questions about the functional significance of this association (den Hertog et al, 1994). The sequences in the vicinity of Y789, FSDYANFK, are identical to those surrounding PTPs Y676, making this an obvious candidate site for GRB2 association with PTPs. Further investigation either through in vitro mutagenesis of this putative GRB2 binding site or through cDNA truncation analysis would identify the region of the PTPase responsible for the association.  62  3.9 Summary Using PCR mediated differential hybridization PTPs was identified as a candidate PTPase mRNA upregulated by pro-inflammatory stimuli. Northern blotting analysis confirmed that the message for this PTPase was induced by a limited number of stimuli in cells of the monocyte/macrophage lineage. With the development of highly specific polyclonal antibodies, PTPs was observed as a 72/74 kDa doublet. p72/74PTPs was found to possess PTPase activity in vitro, predominantly within the cytosol, and its expression was observed to be regulated during cellular differentiation. Further characterization of PTPs indicated that the PTPase was phosphorylated  and  phosphotyrosine could be induced in vivo using pervanadate. Tyrosine phosphorylation was found to induce the association of the small adapter protein GRB2 with PTPs in pervanadate treated cells. However, the stoichiometry of the association appears low to the weak signals obtained The observations provided here and others point to a novel isoform of the previously described transmembrane PTPs PTPase. Indeed, recent observations published by Elson and Leder, 1997 characterize the nucleic acid sequence of the novel isoform, as shown if figure 3.9.1. Studies in chapter 5 provide a more indepth characterization of transmembrane PTPs, as well as the novel p72/74 isoform.  63  EC  TM  PTP domain 1  PTP domain 2 Transmembrane PTPe  Transmembranal PTPe MF.PFCPI:I.rASFSLSLARAGOGNDTTPTESNWTSTTAGPPDPGASQPLLTWLLPLLLLLFLLAAYFFRFRKORKAWSSNDKKMPNGILEEOEQRVM  Cytoplasmic PTPe  MSSRKNFSRLTWFRKQRKAVVSSNDKKMPNIGLEEQEQQRVM  Figure 3.9.1 Nucleic acid sequence comparison of transmembrane and cytosolic  PTPe. E C denotes the extracellular domain, T M indicates the transmembrane domain while PTPase domains 1 and 2 are labelled. Underlined residues indicate the transmembrane domain of the transmembrane PTPe. The two boxes contain the sequences of the the N-terminus of the respective isoforms  64  Chapter 4  Bacterial Expression and in vitro substrate specificity of PTPs and Substrate specificity of native PTPs  4.1 Bacterial expression ofPTPases  Bacterial expression of proteins is a powerful tool for obtaining large quantities of relatively pure recombinant material. Using the glutathione S-transferase fusion protein expression system, large quantities of different PTPases were purified for further use in comparison and characterization (Smith and Johnson 1988). cDNA fragments encoding the cytosolic region containing the catalytic domains, were cloned into the pGEX-2T expression vector (figure 4.1.1). The pGEX expression system consists of a prokaryotic expression vector containing the tac promoter under the control of the lad repressor which is also encoded on the expression vector. The glutathione S-transferase gene is thus under the control of this expression unit and contains at its C-terminus a thrombin  65  cleavage site followed by restriction endonuclease sites for inframecloning of the cDNA of interest and 3 stop codons (Smith and Johnson 1988). Expression of the gene of interest involves transformation of E. coli bacteria with the pGEX expression vector containing the cDNA. Expansion of the cultures is followed by de-repression of the translation unit with the addition of the galactose analog IPTG to the cultures for up to 24 hours. The bacteria are then harvested and the PTPases purified from crude lysates by affinity column chromatography using glutathione Sepharose affinity matrix. The PTPase polypeptide sequences are liberated by batch thrombin digestion of the affinity matrix coupled to the fusion protein. Using this purification scheme approximately 500 pg/L CD45 and PTPa active catalytic domains were expressed to homogeneity as determined by Coomassie stained SDS-PAGE gels (data not shown) and stored at -80°C in 50% glycerol for future use.  Psll  L»» AAA  Scr Asp Pto A t j C l u Pli« H i V i i The A i p • • « TCC CAT CCC CGG GAA TTC. ATC CTC ACT CAC TCA CCA TCT G IciiKl  lyi AAA  S«i A i p K t V a l f i o Ar«* Gl> S « H o C l y I U B l i A n Asp • • • TCG GAT CTG GTT CCG CCT CCA TCC. CCG CCA ATT O T CCT CAC TCA CTC ACC ATC  TC  Figure 4.1.1 Prokaryotic expression vector p G E X - 2 T  66  4.2 Bacterial expression of the cytosolic region of PTPs  For bacterial expression of the catalytic domains of PTPe, mRNA was purified from HL-60 cells as described previously. A cDNA pool was prepared from the mRNA using MuMLV reverse transcriptase and random hexamer oligo-nucleotides as primers. The resulting cDNA reaction was used as the substrate in PCR amplifications using the synthetic oligo-nucleotides ptpel5 and ptpe3 and Vent DNA polymerase. Ptpel5 contains the restriction endonuclease site BamHI, preceded by the sequence CTC and followed by the  complementary  PTPe  sequences  CTCGGATCCCCC ATGAGGAAGC AGAGGAAAGCTGTGGTC-3'.  5'This  strategy  yielded a PCRfragmentcontaining a 5' end that when cleaved with BamHI and ligated into pGEX-2T provided a continuous open reading frame that extended into PTPe coding sequences originating from the most N-terminal cytosolic amino acid proximal to the transmembrane domain (figure 4.2.1).  Muni  BamHI PTPe catalytic domains  I 5'  Figure 4.2.1 Construction of p G E X . 2 T - P T P e  67  The preceding sequences CTC were included to extend the 5' region of the BamHI site to facilitate efficient digestion with the restriction endonuclease. Ptpe3 contains the restriction endonuclease sites NotI, Muni and BamHI, preceded by the sequence CTC and  followed  by  the  complementary  PTPe  sequence  GAGGGATCC AATTGCGGCCGCTC ATTTGAAATT AGC ATAATC AGA-3'.  5'PCR  amplification with this primer yielded a PCR fragment containing full length PTPe and the native PTPe stop codon.  4.3 Purification of PTPases  To generate sufficient quantities of recombinant PTPe for kinetic analysis large scale expression and purification of PTPa, CD45 and PTPe was carried out as described in chapter 2. pGEX2T expression vectors containing PTPa, CD45 or PTPe were introduced in to XL-1 Blue E. coli bacteria. Cultures were initiated and bacterial pellets harvested from one liter cultures in log phase growth after 4 hours stimulation with one millimolar final concentration of IPTG at 23°C. Crude lysates were prepared by sonicating pellets resuspended in TX100 lysis buffer A and insoluble material removed by centrifugation at 100,000 x g. Fusion proteins were purified by affinity column chromatography using glutathione Sepharose beads. Recombinant PTPase catalytic domains were released by limited thrombin digestion and stored in glycerol. Recombinant PTPases used in the following experiments were estimated to be greater  68  than 95% pure by SDS-PAGE electrophoresis and coomassie or silver staining (figure 4.3.1). Approximately 350 ug/L of recombinant PTPs was expressed using this scheme.  • i  -  »• —  Recombinant PTPe  Figure 4.3.1 S D S - P A G E analysis of recombinant PTPe. Approximately lpg of G S T PTPe was subjected to thrombin cleavage with increasing amounts of thrombin. (Lane 1) 0 ng, (lane 2) 50 ng thrombin, (lane 3) 100 ng thrombin, (lane 4) 200 ng thrombin, (lane 5) 500 ng thrombin, for 20 minutes at 23 degrees Celcius.  PTP  CD45  LAR  PTPa  Figure 4.3.2 S D S - P A G E analysis of purified recombinant PTPases. After affinity purification with Glutathione-Sepharose and thrombin cleavage (as described in sections 2.7 and 2.8). Approximately lug of eluted proteins were analyzed on a 10% S D S - P A G E gel for purity.  69  4.4 Synthetic substrate preparation  .  In vitro substrate analysis was carried out using six, 13 residue oligopeptides. The oligopeptides were synthesized containing phosphotyrpsine residues corresponding to sites of in vivo phosphorylation centrally within the peptide. Since little information exists with respect tothe essential features of phosphorylation sites, it was felt that while 13 residue phosphopeptides may not retain the three-dimensional conformation of phosphorylation sites, the flanking six amino acids would maintain the primary sequence while minimizing non-specific secondary structures. The aim of these experiments was to shed some light on the possible in vivo substrates of PTPe, define a theoretical optimum substratefromthe characteristics of a number of suitable in vitro substrates, as well as to compare the substrate specificity of PTPe with that of LAR and CD45.  The six peptides synthesized and employed in this study were: the C-terminal region of the protein tyrosine kinase src; this peptide contains the negatively regulating tyrosyl residue Tyr 527; peptides corresponding to phosphorylation sites within the pchain of the PDGF receptor, Tyr 1021, Tyr 1009 and Tyr 857. Upon growth factor mediated dimerization the intrinsic tyrosine kinase activity of the PDGF-R results in phosphorylation of these residues providing docking sites for a number of signaling proteins including PI-3 kinase, SHP2 and PLCy (Koch et al, 1991, Cantley et al, 1991); peptides corresponding to three phosphorylation sites within the cytosolic region of the CSF1 receptor, Tyr 699 and Tyr 708 in a similar fashion to that described in the case of the PDGF-R the intrinsic kinase acitivity of the CSF1-R results in phosphorylation of  70  these tyrosyl residues which may play a role in CSF1 signal transduction (van der Geer and Hunter ef al, 1990, Reedijk ef a/., 1992).  4.5 In vitro characterization and substrate specificity of PTPs  To quantify free phosphate produced in the PTPase reactions, the modified malachite green colorimetric assay was used. In the presence of inorganic phosphate the color of malachite green changes from orange to dark green. This phosphate detection system had been previously used in the detection of inorganic phosphate in the analysis of calcineurin (Lanzetta et al, 1979). With modifications, most importantly the addition of 0.01%  Tween 20, the sensitivity of the assay was increased to detect inorganic  phosphate with a sensitivity of 300 pmol. In addition, the reaction can be carried out in a microtitre plate (Harder et al, 1994).  Before substrate preferences and enzyme comparisons could be carried out, a buffer analysis and pH profile of PTPe was completed. Figure 4.5.1 shows the results of the pH optimization, where PTPe had a pH optima of 5.6 using KOAc buffered reaction conditions (50mM NaCl, 50mM KOAc pH 6.0, 0.2mM DTT). The apparent low pH optima, compared to physiological conditions where pH is 7, is probably due to the requirments within the catalytic site where the catalytically essential cysteine residue prefers its R group as an anion (—S") vs. the protonated form (-SH). In situ, associated  71  proteins may serve to lower the local pH mimicking the pH optima experimentally determined.  Using the reaction conditions developed in earlier experiments, a substrate preference analysis was carried out using PTPs, with PTPa and CD45 included for a comparative analysis of substrate preference between related PTPases. PTPase reactions were carried out in a standard KOAc buffered PTPase assay cocktail at pH6.0 (50mM KOAc pH6.0, 50mM NaCl, 0.2mM DTT). For pH ranges 4 to 6.5 KOAc was the buffer of choice, for ranges 6.5 to 8.0 Mes buffer was used and for ranges 8.0 and above TrisHCl was used. In the case of PTPa and CD45, pH 6.5 and pH 7.0 was used, as these were the observed pH optima in these experiments and confirmed in the literature (data not shown). The pH of 6.0 was chosen for PTPs, instead of the experimentally determined 5.6, since all substrates were prepared in a mildly acidic buffer to ensure their solubility. With the addition of the substrate the final reaction pH approaches the optimum.  0.5 0.4  8 0.3 NO  § 0.2  0.1 0 5  5.5  6  6.5  7  7.5  8  8.5  9  9.5  pH Figure 4.5.1 p H optimum of PTPe using P D G F - R  Y 1 0 2 1  72  Tabulated in figure 4.5.2 are the results of the in vitro analysis. In comparison to LAR and CD45, PTPe showed overall lower affinity for all synthetic substrates tested. The K value of 224 uM for src  Y521  m  LAR had K  is similar to that of PDGF-R  Y1009  , 250 u.M for PTPe.  values of 159 uM and 91 uM respectively for src  and PDGF-R  Y52J  m  CD45 had K  values of 101 uM for src  Y52J  m  and 107 uM for PDGF-R  Y1009  Y1009  .  . Those  substrates which appeared to be the prefered substrates for LAR and CD45, were also those synthetic peptides with the highest activities for PTPe.  Substrate  km  v v  b  max  CD45  LAR  PTPe V ax /k  km  m  Vmax  Vmax /k  m  k  m  V ax  V ax  m  m  /k  m  m  224+/-15  534+/-17.  2.4  159 +1-6  290+/-11  1.8  101 +/-8  897 +1-1  8.9  147 +/-4  615 +1-9  4.2  11 +/-3  961 +1-1  12.5  260 +/-11  843 +-11  3.2  250 +1-1  625 +A4  2.5  91 +1-9  891 +/-11  9.8  107+/-13  888 +/-3  8.3  PDGF-R™ '  NC  NC  -  124 +/-14  865  6.9  115+/-19  403 +1-1  3.6  CSFl-R  NC  NC  -  NC  NC  -  193 +/-22  119+/-24  0.6  1275 +/-34  187+/-26  0.2  239+/-14  177 +/-16  0.7  188 +/-19  65 +1-6  0.4  Src™' . 1  PDGF-R  Y,U21  PDGF-R  vlooy  1  Ybyv  CSF1-R  Y/US  Table 4.5.2 Kinetic analysis of  PTPs, PTPa and CD45  +/-3  a-pM  b-umole/min/mg NC-Not computed  73  4.6 Comparison of substrate preference of recombinant vs. native PTPe  While kinetic measurements made with recombinant  enzymes are easily  performed and reliable, comparison studies were also carried out using native enzyme immunoprecipitated from differentiated HL-60 cells. These studies were carried out to determine whether post-translational modifications such as serine, threonine or tyrosine phosphorylation might modify the substrate specificity or enzyme activity of PTPe.  HL-60 cells were allowed to differentiate towards the monocyte/macrophage lineage with PMA for 48 hours, then they were allowed a 24 hour recovery period in the absence of PMA. The recovery period was to permit PKG levels to return to normal, since prolonged PMA exposure results in down-regulation of PKC (Hsu et al, 1998). HL-60/mac protein lysates were prepared in TX100 lysis buffer with the omission of orth-vanadate and in the presence of 1.0 mM DTT to prevent oxidation of the essential cysteine residue within the active site. To quantify the specific activity of immunoprecipitating antibody PTPe was immunoprecipitated from various amounts of protein using 1 pg of PTPel5ic antibody and PTPase assays carried out using PDGFR pY  1021  as the substrate. A standard curve was obtainedfromthis experiment and the point  at which anti-PTPe antibodies immunoprecipitate saturating amounts of PTPase activity was determined. The arbitrary number of 12.0 units of PTPe PTPase activity was made equivalent to the amount of phosphate  released from the amount of protein  immunoprecipitated with 1 pg of PTPel5ic antibody, using saturating quantities of  74  protein lysate. These results provided a basis for the substrate specificity analysis using native PTPe immunoprecipitated from 100 pg of HL-60/mac cell lysate.  