Functional Interactions of Protein Tyrosine Phosphatase Alpha (PTPα) and Src in Mouse Development and Integrin Signaling: Investigation of Double PTPα/Src-Deficient Mice and Cells CHEN MIN NATIONAL UNIVERSITY OF SINGAPORE 2007 Functional Interactions of Protein Tyrosine Phosphatase Alpha (PTPα) and Src in Mouse Development and Integrin Signaling: Investigation of Double PTPα/Src-Deficient Mice and Cells CHEN MIN (M.Sc., Shanghai Medical University) (B.Med., Shanghai Medical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 i Acknowledgements I would like to take this opportunity to express my sincere gratitude to my supervisor, Professor Catherine J PALLEN, although word is not always enough Thanks to her great scientific guidance, encouragement, and endless patience, I can go through my graduate study, and complete my thesis with her kind help and careful proofreading I am thankful for my supervisory committee members, Dr Pauline JOHNSON and Dr Michael COX, in Canada; Dr Xinmin Cao, Dr Kongpeng LAM, and Dr Borluen Tang in Singapore Their thoughtful ideas and broad knowledge helped me a lot for the progress of my project I was so luck to have a chance to work with such nice people either in Singapore or in Canada I am thankful for their generosity for sharing the reagents and information, and their wisdom and broad knowledge made my life in the lab more interesting I don’t think I have guts to pursue the degree without the persistent and strong support from my husband, Yan XU I want to let him know that his understanding is always precious, and I really appreciate that I am forever indebted to my parents I want to say thanks to them for their understanding and encouragement I am so grateful for my parents-in-law to take care of my adorable son without any complaint ii Table of Contents Acknowledgements…………………………………………………………………… i Table of contents……………………………………………………………………….ii List of tables………………………………………………………………………….viii List of figures……………………………………………………………………… ix Abbreviations………………………………………………………………………… xi Summary…………………………………………………………………………… xiii CHAPTER1 Introduction ……………………………………………………………1 1.1 Protein phosphorylation……………………………………………… 1.2 Protein tyrosine phosphatase (PTP) superfamily 1.3 Catalytic mechanism of protein tyrosine phosphatases………… 1.4 Receptor-like PTPs and their features ……………………………… 1.5 Receptor-like tyrosine phosphatase alpha (PTPα) ……………………8 1.5.1 Overview of PTPα …………………………………………… 1.5.2 Biological properties of PTPα ……………………………… 10 1.5.2.1 Substrates of PTPα.………………………………10 1.5.2.2 Biological functions of PTPα …………………… 10 1.5.3 Combinatorial regulation of PTPα catalytic activity and specificity …………………………………………………….13 1.5.3.1 1.5.3.2 1.5.3.3 1.5.3.4 1.5.3.5 1.6 Dimerization………………………………… 13 Phosphorylation…………………………………….14 Protein-protein interactions…… ………………… 16 Proteolysis …………….………………………… 18 Oxidation……………………………………………18 PTPs involved in regulating Src family kinases (SFKs)………… .18 1.6.1 Structure and regulation of SFKs …………………………….19 1.6.2 Regulation of SFK activity by PTPs ……………………… 23 iii 1.6.2.1 PTPα… …………………………………………….23 1.6.2.2 CD45……………………………………………… 24 1.6.2.3 PTP1B………………………………………………26 1.6.2.4 SHP1 and SHP2…………………………………….27 1.7 Integrin signaling…………………………………………….…… 28 1.7.1 Kinases in integrin-mediated signaling……………………… 31 1.7.1.1 Focal adhesion kinase (FAK)…………………….…31 1.7.1.2 SFKs in integrin signaling……………………….….34 1.7.2 PTPs in integrin signaling………………………… ……… 36 1.7.2.1 PTPα …………………………………………….