Figure 3.4.1 on page 50 shows the results of the specificity analysis using a small panel of synthetic phosphopeptides consisting of src  Y521  (TATESQpYQQQP),  PDGF-R  Y1021  Y501  (TSTEPQpYQPGENL), hck  (NEGDNDpYIIPLPD),  Y440  IFNy-Ra  Y701  (APTSFGpYDKPHVL), STAT91  (GPKGTGpYDCTELI). It is clear that native PTPe  exhibits some substrate specificity since PDGF-R src ,  hck  Y527  Y501  and the src family members  appear to be superior substrates in vitro in comparison to the  IFNy-Ra ° and STAT91 Y44  Y1021  Y701  .  Recombinant PTPe prepared as described earlier was subjected to substrate specificity analysis similar to that undertaken with native PTPe. Five nanograms of rPTPe was used in enzyme assays carried in 96 well plate format using standard PTPe PTPase assay buffer (50mM NaCl, 50mM KOAc pH6.0, 0.2mM DTT). Liberated free phosphate was measured by the addition of 2 volumes of malachite green reagent (MG reagent) which also terminated the enzyme activity. Analysis was carried out within the linear portion of the MG reagent response curve and spectrophotometer measurements at 620nm converted to free phosphate concentration by calibration of the curve with standards of known phosphate concentration. Table 4.5.3 shows in tabular form the k and V  m a x  m  values obtained from the analysis. The substrate preferences of the recombinant  enzyme appear to mirror the preferences of the endogenous enzyme shown in figure 3.4.1.  75  Substrate PDGF-R  K  Y1021  Y527  src hck  Ym  Stat91  Y701  IFN -R  Y440  (LIM)  m  v  V x/k ma  max  147 +/-4  615+/-9  4.2  224+/-15  534 +/-17  2.4  532 +/-7  446 +/-11  0.8  1275 +/-21  187 +/-13  0.14  -  -  •  Y  V  -  m  Table 4.5.3 In vitro substrate analysis of recombinant PTPe  ;  The  V  m a x  /km  values indicate that PDGF-R  Y1021  (4.2) of this small panel of  substrates is the most preferred with the src peptide (2.4) also showing characteristics of a resonable. The nature of the observed k values and the V m  m  a  x  in general shows no striking substrate preference. The IFNy-R  values indicate that PTPs Y440  substrate was included,  however no kinetic parameter could be calculated due to the poor nature of substrate turnover in those reactions.  Native p72/74PTPs appears to exhibit activity towards a broad range of synthetic substrates. PDGF-R  Y1021  appears to be the best substrate of the panel of 5 substrates tested  with src \ hck , Stat91 , IFNy-R™ following in descending order. Y52  Y50]  Y701  40  76  4.7 Summary  In vitro analysis of recombinant PTPe revealed a pH optima of 5.6. While capable of liberating inorganic phophate from all substrates tested, PTPe did exhibit some degree of substrate specificity, especially when compared to other recombinant PTPases such as CD45 and PTPa (the two PTPases used for comparison in this study). While PTPe appeared to prefer the same-substrates as. other-transmembrane PTPases, the order of preference and general catalytic activity towards the substrates suggested that it might have a specific profile of substrate specificities. The results obtained in the in vitro analysis may be useful in providing clues with respect to the direction of characterization to be taken in the following in vivo analysis described in more detail in chapter 5. From the small number of peptides sampled Src is a plausible candidate for an in vivo substrate when compared to the kinetic parameters of I F N Y - R  Y 4 4 0  or STAT91  Y770  peptides.  77  Chapter 5  Over-expression of transmembrane and non-transmembrane PTPe in mammalian cells and the role of a putative SH3 binding motif in PTPa/PTPefamily  of PTPases  5.1 Expression of PTPe in HEK 293 cells  Over-expression of genes is a commonly used method of characterizing the protein products of specific genes. A number of well described model systems exist for studying proteins of interest in mammalian cell lines including CHO cells, PC 12 cells, A431 cells, NIH3T3 cells and BaF/3 cells. The HEK 293 human embryonal kidney cell line was chosen for the ease with which foreign genes may be introduced and for the presence of the adenovirus El a protein which efficiently activates transcription from  78  CMV-based expression vectors. Preliminary experiments had revealed that PTPe was not expressed in this cell line, and that only moderate levels of PTPa were present.  To drive expression of PTPe in HEK 293 cells, the murine cDNA encoding full length transmembrane  PTPe was cloned into the eukaryotic expression vector  pBCMGSneo. pBCMGSneo accomplishes gene expression through the Cytomegalovirus (CMV) promoter and confers resistance to cytotoxic compound G418 by also containing the neo cassette (figure 5.1.1). In addition pBCMGSneo contains -69% of the Bovine r  Papilloma Virus which posses the necessary sequences to maintain extra-chromosomal replication of the vector. The potential to maintain many copies of the gene of interest within the transfected cell results in increased expression.  Since the discovery of the catalytically essential cysteine residue within the active site of the PTPases (Charbonneau et al, 1989), its modification, by in vitro mutagenesis to either a serine or alanine residue results in the abolition of detectable PTPase activity. Figure 5.1.2 illustrates the crystal structure of PTP1B. Since the 1 catalytic domain of st  tandem catalytic domain PTPases show high sequence identity with PTP IB, the crystal structure  79  3ac)  Figure 5.1.1  pa  The p B C M G S n e o - P T P e expression vector. After Xhol NotI digestion to remove the stuffer fragment,  various PTPe cDNA's were cloned in to the linearized vector.  Human Protein Tyrosine Phosphatase I B (E.C.  Figure 5.1.2 Crystal structure of Human PTP1B. Arrow B indicates the location of the central helix and arrow A illustrates the active site. The enzyme was crystalized in the presence of Tungstate. The ion can be seen covalently bound to the active site cysteine (arrow A).()  80  of PTP IB is analogous to the first catalytic domain of PTPa and other dual catalytic domain transmembrane PTPases. The crystal structure of PTP IB can then be seen as a template for other catalytic domains. As is evident infigure5.1.2, the aforementioned cysteine residue (arrow A) is located at the end of the central helix (arrow B) within the substrate pocket of the enzyme. Catalytically inactive forms of PTPases, generated by alanine or serine substitutions of the cysteine residue have been a useful tool in uncovering their roles in biological signal transduction pathways (Pannifer et al, 1998, Jai et al, 1995). Recent evidence indicates that these mutants, to varying degrees, trap substrates within the active site of the PTPase domain (Pannifer et al, 1998). This is achieved because residues within the pocket of the active site serve to stabilize % electron distribution of the aromatic ring as well as charge distribution of the highly polarized phosphate group. Enzyme-substrate interactions provide  sufficient electrostatic  interactions for substrate binding in spite of the absent covalent phosphothiol ester linkage provided by the thiol anion the catalytic cysteine forms within the low pH conditions of the active site. This phenomenon may result in the sequestration of PTPase substrates. The result, inhibition of wild-type function resulting in dominant negative phenotypes or inhibition of substrate function within its pathway. This potentially culminates in a phenotype.  Figure 5.1.3 shows the strategy employed for the creation of PTPe mutants C334A, C629A, and C334A/C629A. The conversion of cysteine 334 to an alanine residue was accomplished with the use of complementary oligonucleotides Cl>Aa (5'CCCACGCCCGCGCTAGCGTGAACCACAATGGG-3')  and  Cl>As  (5'-  81  CCCATTGTGGTTCACGCTAGCGCGGGCGTGGGT-3')  with  the  underlined  nucleotides forming the mispaired regions resulting in the codon change. The conversion of the cysteine 629 to an alanine residue was obtained with the use of complementary oligonucleotides C2>Aa (5'- CCCTGCTCCCGCCGTAGCGTGCACGGTGATGGG-3') and C2>As (5 '-CCCATCACCGTGACAGCTAGCGCGGGAGCAGGG-3'). As outlined in figures 5.1.3a and 5.1.3b, overlap extension PCR was used to create the mutant cDNAs. This method was utilized for speed, since the process requires the length of time necessary to complete two PCR amplifications, as well as accuracy because >99% of the PCR product will contain the desired mutation. Utilization of overlap extension PCR negated the need for extensive screening of clones to identify those carrying the desired mutation. Both mutagenized cDNAs were  Figure 5.1.3 Overlap extension P C R mutagenesis of C334 and C629 of PTPe (Panel A) The 5 prime PCR fragment (lane 1) and 3 prime PCR fragment (lane 2) of the C334A overlap extention reaction. (Panel B) The 5 prime PCR fragment (lane 1) and 3 prime PCR fragment (lane 2) of the C629A overlap extension reaction.  sub-cloned into pSSBS for mapping and partial sequencing. The final PTPs catalytic mutant PTP&C334A/C629A, which has both domains inactivated, was constructed by  82  cloning the 1.7 kb Styl fragment of pSSBS-PTPeC334A into the Styl site of the purified 3.1 kb fragment of pSSBS-PTPsC629A. This simple swapping strategy exchanges the wild-type Styl fragment for the C334A mutant fragment conserving the open reading frame of the cDNA. The resulting chimeric cDNA containing both C334A and C629A mutants was mapped for integrity then shuttled into the pBCMGSneo expression vector via the NotI cloning site.  Introduction of the eukaryotic expression vectors into HEK 293 cell line was accomplished by transfection using Lipofectamine (GIBCO-BRL). Briefly, lipofection consists of the emulsification of DNA with a specialized ionic lipid resulting in the  1  2  3  4  5  6  7  Is  Figure 5.1.4 Transient expression of P T P E in H E K 293 cells. HEK 293 cells were transiently transfected with various amounts of wild-type PTPs expression vector. PTPe expression was analyzed by immunoprecipitation and immunoblotting with PTPsl5ic. Lane 1 transfection with control vector. Lane 2 0.5 ug. Lane 3 1.0 ug. Lane 4 2.0 pg. Lane 5 5.0 pg. Lane 6 10.0 pg. Lane 7 20.0 pg.  formation of liposomes capable of delivering DNA within the cell. Preliminary experiments titrating the quantity of DNA transfected revealed that, with this cell type, target DNA can be transfected with efficiency approaching 90% when PTPs expression was determined by immunoflourescence. Transient transfection experiments revealed that  83  the introduction of ~2 pg of pBCMGSneo-PTPs plasmid resulted in optimal expression levels after an overnight (-18 hour) recovery period (figure 5.1.4). However, one modification in particular resulted in much greater efficiency. The introduction of a single step coating of the tissue culture plates with bovinefibronectin20 pg/ml prior to cell seeding resulted in enhanced adhesion of the cells to the substratum. This proved to be essential since HEK 293 cells in the presence of Optimem serumfreemedium, which is required for efficient transfection, exhibited greatly reduced adhesion to tissue culture plates. The resulting transfections were of both higher efficiency and protein yield since 100% of the seed cells were harvested (data not shown).  Transfection of full length PTPs (pBCMGSneo-PTPs) into HEK 293 cells had no obvious effect on cell survival as compared with control vector (figure 5.1.5). Protein lysates prepared with TX100 lysis buffer were subjected to immunoprecipitation and immunoblotting with PTPsl5ic polyclonal antibody. Figure 5.1.5 shows that introduction of pBCMGSneo-PTPs in to HEK 293 cells resulted in expression of a 100/110 kDa species migrating with the characteristics of a doublet. The presence of a 100/110 kDa protein with reactivity to anti-PTPs antisera indicates that immature PTPs undergoes post-translational modifications resulting in an apparent molecular weight substantially higher than the predicted 78 kDa.  Transfections of the mutant forms of PTPs were carried out using the same conditions employed for pBCMGSneo-PTPs. Introduction of pBCMGSneo-PTPsC334A, pBCMGSneo-PTPsC629A  or pBCMGSneo-PTPsC334A/C629A  resulted  in no  84  detectable change in cell survival as compared to vector control. Figure 5.1.5 shows that the transfection of the various catalytic mutants of P T P E into  H E K 293  identical migration patterns of PTPs, as observed when wild-type  PTPE  cells resulted in was introduced,  indicating that the full-length protein was being expressed.  1  2  3  4  5  6  7  8  9  10  11  12  ppllO pplOO PTPsl5ic immunoprecipitates Figure 5.1.5 Expression levels of wild-type and various mutants in transfected H E K 293 cells. H E K 293 cells were transfected with empty control vector (lane 1 and 7), PTPs (lane 2 and 8), PTPeC334A (lane 3 and 9), P T P E C 6 2 9 A (lane 4 and 10), PTPeC334A/C629A (lane 5 and 11), PTPEF112P  (lane 6 and 12) and selected for stable expression. TX100 lysates were prepared and  either directly western blotted (lanes 1 through 6) or subjected to P T P E immunoprecipitation then blotted (lanes 7 through 12) with P T P £ l 5 i c polyclonal antibody.  While expression of P T P E cDNA had no effect upon cell survival, there was a noticeable difference in cell morphology in cells expressing wild-type PTPs, PTPEC334A/C629A  and control vector. Figure  5.1.6  shows that cells expressing wild-  type PTPs although very similar in shape to cells transfected with control vector did posses an increased number of cellular protrusions in addition these cells seemed to have a lower number of cell-cell contacts. They tended to spread out in culture and cover the entire substratum. In addition, cells expressing the wild-type construct were able to adhere to the substratum much better than control cells (data not shown). These characteristics were much more apparent when cells expressing PTPs were compared to  85  cells expressing PTPeC334A/C629A. Cells transfected with the dominant negative mutant of the PTPase, had very few cellular protrusions, and maintained a very high number of cell-cell contacts and were often observed to grow in clumps. Additionally cells expressing PTPeC334A/C629A did not spread out in culture and cover the entire substratum but instead grew in long cellular tracts always maintaining very tight cell-cell adhesion while not adhering to the substratum efficiently. These results indicated that PTPe may play a role in cell-cell and/or cell-substratum adhesion.  86  Control  PTPs  Figure 5.1.6 Morphology of cells expressing PTPe. H E K 293 cells expressing various forms of PTPe in a stable manner were seeded in culture at identical concentrations then photographed under phase contrast microscopy at 200x magnification. Upper right panel shows 293 cells transfected with the empty control vector. Upper left panel shows 293 cells transfected with wild-type PTPe. Lower left panel shows the morphology of 293 cells transfected with PTPeC334A/C629A  To confirm that p 100/110 possessed PTPase activity, immunoprecipitations were carried out using PTPsl5ic polyclonal antibodies from protein lysates of transiently transfected HEK 293 cells. The lysates were prepared in the presence of 1.0 mM DTT to maintain the active site cysteine in a reduced state ensuring preservation of PTPase  87  activity. Figure 5.1.7 shows PTPase activity associated with the immunoprecipitates. It can be seen that ectopically expressed PTPe was catalytically active while PTPeC334A mutant exhibited no catalytic activity. In addition, PTPeC629A was observed to be catalytically acitve while the double mutant, PTPeC334A/C629A, predictably was not (data not shown). These results indicated the first catalytic domain of PTPe was responsible for the majority of the detectable PTPase activity. The second catalytic domain either possessed little PTPase activity, had high substrate specificity and Y1021  therefore  unable  to dephosphorylate  the synthetic  substrate  PDGF-R  (NEGDNDpYIIPLPD), or the acitivity of 1 catalytic domain was required for activity. st  time (rrin) Figure 5.1.7 Protein tyrosine phosphatase activity of ectopically expressed PTPe and mutants. Whole cell lysates prepared from 293 cells exhibiting stable expression of A PTPeC334A, • Control and • PTPe were subjected to immunoprecipitation with PTPel5ic polyclonal antibody. Immunoprecipitates were then subjected to M G PTPase assays. O D values were converted to pmols of P 0 using a inorganic P Q calibration curve. 4  4  88  The observation of the pi00/110 doublet was of great interest since the higher molecular weight band appeared somewhat fuzzy (figure 5.1.5). This characteristic is suggestive of heterogeneous glycosylation of a pre-processed "core" protein. Due to the presence of three potential N-linked glycosylation sites and three O-linked glycosylation sites within the extracellular domain of PTPs the addition of carbohydrate moieties was the most likely explanation for the observed molecular weight. To investigate this possibility further, protein lysates were prepared from HEK 293 cells with stable expression of PTPs and PTPe immunoprecipitated with PTPel5ic. Immunoprecipitates were subjected to deglycosylation using N-glycanase which selectively removes carbohydrate moieties from N-linked sites. Figure 5.1.8 indicated that the heterogeneous pi 10 isoform of PTPe resulted from N-linked glycosylation as denoted by the disappearance of the pi 10 band with increased exposure to N-glycanase. These results also suggest that the increased molecular weight of premature pi00 isoform over the predicted molecular weight is due to glycosylation at the numerous potential O-linked sites within the extracellular domain of PTPs.  1  3  4 pllO plOO  Figure 5.1.8 De-glycosylation of P T P E expressed in H E K 293 cells. H E K 293 cells with stable wild-type expression of P T P E were either left untreated (lane 1), or treated with N-glycanase for 1 hour (lane 3), or 16 hour treatment (lane 4). P T P E was visualized by immunoblotting with PTPEl5ic.  89  5.2 Phosphorylation of PTPs expressed in HEK 293 cells and total cell lysate phosphotyrosine profiles  Phosphorylation is a prevalent post-translational modification event with the ability to regulate the function of proteins. Since the over-expression of an active PTPase may indeed have a detectable impact on endogenous substrates, TX100 lysis buffer solublized lysates from HEK 293 cells over-expressing either wild type PTPs or various mutants were probed with the anti-phosphotyrosine antibody 4G10. Phosphotyrosine profiles revealed little change in the relatively abundant proteins whose phosphotyrosine content can be measured in this method.  1 200 kDa  ^  97.4 kDa  >•  69 kDa  46 kDa 30 kDa  2 3  4  5  6  •  y  <  P55  >  Figure 5.2.1 Phosphotyrosine profiles of H E K 293 cells expressing PTPe and mutant forms. Cell lysates from HEK 293 cells expressing either empty vector  (lane 1), wild-type PTPe (lane 2), PTPeC334A (lane 3), PTPeC629A (lane 4), PTPeC334A/C629A (lane 5), or PTPeF112P (lane 6) were subjected to SDSPAGE. Immunoblots were probed with the anti-phosphotyrosine monoclonal antibody 4G10.  90  The exception is a group of proteins migrating with an apparent molecular weight of approximately 55 kDa (figure 5.2.1) whose phosphotyrosine content appeared to decrease with the expression of an active PTPe membrane proximal catalytic domain. Figure 5.2.1 shows the correlation between the active catalytic domain of PTPe and pp55 tyrosine phosphorylation.  To investigate the amount of tyrosine phosphate found in PTPe, HEK 293 cells with stable expression of PTPe, PTPeC334A, PTPeC629A, or PTPeC334A/C62?A protein lysates were prepared from resting cells or cells stimulated with pervanadate. The covalent binding of pervanadate to the essential cysteine within the active site of PTPases results in irreversible inhibition of PTPase activity leading to deregulated tyrosine phosphorylation  of various substrates by  PTK's. After  immunoprecipitation,  immunoblotting with anti-phosphotyrosine antibodies revealed the extent of PTPe tyrosine phosphorylation. As shown in figure 5.2.2 PTPe immunoprecipitated from cells expressing the wild-type PTPase as well as various catalytic mutants show increased amounts of phosphotyrosine in proteins of molecular weight pp40, pp55, pp85, pplOO, ppl 10, and ppl60.  91  1 200 kDa  •  97.4 kDa  •  69 kDa  •  46 kDa  •  2  •  4  m  .  m iii  IH ^•••p*  m 97.4 kDa  3  9  mm  ^^W^^ ?BPWH^  A  ppl60  <«  ppl 10 pplOO  •4  pp85  +  PP55  *  IgH  -<  pp40  Wt  ii  •^w *^mr °mw  t  plOO/HOPTPe  F i g u r e 5.2.2 Pervanadate t r e a t m e n t of H E K 293 cells expressing PTPe c D N A s . (Upper panel) Cell lysates prepared from H E K 293 cells with stable expression of wild-type PTPe (lanes 1 and 2) or PTPeC334A (lanes 3 and 4) were either not stimulated (lanes 1 and 3) or stimulated (lanes 2 and 4) with lOOnM pervanadate for 10 minutes. Various isoforms of PTPe were immunoprecipitated with PTPel5ic polyclonal antibodies and phosphotyrosine content determined by immunoblotting with anti-phosphotyrosine monoclonal 4G10. (Lower panel) the upper panel stripped and re-probed with the anti-PTPe antibody PTPel5ic.  Stripping and re-probing of immunoblots with PTPsl5ic pplOO/ppllO  were PTPs polypeptides (figure  suggested that  5.2.2 lower panel).  The strong  phosphorylation observed with pervanadate stimulation suggested that PTPs might be associated with tyrosine kinases that were normally regulated by PTPase activity. The presence of pp40, pp55, pp85, and ppl60 were unexpected and suggested that PTPs may  92  be able to associate with a number of PTK substrates. The identities of the associated proteins observed are unknown.  GRJ32 was reported to associate with tyrosine phosphorylated PTPa (Su et al., 1994; den Hertog et al., 1994). Anti-GRB2 antibodies were used to determine whether GRB2 would associate with PTPe in a similar fashion. PTPe was immunoprecipitated, using PTPel5ic antibody, from TX100 protein lysates of HEK 293 cells exhibiting stable expression of PTPefromthe pBCMGSneo expression vector. With the use of anti-GRB2  1  2  3  4  GRB2/Sem5  Figure 5.2.3. GRB2 association with PTPe. TX100 lysates prepared from HEK 293 cells transfected with PTPe and selected for stable expression. PTPe was immunoprecipitated with PTPel5ic and probed with anti-GRB2 antibodies. Lane 1 HEK 293 lysate. Lane 2 cells transfected with empty control vector. Lane 3 cells expressing PTPe. Lane 4 cells expressing PTPe and stimulated with 0.1 uM pervanadate for 10 minutes.  monoclonal antibodies, it was determined that GRB2 was present in anti-PTPe immunoprecipitatesfromboth resting and pervanadate stimulated PTPe expressing HEK 293 cells (figure 5.2.3).  93  5.3 Src family kinases  The phosphotyrosine profiles generated in figure 5.2.1 suggested that overexpression of P T P E resulted in the decrease of phosphotyrosine content of a group of proteins with an apparent molecular weight of approximately 55 kDa. In addition, reports have implicated PTPa in dephosphorylation and activation of src family kinases in rat embryo fibroblasts (Zheng et  al,  1992) and P19 cells (den Hertog et  being a close relative to PTPa already shares in  vitro  al,  1993).  PTPE  substrate specificity with PTPa as  shown in chapter 4. To test whether src kinase family members were regulated by PTPs, Vp6<f  rc  and PP59 " were immunoprecipitated from HEK 293 cells over-expressing wild6 1  type PTPs, P T P S C 3 3 4 A ,  PTPEC629A,  or PTPsC334A/C629A. The immunoprecipitates  were immunoblotted with the monoclonal antibody 4G10 to assess the phosphotyrosine content of fyn and src. Figure 5.3.1 shows the results of a single representative experiment which revealed that wild-type P T P E over-expressed in HEK 293 cells resulted in an 82% +/- 7 (the results of five independent experiments) decrease in src phosphotyrosine levels.  94  1  2  3  4  5  4  P P  «  IgH  *  Figure 5.3.1  6(T  pp6(T  c  Phosphotyrosine content of pp60src in H E K 293 cells expressing wild-type PTPe or  PTPe mutants.  {Upperpanel) Triton X-100  cell protein lysates were prepared from H E K 293 cells with  stable expression of empty expression vector (lane 1), PTPe (lane 2), PTPeC334A (lane 3), PTPeC629A (lane 4), PTPeC334A/C629A (lane 5). pp60 antibodies and  src's phosphotyrosine  jrc  was immunoprecipitated with anti-src polyclonal  content assessed with 4G10.  (Lower panel)  The immunoblot in the  upper panel was stripped and reprobed with the anti-src monoclonal antibody G D I 1.  As seen in lane 2 of figure 5.3.1, src dephosphorylation is dependent upon PTPase activity. However, lanes 3 and 4 indicate that for efficient dephosphorylation of pp60  5rc  both catalytic domains of PTPe must be active. The PTPeC334A expressing cells showed a 50%+/-9 decrease in src tyrosine phosphorylation while the PTPeC629A expressing cells exhibited a 10%+/-5 decrease in src tyrosine phosphorylation. In contrast, cells expressing PTPeC334A/C629A showed at modest but reproducibile 7%+/-3 increase in src tyrosine phosphorylation suggesting absence of PTPe PTPase activity may enhance src kinase activity. These results indicate that although the membrane distal catalytic domain of PTPe may not possess detectable PTPase activity in vitro, it likely does play some undefined role in substrate dephosphorylation. Figure 5.3.1 (lower panel) shows  95  that a similar amount of src was immunoprecipitated in each case. However, it was unclear whether PTPe was capable of mediating a substantial decrease in the phosphotyrosine content of lyn, (figure 5.3.2 lanes 7 and 8). Figure 5.3.2 shows the observations from one of three repeated experiments which suggested that lyn may be a less optimal substrate than fyn or src. While PTPe was capable of decreasing the phosphotyrosine content of src and fyn by 80%+/-3 in these experiments the PTPase was capable of producing a 30%+/-13 decrease in the phosphotyrosine content of lyn.  Anti-src 1  2  Anti-yes 3  4  Anti-fyn 5  6  Anti-lyn 7  IgH  +  -  +  -  +  - +  8  <  Src kinases  <—  lyn kinase  PTPe transfection  Figure 5.3.2 Decrease in phosphotyrosine content of other src family members in H E K 293 cells expressing PTPe. H E K 293 cells with stable expression of either empty expression vector (lanes 1, 3, 5, 7) or wild-type PTPe (lanes 2, 4, 6, 8) were subjected to immunoprecipitation with anti-src (lanes 1 and 2), anti-yes (lanes 3 and 4), anti-fyn (lanes 5 and 6) or anti-lyn (lane 7 and 8). Phosphotyrosine content of immunoprecipitated src kinases was observed with 4 G 1 0 .  If PTPe is capable of directly or indirectly mediating the dephosphorylation of the C-terminal regulatory phosphotyrosine of src family members, then the measurable kinase activity of pp60 should be increased in HEK 293 cells over-expressing active src  96  PTPs, but not in those cells expressing mutant forms of the PTPase. Src activity was assessed in cells expressing wild-type as well as mutant forms of PTPs by immunoprecipitating the kinase and measuring auto-phosphorylation in the presence of y32  P-ATP followed by SDS-PAGE and autoradiography. Kinase activity was measured by  scanning densitometric analysis of the autoradiographs. Figure 5.3.3 shows results of one representativefromfour independent experiments. Figure 5.3.3 indicates that PTPs is capable of activating pp60" by 2-fold (+/-0.3). Lanes 3 and 4 indicate again, that the r  presence of two active catalytic domains is necessary for the maximal activation of src kinases by PTPs. Lanes 3 and 4 reveal a smaller activation (1-fold +/-0.4) of pp60 by 5rc  mutants PTPsC334A and PTPsC629A respectively.  pp60"  Figure 5.3.3 Activation of pp60src in H E K 293 cells expressing wildtype PTPe and PTPe mutants. Src immunoprecipitates were subjected to auto-kinase assays with P y A T P and phosphorylated kinase analyzed by S D S - P A G E . (lane 1) 293 cells transfected with control vector, (lane 2) 293 cells transfected with expression vector containing wild-type PTPe. (lane 3) 293 cells transfected with BCMGSneoPTPeC334A. (lane 4) 293 cells transfected with BCMGSneo-PTPeC629A. (lane 5) 293 cells transfected with BCMGSneo-PTPeC334A/C629A. 32  Expression  of  the putative  dominant  negative  form  of  PTPs  (PTPsC334A/C629A), figure 5.3.3 lane 5, caused a 23%+/-8 decrease in pp60 " activity c  rc  as compared to control. This observation suggested that PTPe was either involved in the regulation of an upstream regulator of src family kinases or alternatively that PTPe is  97  directly responsible for modulation of src phosphorylation and kinase activity. If the effect of PTPe on pp60" were direct, one might predict a physical association between r  the two proteins. To investigate this, immunoprecipitations of c-src were probed with anti-PTPe antibodies. Figure 5.3.4 shows that a 110 kDa protein reacting with the antiPTPe polyclonal antibody co-immunoprecipitated with c-src from HEK 293 cells expressing wild-type or mutant forms of PTPe, but not when transfected with vector alone. These results suggest that the observed dephosphorylation and activation of pp60  src  is via a direct  1  2  3  4  5  4  PTPs  IgH  <—  w  PTPs  Figure 5.3.4 Co-immunoprecipitation of pp60src with wildtype P T P s and mutants in H E K 293 cells. H E K 293 cells transfected with various mutants of PTPs were subjected to immunoprecipitation with the anti-src monoclonal antibody GD11. (lane 1) 293 cells transfected with control vector, (lane 2) 293 cells transfected with expression vector containing wild-type PTPs. (lane 3) 293 cells transfected with BCMGSneo- PTPsC334A. (lane 4) 293 cells transfected with BCMGSneo-PTPeC629A. (lane 5) 293 cells transfected with BCMGSneo-PTPsC334A/C629A.  98  interaction, with src being an in vivo substrate of PTPe. It should be noted that when the reciprocal experiment was carried out, immunoprecipitating PTPe and immunoblotting with the c-src monoclonal antibody GDI 1, c-src could not be detected (data not shown).  