….36 1.7.2.2 SHP2……………………………………………… 38 1.7.2.3 PTP1B…………………………………………… 39 1.7.2.4 PTP-PEST……………………………………… 41 1.7.2.5 PTEN……………………………………………….42 1.7.3 Summary of integrin-induced signaling events… ………….43 1.8 Research rationale and objectives…………………………………… 44 CHAPTER 2: Materials and Methods…………………………………………… 47 2.1 Mouse genotyping……………………… 47 2.1.1 DNA extraction from mouse tail tips or embryonic yolk sacs………………………………………………… 47 2.1.2 Genotyping for PTPα and Src ……………… 47 2.2 Generation of PTPα/Src double mutant mice………………….… .48 2.3 Embryonic dissection………………………………………….…… 50 2.4 Mouse growth observation………………………………………… 50 2.5 Histological staining……………………………………….………….50 2.6 Experiments with mouse embryonic fibroblasts…………………… 51 2.6.1 Derivation of mouse embryonic fibroblasts………………… 51 2.6.2 Other mouse embryonic fibroblasts………………………… 52 2.6.3 Cell proliferation assay……………………………………… 52 iv 2.6.4 Cells stimulation with extracellular matrix (ECM) components………………………………………………………… 53 2.6.5 Cell adhesion, spreading and migration assays……………….53 2.6.5.1 Cell adhesion assay ……………………………… 53 2.6.5.2 Cell spreading assay …………………………… ….54 2.6.5.3 Cell migration assay……………………………….54 2.7 Immunofluorescent staining……………………………….………….55 2.8 Protein analysis……………………………………………………….56 2.8.1 2.8.2 Determination of protein concentration …… …….……… 56 2.8.3 Immunoblotting……………………………………… … 57 2.8.4 Immunoprecipitation …………………………… …… .57 2.8.5 2.9 Cell lysis……………………………….………… …………56 Quantification of proteins…………………………… … .58 Transient transfection…………………………………….………… 58 2.9.1 2.9.2 2.10 Plasmids amplification and purification ……… …….………58 Cell culture and transient transfection………………………59 Treatment of cells with inhibitors……………………………… .60 2.10.1 2.10.2 2.11 PP2 and PP3 treatment………………………… ……… 60 Cytochalasin D treatment…………………… … ……… 60 PTPα adenovirus expression system…………………… .61 2.11.1 Generation of pKS-PTPαY789F (∆ Pac) ………………….61 2.11.2 pAdEasy transfer vector (pShuttleCMV) subcloning …… 62 2.11.3 Generation of pAdEasy recombinant plasmids in bacterial cells …… .63 2.11.4 Transfection of recombinant pAdEasy plasmid into Qbi-293A cells……… …………………………………………… 63 2.11.5 Confirmation of adenoviral-mediated PTPα expression… 64 2.11.6 Amplification of viral particles …………… ………… 64 v 2.11.7 Cesium chloride (CsCl) purification of recombinant adenovirus ……………………….……………… …… 65 2.11.8 Viral particles titration.……….………… ……………….66 2.11.9 Re-introduction of wild type and mutant PTPα into PTPα-/cells by recombinant adenovirus infection …… ……… 67 CHAPTER 3: Characterization of PTPα-/-Src-/- Double Knockout Mice …… 68 3.1 Overview …………………………………………………………… 68 3.2 Results ……………………………………………………………… 68 3.2.1 3.2.2 Post-natal survival and growth of double mutant PTPα-/-Src-/mice ……………………………………… ………… .72 3.2.3 3.3 Combined ablation of PTPα and Src does not result in embryonic lethality … ………………………………… 69 The combined ablation of PTPα and Src does not affect organogenesis ………………………………………… .76 Discussion…………………………………………………………….81 CHAPTER 4: Phenotypes of PTPα/Src Double Mutant Mouse Embryonic fibroblasts (α/s DKO)……… 86 4.1 Overview ……………………………………………………….… 86 4.2 Results …………………………………………………………… .87 4.2.1 4.2.2 α/s DKO cells are defective in fibronectin-induced cell adhesion and spreading ………………………………… 91 4.2.3 Integrin-induced cytoskeletal organization is altered in α/s DKO cells …………………………………… ………… 95 4.2.4 Integrin-induced FAK tyr397 phosphorylation is not affected in α/s DKO cells …… … ……………….101 4.2.5 4.3 Mouse embryonic fibroblasts deficient in both PTPα and Src display a distinctive morphology after spontaneous immortalization … .87 Constitutive activation of Erk is a consequence of the combined absence of PTPα and Src……………………….104 Discussion………………………………… 105 vi CHAPTER 5: Integrin-induced PTPα Tyrosine Phosphorylation is Required for cytoskeletal Reorganization and Cell Migration ……………… 112 5.1 Overview ……………………………………………………………112 5.2 Results ………………………………………………………………113 5.2.1 5.2.2 SFKs are essential for FN-induced PTPα tyrosine phosphorylation ………………………………….