5.4 Crkllphosphorylation in cells expressing transmembrane PTPs  Small adapter proteins have been shown to play an important role in intra-celmlar signal transduction especially in the activation of small GTP binding proteins by recruiting their activators to signal transduction complexes at the membrane. Since earlier studies have indicated that the small adapter protein GRB2 complexed with PTPe, a limited screen was launched to uncover other members which were either regulated by PTPe or formed complexes with PTPe.  Preliminary experiments had indicated that one member of the small adapter family, Crkll, but not SHC, or Nek, appeared to show changes in its phosphotyrosine content when PTPe was over-expressed in HEK 293 cells. Crkll is a 40 kDa phosphoprotein consisting of a single SH2 domain followed by two SH3 domains. While GRB2 has not been shown to be phosphorylated on tyrosyl residues Crkll is a  99  phosphotyrosine containing protein. Recent studies have shown that Crkll is phosphorylated in response to a number of stimuli including EGF, insulin, IGF-I and Bcell receptor activation (Hashimoto et al, 1998, Okada et al, 1998, Koval et al, 1998, Kiyokawa et al, 1997). Figure 5.4.1 shows a representative blot of five independent experiments and as can be seen in lane 2 expression of wild-type PTPe resulted in a 92%+/-2 reduction in the phosphotyrosine content of Crkll. The  diminished  phosphorylation of Crkll in PTPs over-expressing HEK293 cells appears to be dependent upon the  1 200 kDa  91A  kDa  2  4  5  +  •  69 kDa  •  46 kDa  •  4 w*^'  «»~ 30 kDa  3  +  Wm  mm?  ppl20/130  IgH  ^IPP  mmm  •<  Crkll  • j j h | ^ j ^ ^ m m *  *  Crkll  Figure 5.4.1 Modulation of C r k l l phosphorylation in H E K 2 9 3 cells expressing PTPe and P T P E mutants. H E K 293 cells transfected with various mutants of P T P E were subjected to immunoprecipitation with anti-crk monoclonal antibodies.(Upper panel) (lane 1) 293 cells transfected with control vector, (lane 2) 293 cells transfected with expression vector containing wild-type P T P E . (lane 3) 293 cells transfected with BCMGSneo- P T P E C 3 3 4 A . (lane 4) 293 cells transfected with B C M G S n e o - P T P £ C 6 2 9 A . (lane 5) 293 cells transfected with BCMGSneoP T P E C 3 3 4 A / C 6 2 9 A . Upper panel probed with anti-phosphotyrosine. Lower panel is the blot in upper panel stripped and reprobed with anti-crk antibodies.  100  presence of two active catalytic domains, as shown in figure 5.4.1 lanes 3 and 4, where Crkll tyrosine phosphorylation was essentially unchanged. The expression of the putative dominant negative mutant of PTPe may have resulted in a modest "protective" effect. The phosphotyrosine content of Crkll being increased to levels, 22%+/-9 above those of the controls in repeated experiments (figure 5.4.1 lane 5).  The arrows in figure 5.4.1 labeled pi 10/120 indicate the presence of high molecular weight proteins that appear to associate with Crkll. Since this immunoblot was probed with the antiphosphotyrosine antibody 4G10, this result indicated that pi 10 and pi20 are phosphorylated on tyrosine. As can be seen in lanes 2, 3, and 4 of figure 5.4.1 the phosphotyrosine content of pi 10 and pi20 are modulated by the presence of PTPe. To attempt to identify the associating proteins, Crkll immunoprecipitates were probed with antibodies against a number of proteins known or hypothesized to associate with Crkll, these included pl30Cbl, c-Abl, Cas, and C3G (data not shown). However, none of the immunoblots were able to identify the associating proteins.  Like src,  Crkll possesses multiple protein-protein interaction domains. To  investigate whether like pp60* , Crkll was capable of direct association with PTPe, myc /r  tagged rat Crkll was over-expressed in HEK 293 cells already expressing wild-type PTPe and its various mutants in a stable manner. The expression vector pCAGGS-rCrkII~myc was introduced transiently into cells seeded onto fibronectin coated plates using lipofection. Figure 5.4.2 shows the expression levels of Crkll in HEK  293 expressing  PTPe (lane 2), PTPeC334A (lane 3), PTPeC629A (lane 4), and PTPeC334A/C629A (lane  101  5) 24 hours after transfection using the anti-myc monoclonal antibody 12CA5. As can be seen, large amounts of a ~40 kDa protein reacting with anti-myc antibodies was expressed in transfected cells but not cells transfected with the empty vector. This protein is also reactive with antibodies specific for Crkll (data not shown). However, figure 5.4.5 panel B reveals that PTPe Cannot be observed in 12CA5 immunoprecipitates from cells transiently over-expressing. 12CA5-tagged Crkll. This observation suggested that PTPe does not form a stable association with Crkll.  102  A  1  2  3  4  5  Figure 5.4.2a Over-expression of Crkll-myc with PTPe. HEK 293 cells expressing PTPe (lane 1 and lane 2), PTPeC334A (lane 3), PTPeC629A (lane 4), PTPeC334A/C629A (lane 5) were transiently transfected with either control vector (lane 1) or myc tagged Crkll (lanes 2 through 5).  B 1  2  3  Figure 5.4.2b Association of Crkll with PTPe. HEK 293 cells expressing PTPe (lane 1), PTPeC334A/C629A (lane 2), PTPeF112P (lane 3) were transiently transfected with myc tagged Crkll. Solubilized proteins were subjected to immunoprecipitation with the 12G5 anti-myc monoclonal antibody and immunoblotted with the anti-PTPe antibody PTPel5ic.  The investigation of the effect of PTPe over-expression on HEK 293 cells suggested that this PTPase might be specific in its effects, regulating src family kinases and Crkll. This suggests that although PTPases in general appear to have broad substrate specificity in vitro, in vivo dephosphorylation may occur only on very specific targets, most likely governed by intracellular localization of the PTPase. This hypothesis makes investigation of the non-transmembrane isoform of PTPe of great interest since a signal  sequence, transmembrane domain, glycosylation and co-localization with membrane associated signaling molecules are precluded in the case of the non-transmembrane isoform.  Expression of the non-transmembrane isoform of PTPe (p74PTPe) was achieved in HEK 293 cells with the use of the pcDEF3 expression vector. This vector utilizes the elongation factor -1 alpha promoter (EF-la) to obtain high level expression of PTPe as can be seen in figure 5.4.3 (upper panel lane 2). This figure also shows that PTPe expressed from the cDNA results in a single polypeptide migrating with an apparent molecular weight of 74 kDa. The conspicuous absence of the second species, observed in immunoprecipitates from HL60/mac cells (figure 3.3.5 lane 2), suggests that the second polypeptide may arise from a yet uncharacterized messenger RNA species. To rule out the possibility the second band of the doublet may be due to post-translational modification of the 74 kDa protein, the cDNA encodingrion-transmembranePTPe was introduced into two hemopoietic cell lines Jurkat and BaF/3. Both cell lines already express p72/74PTPe thus are capable of any post-translation modifications necessary for production of the p72 band in the p72/74 doublet. Figure 5.4.3 shows when pcDEF3cytoPTPe is expressed in either Jurkat or BaF/3 cells an increase in the 74 kDa band of the p72/74 doublet is observed. This again suggests that the 72 kDa band of the p72/74PTPe doublet arisesfroman as yet uncharacterized mRNA species.  104  1  2  3 =  69kDa  P  72r74  p74  69 kDa 1 2  3  4 p74  69 kDa  Figure 5.4.3 Transient expression of non-transmembrane PTPe in Jurkat and BaF/3 cells. (Upper panel) HL-60/mac cell lysate (lane 1) or anti-PTPe immunoprecipitation (lane 3), H E K 293 cells transiently transfected with non-transmembrane PTPe (lane 2) were immunoblotted with PTPel5ic anti-PTPe polyclonal antibody. (Middle panel) Jurkat T cell line or Baf/3 cells (Lower panel), were transiently transfected with non-transmembrane PTPe (lanes 3 and 4) cell lysate (lanes 1 and 2) and PTPel5ic immunoprecipitates were immunoblotted with anti-PTPe.  Expression of plOO/HOPTPe resulted in a modulation in the phosphotyrosine content of a number of signaling proteins when expressed in HEK 293 cells. To compare the impact on overall tyrosine phosphorylation of p74PTPs, anti-phosphotyrosine immunoblots were carried out on cells transiently expressing the PTPase. To assess the impact of p74PTPs on pp60  5rc  a demonstrated substrate of pi00/1 lOPTPs, src was  immunoprecipitated from HEK 293 cells expressing p74PTPe and subjected to both autokinase analysis and immunoblotting with the anti-phosphotyrosine antibody 4G10. Figures 5.4.4 and figure 5.4.5 reveal that src autokinase activity was undiminished when p74PTPe was expressed. In addition, the phosphotyrosine content of src remained unchanged. The inability of p74PTPs to exhibit any effect on pp60* indicates that rc  membrane localization may be essential in determining plOO/HOPTPe substrate preference for src. In addition, these results suggest that p72/74PTPe may regulate the  105  phosphotyrosine content of a completely different set of substrates. This observation effectively describes p72/74PTPe and plOO/HOPTPe as two different isoforms of the enzyme originating from a single gene. In light of these results, earlier observations describing the restricted, and mutually exclusive expression patterns of p 100/110 vs p72/74 PTPs indicates a highly regulated system controlling the presence of the two very different PTPase activities. To better understand the role of p72/74PTPs its intracellular localization must be explored to determine its potential sites of action in intracellular signaling processes.  1 2 p72PTP£  3  4  *  -«*  <  pp60  «  IgH  irc  Figure 5.4.4 Phosphotyrosine content of pp60src in HEK 293 cells expressing non-transmembrane PTPe. HEK 293 cells were transiently transfected with nontransmembrane PTPe (lanes 2 and 4) or control vector (lanes 1 and 3). pp60 was immunoprecipitated (lanes 3 and 4) anti-src polyclonal antibodies and immunoblotted with the anti-phosphotyrosine monoclonal antibody 4G10. Lanes 1 and 2 were immunoblotted with PTPel5ic. src  106  Figure 5.4.5 pp60src activity in HEK 293 cells expressing nontransmembrane PTPe. HEK 293 cells were transiently transfected with non-transmembrane PTPe (lanes 2) or control vector (lanes 1). pp60 was immunoprecipitated with anti-src polyclonal antibodies and immunoprecipitates were subjected to auto-kinase assays with PyATP. The phosphorylated kinase was analyzed by SDS-PAGE and autoradiography.  OT  32  5.5 Localization of PTPe expressed in HEK 293 cells  The importance of intracellular compartmentalization in the understanding of cellular processes has grown significantly in recent years. Localization of proteins within the membrane is crucial to many processes ranging from the assembly of focal adhesions to aggregation, capping and internalization of the antigen receptor complexes of B-cells and T-cells (Holowkwa et al, 1996, Hausen et al, 1997, Kwan et al, 1998, Kwiatkowska et al, 1999). Identification of the molecular weight of PTPs found at the plasma membrane was determined by surface biotinylation, which labelled all transmembrane proteins located at the plasma membrane. Since PTPs was observed as a high molecular weight protein migrating with the characteristics of a glycoprotein this experiment was designed to shed some light on which isoform was actually found at the membrane. HEK 293 cells exhibiting stable expression of PTPs were grown to 80%  107  confluence then surface proteins labeled using NHS-LC-Biotin (N-HydroxysuccinimidylBiotin) which labels primary amine groups with biotin allowing visualization with labelled avidin. After extensive neutralization of reactive biotin and repeated washing, TX100 lysates were prepared. To visualize the presence of PTPs at the outer membrane PTPs was immunoprecipitated using PTPsl5ic antibodies and subjected to SDS-PAGE. After blotting to PVDF membranes, biotinylated PTPs was visualized by probing with horseradish  peroxidase  linked  streptavidin  and  visualized  with  enhanced  chemiluminescence. Figure 5.5.1 reveals that the majority of surface PTPs consists of a gplOO glycoform along with a small portion present as a possibly fully glycosylated gpl 10 form of PTPs. This result suggests that gplOO and gpl 10 isoforms of PTPs are both found on the surface of these cells.  1  2  Figure 5.5.1 Surface biotinylation of H E K 293 cells expressing PTPe. H E K 293 cells expressing either wild-type PTPe (lane 1) or PTPeC334A/C629A (lane 2) were surface labeled with biotin. Cell lysates were immunoprecipitated with anti-PTPe antibodies and immunoblotted with Streptavidin linked horseradish peroxidase.  108  1  2  M  <  pHO plOO  Figure 5.5.2 Phosphotyrosine content of plOO/110 PTPe . The Western blot in figure 5.5.1 was stripped and reprobed with anti-phosphotyrosine monoclonal antibody 4G10. Lane 1, H E K 293 cells expressing wild-type PTPs . Lane 2 cells expressing PTPsC334A/C629A.  Interestingly, upon re-probing of this blot with anti-phosphotyrosine antibodies it appeared that only the mature gpllO was phosphorylated on tyrosine (figure 5.5.2). Indeed, transient expression experiments carried out in HEK 293 cells visualizing PTPe expression and localization by immunoflourescence revealed that PTPs was found in intracellular vesicular compartments (figure 5.5.3). Immunoblotting lysates from these cells with PTPsl5ic antibodies indicated an abundance of the gplOO isoform (figure 5.1.5). These results suggested that glycosylation is potetially an important step in the biosynthesis of mature PTPs.  While surface biotinylation is a useful tool in determining whether a protein is located at the plasma membrane, and the molecular weight of the species, this technique cannot distinguish the location of the protein of interest at the inner aspect of the plasma membrane. In order to characterize the sub-cellular localization immunoflourescence is useful, cells were seeded onto fibronectin coated coverslips and, afterfixationand  109  permeablization (with Triton X-100), stained with PTPs 15 ic polyclonal antibody. Immunoflourescence analysis of HEK 293 cells with stable expression of PTPs showed PTPase was concentrated at the cell surface in cell-cell junctions (figure 5.5.3). Also seen in figure 5.5.3 is the presence of intense PTPs staining in the perinuclear space and within the endoplasmic reticulum, indicating the presence of a premature form of PTPs not located at the cell surface. Interestingly, the gpllO isoform was the only form of PTPs and PTPsC334A/C629A that was phosphorylated when this mutant was overexpressed in HEK 293 cells (figure 5.5.2). This observation reiterates that not only is the gpl 10 isoform the mature form of PTPs in cells with stable expression of transmembrane PTPs but also in cells transiently expressing transmembrane PTPs. Figure 5.5.3 shows that transmembrane PTPs, as described earlier, was found at cell-cell junctions as well as within the perinuclear space. However, non-transmembrane PTPs, as shown in figure 5.5.3, unexpectedly exhibited the strongest immunoflourescence in the nucleus. This observation is reminiscent of the biochemical signaling of the Notch receptor. In Notch signal transduction, the receptor is cleaved by a protease at a conserved valine residue that lies close to the transmembrane domain (Schroeter et ah, 1998). Upon activation Notch translocates to the nucleus where interaction with the CBF1 transcription factor results in gene regulation (Jarriault et al., 1995). While isoform p72/74PTPs appears to arise from a novel mRNA and not proteolytic cleavage the truncated enzyme may function in a similar manner.  