………115 5.2.3 Catalytically inactive mutant PTPαDM or Tyr789 mutant PTPαY789F is not phosphorylated upon integrin stimulation……………………………………… …… 116 5.2.4 Integrin-induced PTPα phosphorylation is dependent on an intact actin cytoskeleton and FAK …… ……………… 119 5.2.5 A PTPα adenoviral expression system efficiently reintroduces wild type and mutant forms of PTPα into PTPα-/fibroblasts………………………………………… … 120 5.2.6 PTPα phosphorylation at Tyr789 is not required for integrininduced Src/Fyn activation and FAK or paxillin phosphorylation ……………………… …………… 121 5.2.7 PTPα Tyr789 phosphorylation is required for integrin induced cell spreading and cytoskeletal organization…………… 126 5.2.8 PTPα Tyr789 phosphorylation is required for integrinstimulated cell migration …… ………………………… 129 5.2.9 The cell detachment-induced dephosphorylation of PTPα is not due to auto-dephosphorylation ……………………… 130 5.2.10 5.3 Integrin-induced tyrosine phosphorylation of PTPα … …113 SHP2 is not the phosphatase responsible for detachmentinduced dephosphorylation of PTPα …………………131 Discussion ……………………………………………………… 133 CHAPTER 6: GENREAL DISSCUSSION and CONCLUSIONS……………136 6.1 Roles of PTPα and Src in embryonic development ……………… 136 6.1.1 PTPα is not essential for embryonic development, but is required for normal hippocampal development and proper function ………………………………………………… 137 vii 6.1.2 6.1.3 6.2 SFKs play essential but redundant roles in embryonic development ………………….………………………… 139 A combined deficiency in PTPα and Src does not affect mouse embryonic development, but does increase postnatal mortality 141 The roles of PTPα and Src in integrin signaling ……………… …143 6.2.1 6.2.2 Role of PTPα as an activator of SFKs in integrin signaling………………………………………………… 145 6.2.3 6.3 SFKs are required for integrin signaling ………………….143 Additional roles of PTPα-mediated SFK activation in integrin signaling ……………… ………………………….…….146 Regulation of PTPα by integrin stimulation………………….…… 151 6.3.1 PTPα is tyrosine phosphorylated upon integrin stimulation, but this is not required for SFK activation … ….……… 151 6.3.2 PTPα Y789 phosphorylation is required for integrinstimulated cell spreading and migration.………………… 155 6.3.3 Two roles of PTPα in integrin signaling …………….… 156 6.3.4 The reciprocal link between integrin-induced PTPα phosphorylation and cytoskeletal organization may underlie the defects observed in α/s DKO cells ……………….157 6.4 Overall summary ………………………………………………… 159 6.5 Future directions ………………………………………….…………162 Reference ………………………………………………………………………… 164 Publications ……………………………………………………………………… 199 viii LIST of TABLES 2.1 Primer sequences used in PCR reactions for mouse PTPα and Src genotyping…………………………………………………………………….49 2.2 Sequences of primers used to generate the PTPαY789F mutant ……….……62 3.1 Embryos obtained from heterozygous PTPα/Src intercrosses (PTPα+/-Src+/-) x (PTPα+/-Src+/-) ………………………………………………………….…… 70 3.2 Embryos obtained from homozygous PTPα and heterozygous Src intercrosses (PTPα-/-Src+/-) x (PTPα-/-Src+/-) …………………………………… …… 71 3.3 Pups obtained from homozygous PTPα and heterozygous Src intercrosses (PTPα-/-Src+/-) x (PTPα-/-Src+/-)……………………………………………….73 3.4 Weights of organs…………………………………………………………… 79 3.5 Organ weight as a percentage of body weight……………………………… 79 4.1 Integrin-induced structural and morphological properties of wild type, PTPα-/-, Src-/-, and α/s DKO fibroblasts…………………………… ………………100 6.1 Summary of phenotypes of PTPα-/-, Src-/-, and PTPα-/-Src-/- mice ………….142 6.2 Summary of phenotypes