110  A  B  . ^f'  •  •W  •  | '  «[  i  C  D  Figure 5.5.3 Immunoflourescence staining of PTPe in H E K 293 cells expressing PTPe in a stable manner. H E K 293 cells with stable expression of wild-type PTPe (panels A and B) or transient expression of non-transmembrane p74PTPe (panel C and D) were stained with PTPel5ic. PTPe sub-cellular localization was observed with FITC coupled goat anti-rabbit polyclonal antibodies and photographed under immunoflourescence. Panels A and Cshow the corresponding phase contrast photograph.  The strong immunoflourescence observed at cell-cell junctions, and at the edges of cell membranes suggested that the surface biotinylation observations described in figure 5.5.1 are valid. Immunoflourescence staining of transiently transfected HEK 293 cells with TRITC labelled phalloidin, a reageant specific for filamentous actin, revealed that non-transmembrane PTPe staining was associated with phalloidin staining filaments within the cytosol (data not shown). Ill  Localization of an alternative spliced form of a transmembrane-cytosolic PTPase to the nucleus is a novel observation. Perhaps offering an explanation for its nuclear localization, the p72/74 sequence contains a putative bipartite nuclear localization consensus sequence within the N-terminus of the non-transmembrane isoform of PTPs (amino acids 14 to 29 RKQRKAVVSTSDKKMP). If responsible for nuclear localization this sequence is unique in that it lies within a region of the protein which is also found in the transmembrane isoform of PTPe. Thus it is likely that upon expression of the nontransmembrane isoform, due to alternative splicing or a second promoter, the signal and transmembrane domain sequences are not incorporated into the mature message, thus the nuclear localization sequence alters the distribution of the molecule. Further site directed mutagenesis experiments will be required to confirm that amino acids 14 to 29 are responsible for nuclear localization.  112  5.6Significance of polyproline sequences in PTPs  Extracellular signals arriving at the membrane take the form of soluble factors, extracellular matrix components or anchored cell surface associated ligands. With the exception of G protein linked receptors where aggregation is not observed, extracellular signals then interact with receptors on the cell surface resulting in oligomerization and activation. Associated protein kinases then set in motion a cascade of phosphorylation events originating at the membrane. The resulting recruitment of signaling proteins to the site of receptor stimulation is the genesis of a complex interplay of protein-protein interactions that has only begun to be explored. With the discovery of the modular domain structure of signaling proteins, great efforts have been made to elucidate how proteins interact within signaling complexes. The list of protein interaction modules continues to grow including SH2, SH3, WW, PTB, PH, and PDZ domains, each of which maintains a specificity for a certain ligand or range of ligands with similar characteristics.  Two of these domains SH3, and WW, exhibit interesting ligand specificity. Highly specific for polyproline regions within proteins, SH3 and WW domains are able to associate with the unique left handed helix. The helix is formed by the amino acid motif PXXP, where X can be either a proline residue or a hydrophobic amino acid (Adzhubei etal, 1993, Williamson 1994, Renzoni etal, 1996). By careful examination of the amino acid sequences of transmembrane PTPases it was observed that certain  113  transmembrane PTPases possessed a polyproline motif within the membrane proximal region before the first catalytic domain of the PTPase (figure 5.6.1) (Harder, 1996). SH3 domains or "Src homology 3 domains" were first described as regions within the c-src proto-oncogene that displayed significant homology with other members of the src family of protein tyrosine kinases (Sadowski et al, 1986, Koch et al, 1991, Pawson et al, 1993, Schlessinger et al, 1994). With the discovery that the SH3 domain of c-Abl had high affinities for 3BP1 and 3BP2 (Cicchetti et al, 1992) polyproline sequences within proteins were shown to bind to SH3 domains. While the SH3 domain-polyproline interaction serves as a site of docking between proteins, whether this interaction plays a specific role in the regulation of enzyme activity is not completely understood. In the case of the p85 subunit of PI-3 kinase, SH3 binding is associated with enzyme activation (Pleiman et al, 1994). However, in the case of c-Abl, its SH3 domain has been described to inhibit the ability of this proto-oncogene to cause transformation in cells (Jackson et al, 1993). The SH3 domains of Src family kinases have also been shown to play a role in regulation of the kinase in cooperation with the SH2 domain through an intramolecular SH2-phosphotyrosine interaction resulting in "closing" of the active site on the kinase. Interestingly, SH3 domains appear to play a pivotal role in the formation of enzyme complexes (Okada et al, 1993, Howell & Cooper, 1994, Kaplan et al, 1994, Erpel et al, 1995). The NADPH oxidase complex, which is involved in the production of reactive oxygen-intermediates in phagocyte activation, is one such enzyme complex, which relies upon SH3 polyproline ligand interaction (McPhail et al, 1994, de Mendez et al, 1996). The crystal structure of a number of SH3 domains has been solved in recent years allowing fine analysis of the mechanism of polyproline binding (Musacchio et al, 1992,  114  Musacchio et al,  1994, Lim et al, 1994). Figure 5.6.2 shows the crystal structure of the  Hck SH3 domain. The box highlights the binding cleft of the SH3 domain, while arrows. The ligand binding is mediated largely by hydrophobic interactions through aromatic amino acids.  PXXP containing PTPases may  in fact be ligands for SH3 domain^ containing  proteins, possibly signaling proteins. Identification of these signaling proteins could yield some insight into the signal transduction pathways regulated by transmembrane PTPases. The polyproline motifs found in certain PTPases fit the consensus for SH3 ligands and suggest the possibility that certain transmembrane PTPases may  associate with other  signaling molecules through an SH3-PXXP interaction. Figure 5.6.1  shows the  relationship of the juxtamembrane region of a few transmembrane PTPases and  SH3  binding sites of other signaling proteins. Included in the collection of PTPases that possess polyproline motifs are PTPe and PTPa. As described earlier, overexpression of PTPe was shown to result in the dephosphorylation and subsequent activation of pp60  5rc  (figure 5.3.1, figure 5.3.4). Overexpression of PTPa also results in dephosphorylation and activation of pp60"" in a variety of systems (Harder 1996, den Hertog et al, c  Zheng et al,  1993,  1992). These results suggested that the polyproline region within these two  PTPases may play a role in allowing a direct association of c-src with PTPases. This would facilitate a direct dephosphorylation event ending in the observed activation of pp6Cf . However, an interesting difference was observed between the juxtamembrane rc  sequences of PTPe and PTPa. As can be seen in figure 5.6.1, PTPa exhibits a characteristic PPLP motif. PTPa has also been demonstrated to associate with various  115  SH3 domains (Harder 1996). The SH3 domain of pp53/56^-showed the highest affinity, in vitro of those SH3 domains tested. In contrast, PTPs has the sequence FPIP, missing the critical first proline residue within the motif. In theory, the absence of the first proline residue abrogates the formation of the left-handed poly-proline helix required for efficient SH3 domain interaction. Further experiments showed that the polyproline region of PTPs is not capable of binding any of the various SH3 domains tested in vitro (Harder 1996). Comparison of the relative src activation observed while PTPs or PTPa was overexpressed suggested that PTPa may also be more efficient in activating pp60 . The src  PTPa A R S P _ S T N R K Y P P L P | V D K L E E E I N R R M PTPe  S R_S_P.  LAR  S G P K K Y F P I P J V E H L E E E I R I R S  N Y Q T P | M R D H P P I ' P I T D L A D N I E R L K  PTPS  N F Q T P I M A S H P P I P I L E L A D H I E R L K  PTPa  N F Q T P | M L S H P P I P I T D M A E H M E R L K  Blk Tec  S  S H  R K T K  I  P  L P _P T. E E E D Q I L K K P L P P  L F E S S I R I T L P P A P E I K K R R P P P P I P  Figure 5.6.1 Alignments of membrane proximal regions of transmembrane PTPases and SH3 binding sites of other signaling molecules. Abbreviations: PTPa, protein tyrosine phosphatase alpha, PTPe protein tyrosine phosphatase epsilon, L A R , leucocyte common antigen, PTPS, protein tyrosine phosphatase delta, PTPa, protein tyrosine phosphatase sigma, Blk, Bruton's tyrosine kinase, Tec, tyrosine kinase expressed in hepatocellular carcinoma  question may then be asked as to whether the poly-proline region in PTPa bestows increased src dephosphorylation activity on PTPs. Furthermore, could the poly-proline  116  region o f P T P s be modified to match that o f P T P a , and i f so would P T P s behave in a manner indicative o f P T P a pp60  dephosphorylation?  jrc  T o determine whether the poly-proline region within transmembrane PTPases P T P s and P T P a indeed plays a role in  c-src  dephosphorylation and activation, mutant  forms of P T P s were constructed in which phenylalanine 112 ( F l 12) was substituted with a proline residue. The resulting P T P s F l 12P mutant was then introduced in to the H E K 293 cell line and clones exhibiting stable expression o f the mutant PTPase were examined further. The P T P s F l 12P mutant in effect recreates the PPIP region o f the polyproline sequence possessed by P T P a possibly restoring the putative left-handed helix formed in PTPs's close relative PTPa. The ability o f PTPs to activate pp60 assessed by immunoprecipitating or  P T P s F l 12P  mutant  c-src from cells  with  the  anti-src  jrc  was  overexpressing either wild-type P T P s monoclonal  antibody  GDll.  The  immunoprecipitates were subjected to autokinase assay as described in materials and methods  (2.20) and  the phosphate incorporation assessed  by  SDS-PAGE  and  autoradiography. The rationale being P T P s F l 12P would be more efficient in activating csrc with respect to wild-type PTPs.  117  Figure 5.6.2 Space filling model (left), Ribbon model (right) of the SH3 domain of the src kinase family member Hck. The highlighted rectangle defines the peptide binding cleft within the SH3 domain.  5.6.1 Structural basis of F112P mutant  W i t h the structures o f both P T P a catalytic domain 1 and p p 6 0 " recently solved, r  certain predictions can be made with respect to (/) the structure o f PTPe, (ii) the accessibility o f the poly-proline region within the membrane proximal region o f transmembrane PTPases, (iii) the structure o f inactive c-src due to intramolecular folding. A s can be seen in figure 5.6.3 the membrane proximal region o f P T P a forms a bi-helical wedge with the poly-proline region forming the anchor points o f one side o f the wedge (Bilwes et al, 1996). It is likely that PTPe would form a similar structure due to the very  118  high sequence similarity within this region in addition to conservation of side chain characteristics in areas of amino-acid substitution. From the crystal structure of this region of PTPa, it is apparent that the polyproline region is not buried within the protein but appears to be located on a solvent accessible surface of the protein. The crystal structure of PTPa provided evidence suggesting that the wedge formed by this region is capable of inserting into the active site of a second PTPase molecule (Bilwes et al, 1996). Upon insertion, it was hypothesized that the PTPase domain would be inhibited due to displacement of potential substrates (Bilwes et al, 1996). This hypothesis raises an interesting "dimerization leading to PTPase inhibition" model for transmembrane PTPases in which ligand induced dimerization would result in the PTPase in question being "turned o f f (Desai et al, 1993, Bilwes et al,  1996). This would be the opposite of the ligand-mediated  dimerization leading to activation of numerous growth factor receptor kinases and other receptors linked to kinases. The results also suggest an interesting possibility for the interaction of SH3 domains with this region, resulting in a possible conformational dissociation of the wedge from the catalytic site of the regulated PTPase, thus leading to PTPase activition. In vitro evidence suggested the SH3 domain of c-src interacted with the poly-proline region of transmembrane PTPases {personal communication Harder et al). It is possible that the association of pp6Cr" will result in dissociation of the r  inhibitory wedge, inducing PTPase activity. The induction of activity in turn may further activate c-src by dephosphorylating the C-terminal tyrosine residue. This model of PTPase-src interaction may describe the nature of c-src's ability to constituitively coimmunoprecipitate with PTPe as well as PTPa. It may be that localization of PTPe and  119  P T P a to a r e g i o n r i c h i n src f a m i l y kinases results i n association o f a f r a c t i o n o f the kinases w i t h P T P e , a n d P T P a l e a d i n g to activation o f this fraction. It is c o n c e i v a b l e that a certain l e v e l o f constituitive src activity v i a P T P a s e a s s o c i a t i o n i s m a i n t a i n e d  at the  m e m b r a n e at sites r i c h i n transmembrane P T P a s e s , e s p e c i a l l y P T P e , P T P a , a n d C D 4 5 . S i n c e P T P e is l o c a l i z e d to sites o f cell-cell contact, P T P e - s r c f a m i l y kinase c o m p l e x e s m a y regulate c e l l - c e l l adhesion.  Cadherin-Catenin  c o m p l e x e s are reported to regulate c e l l - c e l l a d h e s i o n w i t h  catenin e x h i b i t i n g exceptional  c-src  substrate characteristics  in vivo.  0-  The morphology o f  H E K 293 cells expressing the d o m i n a n t negative mutant o f P T P e , P T P e C 3 3 4 A 7 C 6 2 9 A , i n d i c a t e d that the cell-cell vs. cell-substratum a d h e s i o n e q u i l i b r i u m h a d b e e n altered. Cells expressing  PTPeC334A7C629A  i n a stable f a s h i o n s h o w e d  increased  cell-cell  a d h e s i o n a n d decreased cell-substratum adhesion, c o i n c i d i n g w i t h a modest decrease i n csrc tyrosine p h o s p h o r y l a t i o n a n d activity.  