of PTPα-/-, Src-/-, and α/s DKO (PTPα-/-Src-/-) fibroblasts in response to FN stimulation ………………………………… 150 185 Pallen CJ Protein tyrosine phosphatase alpha (PTPalpha): a Src family kinase activator and mediator of multiple biological effects Curr Top Med Chem 2003; 3: 821-35 Pao LI, Bedzyk WD, Persin C, Cambier JC Molecular targets of CD45 in B cell antigen receptor signal transduction J Immunol 1997; 158: 1116-24 Parsons JT Focal adhesion kinase: the first ten years J Cell Sci 2003; 116: 1409-16 Parsons JT Focal adhesion kinase: the first ten years J Cell Sci 2003; 116: 1409-16 Pawson T Protein modules and signalling networks Nature 1995; 373: 573-80 Pawson T New impressions of Src and Hck Nature 1997; 385: 582-3, 585 Pei D, Lorenz U, Klingmuller U, Neel BG, Walsh CT Intramolecular regulation of protein tyrosine phosphatase SH-PTP1: a new function for Src homology domains Biochemistry 1994; 33: 15483-93 Petrone A, Battaglia F, Wang C, Dusa A, Su J, Zagzag D, Bianchi R, Casaccia-Bonnefil P, Arancio O, Sap J Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation Embo J 2003; 22: 4121-31 Pingel JT, Thomas ML Evidence that the leukocyte-common antigen is required for antigeninduced T lymphocyte proliferation Cell 1989; 58: 1055-65 Pluskey S, Wandless TJ, Walsh CT, Shoelson SE Potent stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of both SH2 domains J Biol Chem 1995; 270: 2897-900 186 Ponniah S, Wang DZ, Lim KL, Pallen CJ Targeted disruption of the tyrosine phosphatase PTPalpha leads to constitutive downregulation of the kinases Src and Fyn Curr Biol 1999; 9: 535-8 Ren XD, Kiosses WB, Sieg DJ, Otey CA, Schlaepfer DD, Schwartz MA Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover J Cell Sci 2000; 113 ( Pt 20): 3673-8 Resh MD Interaction of tyrosine kinase oncoproteins with cellular membranes Biochim Biophys Acta 1993; 1155: 307-22 Saha S, Bardelli A, Buckhaults P, Velculescu VE, Rago C, St Croix B, Romans KE, Choti MA, Lengauer C, Kinzler KW, Vogelstein B A phosphatase associated with metastasis of colorectal cancer Science 2001; 294: 1343-6 Salazar EP, Rozengurt E Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397 J Biol Chem 2001; 276: 17788-95 Salmeen A, Andersen JN, Myers MP, Tonks NK, Barford D Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B Mol Cell 2000; 6: 1401-12 Salter MW, Kalia LV Src kinases: a hub for NMDA receptor regulation Nat Rev Neurosci 2004; 5: 317-28 Sap J, D'Eustachio P, Givol D, Schlessinger J Cloning and expression of a widely expressed receptor tyrosine phosphatase Proc Natl Acad Sci U S A 1990; 87: 6112-6 187 Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K, Sasaki T Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily J Biol Chem 1995; 270: 21206-19 Sastry SK, Rajfur Z, Liu BP, Cote JF, Tremblay ML, Burridge K PTP-PEST couples membrane protrusion and tail retraction via VAV2 and p190RhoGAP J Biol Chem 2006; 281: 11627-36 Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions Proc Natl Acad Sci U S A 1992; 89: 5192-6 Schaller MD, Borgman CA, Parsons JT Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK Mol Cell Biol 1993; 13: 78591 Schaller MD, Parsons JT Focal adhesion kinase and associated proteins Curr Opin Cell Biol 1994; 6: 705-10 Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src Mol Cell Biol 1994; 14: 1680-8 Schaller MD, Otey CA, Hildebrand JD, Parsons JT Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains J Cell Biol 1995; 130: 1181-7 188 Schaller MD, Hildebrand JD, Parsons JT Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases Mol Biol Cell 1999; 10: 3489-505 Schlaepfer DD, Hanks SK, Hunter T, van der Geer P Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase Nature 1994; 372: 786-91 Schlaepfer DD, Hunter T Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases Mol Cell Biol 1996; 16: 5623-33 Schlaepfer DD, Broome MA, Hunter T Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins Mol Cell Biol 1997; 17: 1702-13 Schlaepfer DD, Jones KC, Hunter T Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events Mol Cell Biol 1998; 18: 2571-85 Schoenwaelder SM, Petch LA, Williamson D, Shen R, Feng GS, Burridge K The protein tyrosine phosphatase Shp-2 regulates RhoA activity Curr Biol 2000; 10: 1523-6 Schwartzberg PL The many faces of Src: multiple functions of a prototypical tyrosine kinase Oncogene 1998; 17: 1463-8 Sefton BM, Hunter T, Beemon K, Eckhart W Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus Cell 1980; 20: 807-16 189 Shattil SJ, Ginsberg MH, Brugge JS Adhesive signaling in platelets Curr Opin Cell Biol 1994; 6: 695-704 Sheetz MP, Felsenfeld DP, Galbraith CG Cell migration: regulation of force on extracellularmatrix-integrin complexes Trends Cell Biol 1998; 8: 51-4 Shrivastava P, Katagiri T, Ogimoto M, Mizuno K, Yakura H Dynamic regulation of Srcfamily kinases by CD45 in B cells Blood 2004; 103: 1425-32 Sicheri F, Moarefi I, Kuriyan J Crystal structure of the Src family tyrosine kinase Hck Nature 1997; 385: 602-9 Sieg DJ, Hauck CR, Schlaepfer DD Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration J Cell Sci 1999; 112 ( Pt 16): 2677-91 Sieg DJ, Hauck CR, Ilic D, Klingbeil CK, Schaefer E, Damsky CH, Schlaepfer DD FAK integrates growth-factor and integrin signals to promote cell migration Nat Cell Biol 2000; 2: 249-56 Skelton MR, Ponniah S, Wang DZ, Doetschman T, Vorhees CV, Pallen CJ Protein tyrosine phosphatase alpha (PTP alpha) knockout mice show deficits in Morris water maze learning, decreased locomotor activity, and decreases in anxiety Brain Res 2003; 984: 1-10 Smart JE, Oppermann H, Czernilofsky AP, Purchio AF, Erikson RL, Bishop JM Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) Proc Natl Acad Sci U S A 1981; 78: 6013-7 190 Somani AK, Bignon JS, Mills GB, Siminovitch KA, Branch DR Src kinase activity is regulated by the SHP-1 protein-tyrosine phosphatase J Biol Chem 1997; 272: 21113-9 Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al SH2 domains recognize specific phosphopeptide sequences Cell 1993; 72: 767-78 Sonnenburg ED, Bilwes A, Hunter T, Noel JP The structure of the membrane distal phosphatase domain of RPTPalpha reveals interdomain flexibility and an SH2 domain interaction region Biochemistry 2003; 42: 7904-14 Soriano P, Montgomery C, Geske R, Bradley A Targeted disruption of the c-src protooncogene leads to osteopetrosis in mice Cell 1991; 64: 693-702 Stein PL, Lee HM, Rich S, Soriano P pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells Cell 1992; 70: 741-50 Stein PL, Vogel H, Soriano P Combined deficiencies of Src, Fyn, and Yes tyrosine kinases in mutant mice Genes Dev 1994; 8: 1999-2007 Stetak A, Csermely P, Ullrich A, Keri G Physical and functional interactions between protein tyrosine phosphatase alpha, PI 3-kinase, and PKCdelta Biochem Biophys Res Commun 2001; 288: 564-72 Streuli M, Krueger NX, Hall LR, Schlossman SF, Saito H A new member of the immunoglobulin superfamily that has a cytoplasmic region homologous to the leukocyte common antigen J Exp Med 1988; 168: 1523-30 191 Streuli M, Krueger NX, Thai T, Tang M, Saito H Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR Embo J 1990; 9: 2399-407 Streuli M, Krueger NX, Ariniello PD, Tang M, Munro JM, Blattler WA, Adler DA, Disteche CM, Saito H Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region Embo J 1992; 11: 897-907 Stuckey JA, Schubert HL, Fauman EB, Zhang ZY, Dixon JE, Saper MA Crystal structure of Yersinia protein tyrosine phosphatase at 2.5 A and the complex with tungstate Nature 1994; 370: 571-5 Su J, Batzer A, Sap J Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2 J Biol Chem 1994; 269: 18731-4 Su J, Yang LT, Sap J Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor Dual Src homology (SH) 2/SH3 domain requirement and functional consequences J Biol Chem 1996; 271: 28086-96 Su J, Muranjan M, Sap J Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts Curr Biol 1999; 9: 505-11 Su XD, Agango EG, Taddei N, Bucciantini M, Stefani M, Ramponi G, Nordlund P Crystallisation of a low molecular weight phosphotyrosine protein phosphatase from bovine liver FEBS Lett 1994; 343: 107-8 192 Sudol M, Greulich H, Newman L, Sarkar A, Sukegawa J, Yamamoto T A novel Yes-related kinase, Yrk, is expressed at elevated levels in neural and hematopoietic tissues Oncogene 1993; 8: 823-31 Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN Science 1998; 280: 1614-7 Tamura M, Gu J, Danen EH, Takino T, Miyamoto S, Yamada KM PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway J Biol Chem 1999; 274: 20693-703 Taylor JM, Mack CP, Nolan K, Regan CP, Owens GK, Parsons JT Selective expression of an endogenous inhibitor of FAK regulates proliferation and migration of vascular smooth muscle cells Mol Cell Biol 2001; 21: 1565-72 Tertoolen LG, Blanchetot C, Jiang G, Overvoorde J, Gadella TW, Jr., Hunter T, den Hertog J Dimerization of receptor protein-tyrosine phosphatase alpha in living cells BMC Cell Biol 2001; 2: Tezuka T, Umemori H, Akiyama T, Nakanishi S, Yamamoto T PSD-95 promotes Fynmediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A Proc Natl Acad Sci U S A 1999; 96: 435-40 Thomas JW, Ellis B, Boerner RJ, Knight WB, White GC, 2nd, Schaller MD SH2- and SH3mediated interactions between focal adhesion kinase and Src J Biol Chem 1998; 273: 577-83 Thomas SM, Soriano P, Imamoto A Specific and redundant roles of Src and Fyn in organizing the cytoskeleton Nature 1995; 376: 267-71 193 Thomas ML, Reynolds PJ, Chain A, Ben-Neriah Y, Trowbridge IS B-cell variant of mouse T200 (Ly-5): evidence for alternative mRNA splicing Proc Natl Acad Sci U S A 1987; 84: 5360-3 Thomas ML The leukocyte common antigen family Annu Rev Immunol 1989; 7: 339-69 Thomas ML, Brown EJ Positive and negative regulation of Src-family membrane kinases by CD45 Immunol Today 1999; 20: 406-11 Thomas SM, Brugge JS Cellular functions regulated by Src family kinases Annu Rev Cell Dev Biol 1997; 13: 513-609 Thompson KM, Uetani N, Manitt C, Elchebly M, Tremblay ML, Kennedy TE Receptor protein tyrosine phosphatase sigma inhibits axonal regeneration and the rate of axon extension Mol Cell Neurosci 2003; 23: 681-92 Thuveson M, Albrecht D, Zurcher G, Andres AC, Ziemiecki A iyk, a novel intracellular protein tyrosine kinase differentially expressed in the mouse mammary gland and intestine Biochem Biophys Res Commun 1995; 209: 582-9 Tian SS, Tsoulfas P, Zinn K Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo Cell 1991; 67: 675-80 Tonks NK, Diltz CD, Fischer EH Characterization of the major protein-tyrosine-phosphatases of human placenta J Biol Chem 1988; 263: 6731-7 194 Tonks NK, Neel BG From form to function: signaling by protein tyrosine phosphatases Cell 1996; 87: 365-8 Tracy S, van der Geer P, Hunter T The receptor-like protein-tyrosine phosphatase, RPTP alpha, is phosphorylated by protein kinase C on two serines close to the inner face of the plasma membrane J Biol Chem 1995; 270: 10587-94 Tsai W, Morielli AD, Cachero TG, Peralta EG Receptor protein tyrosine phosphatase alpha participates in the m1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity Embo J 1999; 18: 109-18 Tsuda M, Matozaki T, Fukunaga K, Fujioka Y, Imamoto A, Noguchi T, Takada T, Yamao T, Takeda H, Ochi F, Yamamoto T, Kasuga M Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2 Roles of Fak and Src family kinases J Biol Chem 1998; 273: 13223-9 Turner CE, Schaller MD, Parsons JT Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin J Cell Sci 1993; 105 ( Pt 3): 637-45 van der Sar A, Betist M, de Fockert J, Overvoorde J, Zivkovic D, den Hertog J Expression of receptor protein-tyrosine phosphatase alpha, sigma and LAR during development of the zebrafish embryo Mech Dev 2001; 109: 423-6 van der Sar AM, Zivkovic D, den Hertog J Eye defects in receptor protein-tyrosine phosphatase alpha knock-down zebrafish Dev Dyn 2002; 223: 292-7 Volberg T, Romer L, Zamir E, Geiger B pp60(c-src) and related tyrosine kinases: a role in the assembly and reorganization of matrix adhesions J Cell Sci 2001; 114: 2279-89 195 von Wichert G, Jiang G, Kostic A, De Vos K, Sap J, Sheetz MP RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages J Cell Biol 2003; 161: 143-53 Walton KM, Dixon JE Protein tyrosine phosphatases Annu Rev Biochem 1993; 62: 101-20 Wang Y, Pallen CJ The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities Embo J 1991; 10: 3231-7 Wary KK, Mariotti A, Zurzolo C, Giancotti FG A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth Cell 1998; 94: 625-34 Watt FM, Hodivala KJ Cell adhesion Fibronectin and integrin knockouts come unstuck Curr Biol 1994; 4: 270-2 Wennerberg K, Fassler R, Warmegard B, Johansson S Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A Requirement for threonines 788-789 in receptor activation J Cell Sci 1998; 111 ( Pt 8): 1117-26 Whitney GS, Chan PY, Blake J, Cosand WL, Neubauer MG, Aruffo A, Kanner SB Human T and B lymphocytes express a structurally conserved focal adhesion kinase, pp125FAK DNA Cell Biol 1993; 12: 823-30 Wo YY, Zhou MM, Stevis P, Davis JP, Zhang ZY, Van Etten RL Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart Biochemistry 1992; 31: 1712-21 196 Wu L, Zhang ZY Probing the function of Asp128 in the lower molecular weight proteintyrosine phosphatase-catalyzed reaction A pre-steady-state and steady-state kinetic investigation Biochemistry 1996; 35: 5426-34 Wu L, Buist A, den Hertog J, Zhang ZY Comparative kinetic analysis and substrate specificity of the tandem catalytic domains of the receptor-like protein-tyrosine phosphatase alpha J Biol Chem 1997; 272: 6994-7002 Xing Z, Chen HC, Nowlen JK, Taylor SJ, Shalloway D, Guan JL Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain Mol Biol Cell 1994; 5: 41321 Xiong W, Parsons JT Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase J Cell Biol 1997; 139: 529-39 Xu W, Harrison SC, Eck MJ Three-dimensional structure of the tyrosine kinase c-Src Nature 1997; 385: 595-602 Yanagi S, Sugawara H, Kurosaki M, Sabe H, Yamamura H, Kurosaki T CD45 modulates phosphorylation of both autophosphorylation and negative regulatory tyrosines of Lyn in B cells J Biol Chem 1996; 271: 30487-92 Yang LT, Alexandropoulos K, Sap J c-SRC mediates neurite outgrowth through recruitment of Crk to the scaffolding protein Sin/Efs without altering the kinetics of ERK activation J Biol Chem 2002; 277: 17406-14 Yang Q, Co D, Sommercorn J, Tonks NK Cloning and expression of PTP-PEST A novel, human, nontransmembrane protein tyrosine phosphatase J Biol Chem 1993; 268: 17650 197 Yu DH, Qu CK, Henegariu O, Lu X, Feng GS Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion J Biol Chem 1998; 273: 21125-31 Yu H, Li X, Marchetto GS, Dy R, Hunter D, Calvo B, Dawson TL, Wilm M, Anderegg RJ, Graves LM, Earp HS Activation of a novel