5.6.2 Characterization of HEK 293 cells expressing F112P mutant  T o express P T P e F l 12P i n H E K 293 cells, the mutant c D N A w a s c l o n e d into the eukaryotic v e c t o r B C M G S n e o . T h e expression  vector p B C M G S n e o - P T P e F l 12P w a s  then i n t r o d u c e d into H E K 293 cells b y l i p o f e c t i o n i n s e r u m free media. C e l l s e x p r e s s i n g P T P e F l 12P, u n d e r the control o f the C M V promoter, i n a stable m a n n e r w e r e selected f o r i n G 4 1 8 at a concentration o f 600 ug/ml f o r 7 days at l i m i t i n g d i l u t i o n i n 96 w e l l dishes.  120  Expression of PTPsF112P appeared not to be lethal since clones were readily obtained. Immunoprecipitation PTPase assays with PTPel5ic polyclonal antibodies from lysates prepared from HEK 293 cells expressing PTPeFl 12P showed that the mutant PTPase possessed PTPase activity (figure 5.6.4). This indicates that the F112P mutation did not alter the conformaiton of the PTPase to an extent to hinder the catalytic  Figure 5.6.3 Crystal structure of PTPa. The MP region of PTPa is shown in white, while the analogous N-terminus of PTP1B is shown in grey.  activity. Upon close observation, although HEK293 cells expressing PTPsF112P were viable, their morphological characteristics were different than control HEK293 cells and  121  m)i  04-  - i  0  5  1  1  1  »  15  23  tin^nn) Figure 5.6.4 PTPase activity of P T P e F l 12P. Whole cell lysates prepared from 293 cells exhibiting stable expression of • PTPeFl 12P, • Control and • PTPe were subjected to immunoprecipitation with PTPel5ic polyclonal antibody. Immunoprecipitates were then subjected to M G PTPase assays. O D values were converted to pmols of P 0 using a inorganic P 0 calibration curve. 4  4  from 293 cells expressing wild-type PTPs. While 293 cells are adherent cells with prominent cellular processes as can be seen in figure 5.6.5, cells expressing  PTPEF112P  appeared to have longer, more pronounced processes as compared to those cells expressing wild-type PTPs or control cells. Although subtle, the change in cellular morphology was observed in all 293 clones expressing the F112P mutation observed. Interestingly, a report by Luttrell and Parsons also describes a similar increased prominence of cellular processes in adherent cell lines. The phenomena described was linked to an increase in src family kinase activity (Luttrell et al., 1988).  122  Figure 5.6.5 Cellular morphology of H E K 293 cells. Phase contrast micrographs of H E K 293 cells under normal growth conditions at 200x magnification. (Left Panel) Cells exhibiting stable expression of the control vector B C M G S . (Right Panel) Cells expressing PTPeFl 12P. White arrows indicate spindle like cellular processes. n e 0  To determine whether the substitution of phenylalanine at position 112 for a proline residue had any impact upon the function of PTPe, immunoprecipitations of c-src were probed with anti-phosphotyrosine monoclonal antibody 4G10. Figure 5.6.6 reveals that c-src within cells expressing PTPeFl 12P (lane 6) possessed far less phosphotyrosine than c-src found in cells expressing wild-type PTPe (lane 2) or cells not expressing the PTPase (lane 1). If the dephosphorylation of c-src is a direct effect as suggested in section 5.6.6 this result indicates that the F112P mutation of PTPe resulted in c-src becoming a more prominent substrate of this PTPase. This implies that the restoration of the putative SH3 domain binding site within PTPe somehow altered the ability of PTPe to dephosphorylate c-src, possibly through interaction with src's SH3 domain. To examine whether this phenomenon resulted in increased c-src activity, src immunoprecipitates  123  were assayed for autokinase activity. Figure 5.6.7 shows that PTPeFl 12P expression in HEK 293 cells causes not only a decrease in c-src phosphorylation but also a corresponding increase in src's kinase activity.  1 2  3  Figure 5.6.6 pp60  src  4  5  6  4  pp60  <  IgH  ;  tyrosine phosphorylation in H E K 293 cells expressing  PTPEF112P mutant. H E K 293 cells transfected with various mutants of PTPe were subjected to immunoprecipitation with the anti-src monoclonal antibody GD11. (lane 1) 293 cells transfected with control vector, (lane 2) 293 cells transfected with expression vector containing wild-type PTPe. (lane 3) 293 cells transfected with BCMGSneo-PTPeC334A. (lane 4) 293 cells transfected with  BCMGSneo-PTPeC629A.  BCMGSneo-PTPeC334A/C629A.  (lane  5)  293  cells  transfected  with  (lane 6) H E K 293 cells transfected with  BCMGSneo-PTPeFl 12P.  Since the C-terminal phosphorylation site is responsible for negative regulation of the family of kinases, this result suggested that, in agreement with the in vitro PTPase assays, that the C-terminal phosphorylation site of src family kinases may be an in vivo substrate of PTPe. The dephosphorylation of c-src, observed in figure 5.6.6, must also include the C-terminal regulatory phosphorylation site to bring about the increase in kinase activity observed in figure 5.6.7. It has been shown that proteins binding to the csrc, SH2 or SH3 domains, may also produce conformational changes that increase kinase activity. For example, binding of the c-src SH3 domain to a peptide fragment of the Crkassociated substrate pl30 -related protein, Sin, is sufficient to induce activation of c-src Cas  124  (Alexandropoulos  and  Baltimore  1996).  Similarly,  binding  of  the  human  immunodeficiency virus-type 1 Nef protein to the SH3 domain of the Src family tyrosine kinase Hck increases the specific activity of the kinase in vitro (Moarefi et al, 1997).  2  5  4  3  6 ,src  <  Figure  5.6.7  Activation  PP60'  of pp60src in H E K 293  cells expressing  P T P e F l 12P mutant. RIP A buffer soluble lysates were prepared from H E K 293 cells expressing various isoforms of PTPe in a stable manner. pp60 was immunoprecipitated from: lane 1, cells transfected with control vector, lane 2, cells expressing wild-type PTPe, lane 3, cells expressing PTPeC334A, lane 4, cells expressing PTPeC629A, lane 5, cells expressing PTPeC334A/C629A, lane 6, cells expressing PTPeFl 12P, and subjected to autokinase assay. ire  In this context, the PTPsFl 12P mutant appears to have a two-fold role in the activation of src. First, the restoration of a putative SH3 domain may facilitate displacement of c-src's SH2-SH3 domain from the C-terminal phosphotyrosine of the kinase. Secondly, this allows the PTPase to more efficiently dephosphorylate of the C-terminal phosphotyrosine of src, which in turn results in the kinase remaining in an active state.  In section 5.4 experiments focused upon small adaptor proteins revealed that Crkll  showed decreased phosphotyrosine content in cells expressing PTPs. This result  suggested  that  the  PTPase  may  be  directly  or  indirectly modulating  the  dephosphorylation of C r k l l since the phenomenon was dependent upon the expression of  125  an active PTPase domain. The results observed with respect to c-src indicated the relationship between PTPe and Crkll should be revisited. The phosphotyrosine content of Crkll was assayed by immunoprecipitation of Crkll from cell lysates prepared from cells expressing PTPeFl 12P, wild-type PTPe or other mutants and immunoblotting with the anti-phosphotyrosine antibody 4G10. Figure 5.6.8 shows that indeed the F112P mutant does impact the effect of PTPe on Crkll. Lane 2 reveals that Crkll's phosphotyrosine content decreases with PTPe expression as described earlier. However, lane 6 shows that expression of PTPeFl 12P does not result in the dephosphorylation of Crkll as seen when wild-type PTPe is expressed.  1  2  3  4  5  6  Figure 5.6.8 C r k l l phosphorylation in H E K 293 cells expressing P T P e F l 12P. H E K 293 cells transfected with various mutants of PTPe were subjected to immunoprecipitation with anti-crk monoclonal antibodies. (Upper panel) After immunoblotting the phosphotyrosine content of Crkll was observed with 4G10 immunoblotting. (lane 1) 293 cells transfected with control vector, (lane 2) 293 cells transfected with expression vector containing wild-type PTPe. (lane 3) 293 cells transfected with BCMGSneo-PTPeC334A. (lane 4) 293 cells transfected with BCMGSneo-PTPeC629A. (lane 5) 293 cells transfected with BCMGSneo-PTPeC334A/C629A. Lower panel is the blot in the upper panel stripped and reprobed with anti-Crk antibodies.  These observations suggested that the substitution of phenylalanine residue at position 112 for a proline residue, resulted in restoration of a putative SH3 domain ligand. The substitution also altered the substrate specificity of PTPe while not disrupting, in any measurable way, PTPase activity. While a complete account of the  126  mechanism responsible for this phenomenon is beyond the scope of this thesis. The ability of a single amino acid substitution to significantly alter the phenotype of a PTPase warrants further study.  5.7 Summary By utilizing cDNAs encoding both transmembrane and non-transmembrane isforms of PTPs, the different forms of this PTPase were introduced into mammalian cells. In the case of transmembrane PTPs, various mutants were characterized in vivo. These studies revealed that plOO/HOPTPs is a highly glycosylated membrane protein, found predominantly at cell-cell junctions. While plOO/HOPTPs was capable of associating with and modulating the phosphotyrosine content and kinase activity of the src family kinase members pp60 ~ and pp59*", p72/74PTPs was not. This phenomenon c  src  was probably in large part due to the localization of p72/74PTPs to the nucleus and not the plasma membrane where src family kinases are found. Finally, by the introduction of a proline residue at position F l 12, an S H 3 domain-binding site was reconstituted, thus turning PTPs into a PTPa-like molecule. PTP&F112P exhibited altered substrate specificity showing increased activity towards src family kinases and reduced activity towards the in vivo substrate Crkll. These findings are summarized in figure 5.7.1.  127  PTPe  PTPe C334A  PTPe C629A  Morphological changes ++ Cytosolic Nuclear Membrane associated Src dephosphorylation Fyn dephosphorylation Lyn dephosphorylation Crkll dephosphorylation  PTPeC334A /C629A  PTPe F112P  +++  ++  >-  .:+++  ++ ++ + +++  +++  +++  +  +  +  ,.+++  +++ .  -  +++  -  -  P74PTPe  ++ +++ + .  Figure 5.7.1 Summary of in vivo observations with plOO/llOPTPe , p74PTPs and the various mutants. Plus signs indicate intensity of effect or magnitude of observation on a scale of one to three. Minus sign indicates opposing effect  128  Chapter 6  Discussion  The roles of protein tyrosine phosphatases in the regulation of cell growth, differentiation and cell adhesion, with a few exceptions, are still relatively poorly understood. This thesis has endeavored to shed some light on the roles of PTPases in cellular regulation by asking the question which PTPases are upregulated in cells of the monocyte/macrophage lineage in response to the pro-inflammatory stimuli IFNy. Since cells of the monocyte/macrophage lineage play a large role in the process of inflammation, they may be a prime target system to search for novel methods of intervention for treatment of certain inflammatory diseases. A novel PCR based screening system was developed and PTPs was isolated as a candidate PTPase. The aim of this thesis then focused on the characterization of PTPs in hemopoietic and nonhemopoietic cells. This involved the production of recombinant forms of PTPs to investigate the activity and substrate specificity of PTPs in comparison to PTPa and  129  CD45, PTPases that share relatively high sequence similarity to PTPs. In studying both hemopoietic and non-hemopoietic cells a novel 72/74 kDa isoform of PTPs was discovered in hemopoietic cells. The analysis involved the development of novel techniques including mass spectrometric analysis of tryptic peptides and MS/MS sequencing leading to its isolation and characterization. The study of PTPs in nonhemopoietic cells required the expression of the enzyme in mammalian cells and necessitated the development of novel reagents including antibodies and mutant forms of the enzyme. PTPs expression in mammalian ceils resulted in a number of insights in to the functions of PTPs in cells as well as possible in vivo substrates and signal transduction mechanisms of the PTPase.  6.1 Upregulation of PTPe in cells of the monocyte/macrophage lineage. Using  degenerate oligonucleotides and cDNA prepared from IFNy stimulated RAW 264.7 cells, a murine peritoneal macrophage cell line, PCRfragmentsof the catalytic domains of PTPases were purified. The fragments were immobilized and subjected to hybridization with labeled PCR fragments from unstimulated cells. The screen resulted in the identification of CD45, PTPa, PTP1B and PTPs as candidate PTPases. The identification of PTPs in this screen indicated this PTPase was expressed and possibly regulated in hemopoietic cells. Vanadate treatment also resulted in upregulation of PTPs message. This experiment suggested the involvement an unknown PTK in PTPs message regulation in response to LPS or IFNy. In light of the accepted intracellular signal transduction mechanism of IFNy it may be suggested that Jakl and Jak3 may be the  130  targets of vanadate if indeed the PTPs promoter were found to possess an ISGF3/STAT1 binding site. Future studies will undoubtedly uncover the answers to these questions.  Filter hybridization analysis of mRNA purified from HL-60 cells revealed transcripts of -6.5 kb, -2.1 kb and -1.8 kb hybridizing to the PTPs cDNA sequences (Seimiya et al., 1995; Elson and Leder, 1995b). Similar transcript heterogeneity was observed in MEL cells treated with various stimuli (Kume et al., 1994).  In addition,  evidence of as many as four different transcripts was obtained from analysis of various mouse mammary tumor lines (Elson and Leder, 1995a). Our study has also described the PTPs mRNA heterogeneity as well as regulation of message levels by the proinflammatory stimuli. The numerous PTPs-hybridizing transcripts likely arise as a result of alternative splicing of one gene, as in situ hybridization revealed only a single chromosomal position, 10q26, for PTPRE (Melhado et al, 1996, Maagdenberg et al., 1995)3. LPS and IFNy stimulation regulated PTPs production at the post-transcriptional level in HL-60 cells. In response to IFNy or LPS increased proteins levels were not observed, although message levels showed a 16-fold induction over a twenty hour period. In contrast to LPS, IFNy and MCP-1 other pro-inflammatory stimuli had little effect on PTPs messenger RNA indicating that the PTPs promoter may be restricted in the stimuli that result in induction of message. This was not the case when HL-60 cells were exposed to phorbol esters where both mRNA as well as protein levels increased within 3 hours of stimulation.  