calcium-dependent protein-tyrosine kinase Correlation with c-Jun N-terminal kinase but not mitogen-activated protein kinase activation J Biol Chem 1996; 271: 29993-8 Yuvaniyama J, Denu JM, Dixon JE, Saper MA Crystal structure of the dual specificity protein phosphatase VHR Science 1996; 272: 1328-31 Zeng L, D'Alessandri L, Kalousek MB, Vaughan L, Pallen CJ Protein tyrosine phosphatase alpha (PTPalpha) and contactin form a novel neuronal receptor complex linked to the intracellular tyrosine kinase fyn J Cell Biol 1999; 147: 707-14 Zeng L, Si X, Yu WP, Le HT, Ng KP, Teng RM, Ryan K, Wang DZ, Ponniah S, Pallen CJ PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration J Cell Biol 2003; 160: 137-46 Zeng Q, Si X, Horstmann H, Xu Y, Hong W, Pallen CJ Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome J Biol Chem 2000; 275: 21444-52 Zhang SQ, Yang W, Kontaridis MI, Bivona TG, Wen G, Araki T, Luo J, Thompson JA, Schraven BL, Philips MR, Neel BG Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment Mol Cell 2004; 13: 341-55 198 Zhang ZY, Palfey BA, Wu L, Zhao Y Catalytic function of the conserved hydroxyl group in the protein tyrosine phosphatase signature motif Biochemistry 1995; 34: 16389-96 Zhang Z, Lin SY, Neel BG, Haimovich B Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration J Biol Chem 2006; 281: 1746-54 Zhang ZY, Wang Y, Wu L, Fauman EB, Stuckey JA, Schubert HL, Saper MA, Dixon JE The Cys(X)5Arg catalytic motif in phosphoester hydrolysis Biochemistry 1994; 33: 15266-70 Zhang ZY Protein-tyrosine phosphatases: biological function, structural characteristics, and mechanism of catalysis Crit Rev Biochem Mol Biol 1998; 33: 1-52 Zheng XM, Wang Y, Pallen CJ Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase Nature 1992; 359: 336-9 Zheng XM, Resnick RJ, Shalloway D A phosphotyrosine displacement mechanism for activation of Src by PTPalpha Embo J 2000; 19: 964-78 Zheng XM, Shalloway D Two mechanisms activate PTPalpha during mitosis Embo J 2001; 20: 6037-49 Zheng XM, Resnick RJ, Shalloway D Mitotic activation of protein-tyrosine phosphatase alpha and regulation of its Src-mediated transforming activity by its sites of protein kinase C phosphorylation J Biol Chem 2002; 277: 21922-9 199 List of Publications Chen M, Chen S, Pallen CJ Integrin-induced tyrosine phosphorylation pf protein tyrosine phosphatase alpha is required for cytoskeletal reorganization and cell migration J Biol Chem 2006; 281: 11972-80 Sutherland BW, Kucab J, Wu J, Lee C, Cheang MCU, Yorida EY, Turbin D, Dedhar S, Nelson C, Pollak M, Grimes HL, Miller K, Badve S, Hunstman D, Gills CB, Chen M, Pallen CJ, Dunn SE Akt phosphorylates the Y-box binding protein (YB-1) at Ser 102 located in the cold shock domain and affects the anchorage independent growth of breast cancer cells Oncogenen 2005; 24: 4281-92 Wu L, Bernard-Trifilo JA, Lim Y, Lim ST, Mitra SK, Uryu S, Chen M, Pallen CJ, Cheung NK, Mikolon D, Mielgo A, Stupack DG, Schlaepfer DD Distinct FAK-Src activation events promote alpha5beta and alpha4beta1 integrin-stimulated neuroblastoma cell motility Oncogene 2007; (in press) .. .Functional Interactions of Protein Tyrosine Phosphatase Alpha (PTPα) and Src in Mouse Development and Integrin Signaling: Investigation of Double PTPα /Src- Deficient Mice and Cells CHEN MIN... integrin signaling, and plays a negative feedback role in orchestrating integrin signaling To determine how PTPα is regulated upon integrin stimulation and how a signal emanating from integrin. .. Phosphorylation of PTPα at Ser180 and Ser204 reduces the affinity of Grb2 SH2 binding to phospho-Tyr789 of PTPα without reducing the affinity of Src SH2 binding, resulting in less Grb2 and more Src binding