131  While LPS and IFNy signals originate at the plasma membrane with CD 14 and the IFNy-R respectively, PMA  intercalates into the plasma membrane independent of  receptor binding and activates PKC. LPS and EFNy stimulation result in a defined intracellular signal transduction cascade, giving rise to the respective effects of the two stimuli. PMA  stimulation would result in the activation of a number of different signal  transduction cascades. It could be for this reason that PMA  stimulation provides all the  necessary signals for PTPe upregulation simultaneously, while LPS and IFNy require a sequential signal cascade resulting in the activation of the proper kinases  and  transcription factors for PTPs promoter activation.  The mechanisms of mRNA induction by the various stimuli as well as the posttranscriptional regulation while an interesting phenomenon were beyond the scope of this thesis.  6.2 PTPe, PTPa and CD45. Protein tyrosine kinases have been shown to phosphorylate specific substrates in vivo and in vitro (Tan et al, 1993, Milarski et al, 1993, Lammers et al,  1997, Ahmad et al; 1997). To determine whether PTPases would display substrate  specificity, recombinant PTPs, PTPa, and CD45 proteins were prepared using the pGEX prokaryotic expression system. Using a glutathione affinity matrix, the proteins were purified to greater than 95% purity as judged by SDS-PAGE. The enzymatic activities of the PTPases were examined using a panel of synthetic phosphopeptide substrates compiled from known regulatory phosphorylation sites of signaling proteins. The analysis used excess substrate and enzyme concentrations that allow less than 10% of the  132  substrate to be converted to the non-phosphorylated form to avoid end-product inhibition effects. In addition, reaction times were used that measure the initial rate of substrate turn over. PTPe, PTPa, CD45 and native immunoprecipitated p72/74PTPe were subjected to kinetic analysis. The plot in figure 4.3.1 revealed a parabolic curve of PTPase activity in response to increasing pH. The relatively acidic pH optima indicates that the PTPase was most efficient when an atom within the catalytic site is in a deprotonated state. The pKa of the sulfhydryl group of a cysteine residue is very close to the pH optima of PTPe suggesting that the deprotonation of the catalytically essential cysteine residue within the active site is most likely crucial for catalysis of phosphotyrosine. However, the pH optima curve of PTPe after pH 5.8 does not plateau but enters a phase of decreasing activity with increasing pH indicating that as another atom within the active site becomes protonated it is less able to participate in the catalysis of phosphotyrosine to tyrosine.  The following phosphopeptides, tyrosine 1009 an auto-phosphorylation site of the platelet-derived growth factor receptor (PDGF-R) (van der Geer et (PDGFR  Y1009  ), tyrosine 1021 of the PDGFR (PDGFR  proto-oncogene (src of the p59/64 c  hck  Y527  YI021  al,  1994)  ), tyrosine 527 of the c-src  ) (Cooper and Howell, 1993), and the carboxy-terminal tyrosine  src family PTK (Ziegler et al, 1987), hck  YS01  were found to be suitable  PTPe substrates for PTPe. The aforementioned substrates displayed V  max  /k values of m  2.5, 4.2, 2.5, 0.8. In general, PTPe showed no activity towards a number of peptides which LAR  and CD45 were able to dephosphorylate with high efficiency. Those  substrates which were particularly poor for PTPe, but good for LAR or CD45, include  133  PDGF-R  Y 8 5 7  and C S F 1 - R  Y 7 0 8  . These results suggested that P T P e m a y e x h i b i t m o r e  restricted substrate s p e c i f i c i t y than L A R o r C D 4 5 in vitro, h o w e v e r , analysis o f a larger p a n e l o f synthetic substrates w o u l d be r e q u i r e d to validate s u c h a d e t e r m i n a t i o n . Peptides based o n the interferon-receptor a l p h a - c h a i n (IFNy-Roc) S T A T 1 b i n d i n g site ( F a r r a r a n d Schreiber, 1993), r F N y - R a 1993), S T A T 9 1  Y 7 0 1  Y 4 4  ° , a n d a p h o s p h o r y l a t i o n site o n S T A T 1  ( S h u a i et al.,  were r e l a t i v e l y p o o r substrates i n the phosphatase assay.  villi A released o n l y trace amounts o f phosphate f r o m the S T A T 9 1  Y 7 0 1  Indeed,  phosphopeptide.  W h i l e the n u m b e r o f different phosphopeptides u s e d w a s s m a l l , there w a s sufficient  variation between  the peptide  substrates  that clear trends  r e g a r d i n g the substrate preferences o f p 7 2 / 7 4 . P D G F - R substrate o f the p a n e l o f 5 substrates tested w i t h src \ YS2  Y 1 0 2 1  were  present  appeared t o b e the best  hck \ Y50  Stat91  Y 7 0 1  , FNy-R  Y 4 4  °  f a l l i n g i n the order listed. T h e r a n k i n g o b s e r v e d w i t h the native e n z y m e w a s s i m i l a r to that o f the r e c o m b i n a n t P T P e w h e r e the substrate s p e c i f i c i t y c o u l d b e d e s c r i b e d w i t h k i n e t i c parameters. P T P e appears to prefer n e g a t i v e l y charged residues to the a m i n o t e r m i n a l side o f the p h o s p h o t y r o s i n e m o i e t y , a n d a n absence o f p o s i t i v e l y c h a r g e d residues  at the c a r b o x y l - t e r m i n a l side. S i m i l a r results  were  obtained  from  native  p 7 2 / 7 4 P T P e i n i m m u n o p r e c i p i t a t i o n P T P a s e experiments. . F i g u r e 4.6.3 s h o w s a definite preference for a c i d i c , o r residues w i t h p o l a r side chains c o n t a i n i n g o x y g e n N - t e r m i n a l to the p h o s p h o t y r o s i n e . H o w e v e r , non-polar, or h e l i x b r e a k i n g residues s u c h as p r o l i n e w e r e less tolerated. T h e r e g i o n C terminus to the p h o s p h o t y r o s i n e residue appeared less c r i t i c a l w i t h o n l y b a s i c residues appearing t o have deleterious affects t o P T P a s e a c t i v i t y . T h i s suggests that r e c o m b i n a n t P T P e is a suitable source o f p u r i f i e d p r o t e i n f o r in vitro  134  studies. Such preferences would be consistent with observations made in similar analyses of several different PTPases (Cho et al, 1993; Ruzzene et al, 1993; Zhang et al,  1993;  Harder et al, 1994; Dechert et al, 1994). However, additional studies employing a wider panel of substrates, and quantities of enzyme sufficient to allow kinetic analysis, will be required for a more detailed picture of the in vitro substrate preference of p72/74. The amount of useful information to be gained from further in vitro studies would likely be minimal due to the general ability of PTPases to catalyze the release of phosphate from even the poorest synthetic substrates. In vitro studies to date suggest that PTPases choose substrates not only by the binding preferences of the catalytic site but by other substrate binding sites located elsewhere on the molecule. However, the peptides used lack a definable secondary or tertiary structure and are unable to extend outside of the catalytic pocket of the enzyme. This may provide a partial explanation of generic nature of the synthetic substrates used as determined by the poor k values observed in comparison m  with protein kinases. It may be that protein kinases contain much of the substrate specificity information within the catalytic domain of the enzyme, possibly the substratebinding pocket itself. In contrast the majority of PTPase substrate specificty may  be  determined by sequences outside of the substrate-binding pocket, possibly at adjacent sites within the catalytic domain or other sites of the protein. This may explain the very high amino acid conservation between PTPases within the catalytic site, highlighting the generic nature of the catalytic activity, whereas outside the catalytic domain PTPases diverge quite drastically.  135  6.3 Expression of transmembrane and non transmembrane PTPs. To characterize PTPe  function, a variety of reagents were developed. These included high affinity specific polyclonal antibodies, the cDNA encoding non-transmembrane PTPe and point mutated cDNAs generated by site directed mutagenesis. The point mutated forms of the transmembrane enzyme lack the catalytically essential cysteine residue in either or both of the catalytic domains. In addition, a novel mutant was generated replacing phenyalanine 112 with a proline residue restoring a putative SH3 binding site first observed in P T P a and homologues.  Expression of the wild-type PTPe cDNA in 293 cells gave rise to a 100/110 kDa protein doublet as observed when cell lysates were immunoblotted with anti-PTPe antisera. The 100/110 kDa protein also immunoprecipitated with anti-PTPe antibodies and exhibited PTPase activity when immunoprecipitated. The migration pattern, observed by SDS-PAGE electrophoresis, of plOO/HOPTPe was characteristic of a protein containing significant carbohydrate structures. Indeed, the observed molecular weight of the transmembrane isoform is approximately 50 kDa greater than the predicted molecular weight deduced from the primary proteins sequence. Immunoflourescence microscopy analysis revealed that PTPe was located at the plasma membrane and in high concentration at cell-cell junctions. This cellular localization pattern suggests that PTPe may play a role in regulating the tight junctions or adherens junctions. Indeed, src has been shown to play a role in regulating cadherin function (Matsuyoshi et al, 1992). In light of the in vitro substrate specificity results discussed earlier PTPe may play a role in the regulation of src family kinases.  136  The truncated cDNA proved extremely useful, giving rise to a single 74 kDa band, the p74 isoform of PTPe when expressed in mammalian cells. The absence of the 72 kDa protein observed in HL-60 cells with phorbol-ester induced differentiation, when this cDNA was expressed, indicates that the p72 isoform is probably the product of an additional messenger RNA species. This isoform was localized to the nucleus as well as associated with actin bundles within the cytosol. The subcellular localization of p74PTPe suggests that the two isoforms behave differently in cellular signal transduction. An assessment of pp60 "* tyrosine phosphorylation and kinase activity revealed that c  rc  while expression of the transmembrane isoform resulted in dephosphorylation and increased kinase activity, when the non-transmembrane isoform was expressed there was no change in c-src. This observation highlights the potential complexity of PTPe function in signal transduction systems.  It was hypothesized that a single PTPe gene gives rise to similar proteins with identical catalytic domain sequences that carry out different functions and have different substrates. In the case of PTPases, cellular localization appears to be the determining factor of substrate specificity. A single PTPase may be localized to a variety of potential molecular complexes at a number sites within the cell. At any given site, there may be a completely different population of suitable substrates. This provides one explanation of why in vitro PTPase assays revealed very similar synthetic substrate preference profiles between different PTPases. This also suggests that not only the substrate-binding pocket determines the substrate specificity but also other sequences, and in certain instances  137  perhaps the entire protein plays some role in substrate recognition and association. Thus the paradigm of one enzyme, one function, one substrate may only be true for enzymes in less complex systems such as metabolic pathways. However, this may be invalid for enzymes involved in systems with high degrees of complexity, such as regulatory pathways  within  biological  systems.  Biological systems  require  coordinate  communication between multiple pathways. High degree of cross-talk necessity to achieve the degree of regulation required for the organization a multicellular organism. This may be achieved with an economy of effort by reuse of regulatory proteins in different signal transduction systems.  6.4 PTPs signal transduction. Expression of PTPe and its mutants in eukaryotic cells gave rise to both morphological changes as well as biochemical changes. PTPe appeared to be glycosylated, which seemed to be important for the biosynthesis of the transmembrane isoform of PTPe. An initial glycosylation event gives rise to the nonheterogeneous gplOO isoform or "core protein". In a second glycosylation event the mature heterogeneous gpllO protein is produced and transported to its compartment on the cell surface. At the plasma membrane suitable upstream regulators, including PTKs and substrates, are also co-localized. It well be that glycosylation is utilized as a regulatory step controlling the amount of PTPe available at particular sites within the membrane. Indeed, immunoflourescence experiments showed intracellular vacuoles of PTPe and only the plOO isoform could be detected in transiently transfected HEK 293 cells. A possible explanation for this observation is that the intracellular machinery  138  responsible was not in place for the glycosylation of PTPe resulting in its transport to the cell surface. Expression of wild type PTPe as well as its mutants resulted in six significant observable changes. First, the phosphotyrosine profile of cells expressing PTPe showed decreased phosphorylation of a number of proteins within the 40 kDa and 60 kDa ranges. Secondly, cells expressing wild-type PTPe displayed long spindle-like cellular process and a reduced number of cell-cell contacts. Thirdly, pp60 ~* showed a decrease in c  rc  tyrosine phosphorylation and a modest increase in kinase activity only in those cells expressing wild-type PTPe. Cells expressing mutant forms of PTPe where either catalytic domain was compromised, phosphorylation and kinase activity of c-src showed little change. However, in cells expressing the PTPeC334A/C629A mutant, where both catalytic domains were compromised, there was no detectable change in cell survival as compared to vector control. This indicated that ectopic expression of potential dominant negative forms of PTPe was not toxic to these cells. PTPe may not impinge upon any signal transduction pathways essential for cell survival. While cell survival was normal, intracellular changes were detected, c-src showed a modest but reproducible increase in tyrosine phosphorylation and decrease in respective kinase activity. The family of src protein tyrosine kinases share the same protein domain structure as well as very high sequence similarity. As shown in figure 6.1, src family members also share a tyrosine residue in the C-terminal region of the kinases. A key regulatory site, when phosphorylated  either through auto-phosphorylation  or phosphorylation by csk  dramatically inhibits kinase activity. While all family members share this site, the amino acid sequence around this site is variable, and falls into two subfamilies. The first  139  subfamily contains the sequence PQYQPGENL and includes src, yes, fyn, and to a certain extent fgr. The second subfamily is defined by the sequence GQYQQQP with Ick, hck, lyn, and (to a lesser extent) blk as its family members. PTPe was capable of causing a decrease in the phosphotyrosine content offynto the same extent as src. However, PTPe was not capable of mediating a decrease in the phosphotyrosine content of lyn. This observation.indicated that if indeed the dephosphorylation of src family members is an event mediated directly by PTPe, then this PTPase may be restricted to preferencially regulating the phosphorylation of the PQYQPGENL subfamily of src family kinases. Co-immunoprecipitation  experiments  also suggested that the changes in c-src  phosphorylation and subsequent activity might be a direct effect, revealing a stable association between pp60  csrc  and PTPe. However, when immunoprecipitating PTPe and  immunoblotting for src an association could not be detected. A number of explanations are available to account for the inability of the reciprocal experiment to work. The poor immunoblotting efficiency of the monoclonal antibody may prohibit the approach of  src yes fyn fgr lyn hck Ick blk  T T T T T T T T  S A A S A A A A  T T T A T T T T  E P Q Y Q P E P Q Y Q P E P Q Y Q P E P Q Y Q P E G Q Y Q Q E S Q Y Q Q E G Q Y Q P E P Q Y E L  G G G G Q Q Q Q  E E E D P P P P  N N N  L L L Q T  Figure 6.1 Homology between C-terminal regions of src family members. The tyrosine residue in bold and underlined denotes the C-terminal regulatory phosphorylation site within the src family of protein tyrosine kinases.  140  immunoblotting for c-src. In addition, the actual contact points of the c-src molecule are not known, thus there is a possibility that the anti-PTPe polyclonal antibody may somehow disrupt the interaction. In any event, the functional evidence in cooperation with the detection of PTPe in c-src immunoprecipitates, indicate a strong likelihood that the two proteins do associate in vivo.  Fourthly, the small adapter protein Crkll mirrored the tyrosine phosphorylation of c-src. Cells transfected with wild-type PTPe showed a decrease in Crkll phosphorylation.  However,  cells  expressing  PTPeC334A,  PTPeC629A,  or  PTPeC334A/C629A, which have either catalytic domain mutated or both, showed little change  in  Crkll  tyrosine  phosphorylation.  Moreover,  cells  expressing  PTPeC334A/C629A exhibited a modest but reproducible increase in Crkll tyrosine phosphorylation. Whether this phenomenon was a direct effect of PTPe tyrosine dephosphorylation of Crkll, or an indirect effect of PTPe expression activating an unknown signaling pathway resulting in Crkll dephosphorylation or inhibition of phosphorylation is unclear. Co-immunoprecipitation experiments indicated that PTPe and Crkll, unlike PTPe and c-src, did not form a stable physical association. However, those experiments did not rule out the possibility that Crkll is a physiological substrate of PTPe and the two proteins merely form a transient complex.  The significance of pp60 ' , and Crkll dephosphorylation remains unclear. Src c  src  has been demonstrated to play a role in cellular adhesion (Matsuyoshi et al, 1992) with the participation of PTPa possibly through the regulation of focal adhesions (Harder et  141  al, personal communication). Observations presented here describe a role for PTPe, PTPas close relative, in cell-cell junctions. Src has also been shown to play a role at the sites of cellular contact through phosphorylation of E-Cadherin and P-catenin and subsequent regulation of cell-cell interactions including invasiveness of carcinoma cells while Crkll has not (Behrens et al,  1993). Again the possibility exists that there are  multiple pools of PTPe at the plasma membrane that have different substrates. PTPe may dephosphorylate both c-src and Crkll under certain physiological conditions within various pools, however, the two events are mutually exclusive since the proteins would be separated spatially and involved in different signaling pathways. At any given site, there may be a completely different population of suitable substrates.  The fifth significant difference involved expression of PTPeC334A/C629A, the mutant form of the PTPase in which both catalytic domains have had the essential cysteine residue converted to alanine, resulted in a significant morphological change. Expression of the mutant PTPase incapable of catalyzing the release of free phosphate from its substrates resulted in the mammalian cells adhering poorly to the substratum. Additionally, PTPeC334A/C629A expressing cells adhered very tightly to other cells and appeared in capable of spreading out to populate the entire culture dish. The transfected cells grew in large elongated clumps maintaining cellular contact between clumps. From these observations, it may adhesion. PTPe may  be suggested that PTPe indeed may  possibly be  influence cellular  regulating these cellular functions through  dephosphorylating structural or regulatory proteins found at cell-cell junctions or cellsubstratum adhesion sites. The role of PTPases in cellular adhesion is not a novel  142  concept, recent reports have demonstrated the role of transmembrane PTPases LAR, and RPTPp as well as PTPi^ and PTP8 in homotypic adhesion (Kypta et al, 1996, Peles et al, 1998). The immunoflourescence results indicating a large proportion of PTPe is localized to cell-cell contact points serves to strengthen the argument. When observed closely, cells expressing wild-type PTPe appeared to maintain very few cellular contacts compared to the parental cell line. In addition to few cellular contacts, cells expressing wild-type PTPe appeared to spread out, and populate the entire culture dish. These observations may be interpreted in a number of ways. However, the most likely interpretation places PTPe somewhere at the membrane within cell-cell adhesion complexes. In this light, active PTPe may amplify the normal signal originating from adherens junctions resulting in fewer physical cell-cell contacts yielding the same signal necessary for survival. Alternatively, hyper-expression of active PTPe may result in hypo-phosphorylation of key proteins with complexes located and cell-cell contacts resulting in the dismantling of cell-cell adhesion complexes.  Consistent with this model, the morphology of cell  lines expressing  PTPeC334A/C629A appears to strengthen the hypothesis, suggesting the signal emanating from cell-cell contacts, presumably through cadherin-catenin interaction, was either muted by the interfering dominant negative PTPe mutant or substrates whose phosphorylation results in dissolution of adhesion complexes were protected. Hence, the atypical cell-cell adhesion and clumping morphology may represent a cellular response to provide adequate signaling from cell-cell junctions for survival. A recent link between PTPase activity and cell-cell junction regulation through N-cadherin as well as E-  143  Cadherin has been reported (Burden-Gulley  et al,  1999, Hiscox et al,  1998).  Keratinocytes treated with pervanadate, exhibited a major change in cellular morphology and cadherin/catenin localization. The latter molecules were no longer observed to colocalize with the actin cytoskeleton of cells, and the amount of E-cadherin bound to the cytoskeleton decreased. A more intense phosphotyrosine labeling was apparent at the edges of the treated cells, which suggested that an increase in the phosphorylation rate of some cadherin-catenin complex proteins induced a diminished intercellular adhesion. Immunoprecipitation experiments of the E-cadherin/catenin complex from pervanadate treated keratinocytes revealed an increase in the tyrosine phosphorylation rate of Ecadherin, beta catenin and probably gamma catenin (Soler et al,  1998). This report  suggested an essential role for protein tyrosine phosphatase in intercellular junctions (Soler et al, 1998).  The sixth and final observable difference was potentially the most interesting. Following upon observations previously made in our laboratory the polyproline region found within the juxtamembrane region of PTPe was targeted for further study. While PTPa has a primary amino acid sequence of PPLP within this region PTPe is conspicuously lacking the first proline within this motif, FPIP. Earlier work has shown that the polyproline region within PTPa was a ligand for SH3 domains. Previous work had also shown the absolute requirement for the first proline residue within this sequence for binding of conventional SH3 domains. The question then arises, why has PTPe diverged from PTPa so specifically at the polyproline motif? While this question was beyond the scope of this body of work, the ramifications of this amino acid substitution  144  on the signaling properties were not. To shed some light on the possible functions of this region, the phenylalanine 112 residue within PTPs was converted to a proline residue. This site directed mutant restored the polyproline motif as identified in PTPa but in the context of PTPe. When introduced into mammalian cells PTPeF112P appeared to alter the substrate specificity of PTPs in vivo. The PTPsFl 12P mutant, although fully retaining PTPase activity, did not dephosphorylate Crkll. In contrast, PTPsFl 12P appeared more capable of dephosphorylating pp60"* . The increased activity on c-src resulted in a c  rc  dramatic increase in kinase activity, as observed by in vitro autokinase assays. This result suggested that the polyproline regions within PTPs, and PTPa somehow play a role in substrate determination. While many mechanisms could be postulated, two are most likely. First, the polyproline region serves as a contact point between enzyme and substrate. In the case of pp60 ~*, the polyproline region of PTPs may transiently interact c  rc  with the SH3 domain in the N-terminal region of c-src changing the conformation of the substrate to provide access to the phosphotyrosine. By altering this region, one alters the affinity of the enzyme for the substrate leading to a shift in substrate specificity between two competing substrates. The second is, by altering a putative SH3 binding site the SH3 domain which associates with this site at the plasma membrane, is no longer able to recruit the PTPs to a specific pool where Crkll is localized. The subtle shift in PTPs distribution would result in Crkll retaining the tyrosine phosphate it would have had if PTPs were not expressed and giving the appearance that the substrate specificity of PTPs had been altered by the mutation. Either scenario may be applicable or a combination of both may be applicable. The similarities between PTPs and PTPa are striking, but the regions where amino acid identity break down are equally interesting. The respective  145  extracellular domains and juxtamembrane regions are the two notable regions. The less than 10% similarity of the PTPe and PTPe extracellular domains as well as the amino acid substitution at Fl 12 in PTPe appear to mark an evolutionary fork where two very similar PTPases diverged in their intracellular functions.  To date, only the family of stress induced PTPases such as PAC-1, CL100 and MKP1 have been have been shown to be regulated by similar stimuli and localized to the nucleus. Our observations suggest that PTPe may also be included in this family of induced nuclear PTPases. However it is unlikely that nuclear PTPe regulates similar substrates as the dual specificity PTPases. Identification of the STAT (signal transducers and activators of transcription) family of tyrosine phosphorylated transcription factors, which function in a number of cytokine signal transduction pathways including the interferon signaling pathway (Fu et al., 1992, David et al, 1993), raises the possibility that nuclear PTPases may play active roles in regulating gene expression either through regulating the phosphorylation status of transcription factors or nuclear kinases such as cabl. Whether this level of gene regulation is unique to PTPe remains to be seen, however, the observations described undoubtedly describe a new layer of complexity in PTPase regulation. Numerous attempts to define a role for PTPe in hemopoietic cells in response to various growth factors and cytokines yielded no change in tyrosine phosphorylation or enzyme activity. The only stimulus capable of eliciting a change in PTPe tyrosine phosphorylation  was pervanadate, a stimulus currently characterized as non-  physiological. Introduction of mutant isoforms of p72/74PTPe was also attempted. This  146  possibly would have allowed substrate trap experiments to be carried to determine the •i'  substrates of p72/74PTPe. However, poor transfection efficiencies hampered this line of investigation. In the future perhaps novel gene transfer techniques will facilitate such experiments. Further efforts must be aimed at trying to understand the physiological role of p72/74PTPs, a molecule that may  represent the only protein isoform of PTPs  expressed in hemopoietic cells.  During the course of this study, the expression of the transmembrane isoform of PTPs in mammalian cells indicated a role for the PTPase in the regulation of cell-cell adhesion. Immunoflourescence studies and cellular morphology form the basis for this hypothesis. While preliminary investigation revealed no change in the expression of Pcatenin or cadherins, suitable reagents were not available to assess the tyrosine phosphorylation status of these key cell-cell adhesion proteins. Recent reports however, do implicate PTPases in the regulation of cell-cell adhesion through cadherin-catenin complexes (Soler et al, 1998). In addition, PTPs's apparent activation of pp60 "* forms c  rc  a tangible link to cell-cell adhesion. Expression of an active form of src in mammalian cells leads to hyper-phosphorylation of P-catenin. Cadherin-catenin complexes bearing excess phosphotyrosine begin to dismantle, resulting in a loss of cell-cell adhesion (Behrens et. al,  1993). This was shown by the morphology of cells expressing the  dominant negative mutant of PTPs, where abnormal cell-cell adhesion was observed with a corresponding, though modest, increase in pp60 " c  src  tyrosine phosphorylation and  decrease in kinase activity. Interestingly, it may be that PTPs has numerous substrates at the sites of adherens junctions but the dominant phenotype is the increase in cell  147  adhesion, due to the modest decrease in kinase activity. While these experiments were being carried out, an effort to use homologous recombination to create homozygous null mice at the RPTPE locus was successfully completed (Pownall and Jirik, unpublished). While analysis is ongoing, preliminary experiments revealed no visible phenotype in RPTPE" " mice. This finding may be interpreted in a number of ways. Due to the /  restricted expression pattern of PTPe as described here and by other groups, the phenotype of the PTPe knock-out may be effectively hidden since it is not yet known where PTPe function is most critical. Indeed, PTPa may in some cell types provide a level of redundancy for the loss of PTPe expression if PTPa and PTPe are concurrently expressed. 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