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Regulation of Fibroblast Motility by the Protein Tyrosine Phosphatase PTP-PEST

1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês

10.1074/jbc.274.6.3811

1083-351X

Andrew J. Garton, Nicholas K. Tonks,

Cellular Mechanics and Interactions

The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130Cas as a substrate, bothin vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130Cas than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130Cas is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130Casto the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130Cas with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130Cas, thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130Cas and regulating cell migration. The protein tyrosine phosphatase PTP-PEST is a cytosolic enzyme that displays a remarkable degree of selectivity for tyrosine-phosphorylated p130Cas as a substrate, bothin vitro and in intact cells. We have investigated the physiological role of PTP-PEST using Rat1 fibroblast-derived stable cell lines that we have engineered to overexpress PTP-PEST. These cell lines exhibit normal levels of tyrosine phosphorylation of the majority of proteins but have significantly lower levels of tyrosine phosphorylation of p130Cas than control cells. Initial cellular events occurring following integrin-mediated attachment to fibronectin (cell attachment and spreading) are essentially unchanged in cells overexpressing PTP-PEST; similarly, the extent and time course of mitogen-activated protein kinase activation in response to integrin engagement is unchanged. In contrast, the reduced phosphorylation state of p130Cas is associated with a considerably reduced rate of cell migration and a failure of cells overexpressing PTP-PEST to accomplish the normally observed redistribution of p130Casto the leading edge of migrating cells. Furthermore, cells overexpressing PTP-PEST demonstrate significantly reduced levels of association of p130Cas with the Crk adaptor protein. Our results suggest that one physiological role of PTP-PEST is to dephosphorylate p130Cas, thereby controlling tyrosine phosphorylation-dependent signaling events downstream of p130Cas and regulating cell migration. PTP-PEST is a ubiquitously expressed mammalian, cytosolic protein tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.1The abbreviations used are: PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.that was cloned in our laboratory (1Yang Q. Co D. Sommercorn J. Tonks N.K. J. Biol. Chem. 1993; 268 (Correction (1993) J. Biol. Chem.268, 17650): 6622-6628Abstract Full Text PDF PubMed Google Scholar) and by others (2Charest A. Wagner J. Shen S.H. Tremblay M.L. Biochem. J. 1995; 308: 425-432Crossref PubMed Scopus (62) Google Scholar, 3den Hertog J. Pals C.E. Jonk L.J. Kruijer W. Biochem. Biophys. Res. Commun. 1992; 184: 1241-1249Crossref PubMed Scopus (26) Google Scholar, 4Takekawa M. Itoh F. Hinoda Y. Arimura Y. Toyota M. Sekiya M. Adachi M. Imai K. Yachi A. Biochem. Biophys. Res. Commun. 1992; 189: 1223-1230Crossref PubMed Scopus (34) Google Scholar). Several signaling proteins have been shown to be capable of binding to PTP-PEST, including Shc (5Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar, 6Charest A. Wagner J. Jacob S. McGlade C.J. Tremblay M.L. J. Biol. Chem. 1996; 271: 8424-8429Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), paxillin (7Shen Y. Schneider G. Cloutier J.F. Veillette A. Schaller M.D. J. Biol. Chem. 1998; 273: 6474-6481Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), Grb2 (8Charest A. Wagner J. Kwan M. Tremblay M.L. Oncogene. 1997; 14: 1643-1651Crossref PubMed Scopus (48) Google Scholar), and Csk (9Davidson D. Cloutier J.F. Gregorieff A. Veillette A. J. Biol. Chem. 1997; 272: 23455-23462Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), but the physiological significance of these interactions and their relevance to the function of PTP-PEST is unclear. In order to investigate the physiological substrate specificity of members of the PTP family, we developed a novel method involving expression of substrate-trapping mutant forms of PTPs (10Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 11Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar). These mutants retain a high affinity for substrates but are catalytically impaired. Therefore, they form complexes with appropriate target substrates that are amenable to isolation and characterization. We have used this method to identify substrates of PTP-PEST and thus gain insight into its physiological function. We have shown that the adaptor protein p130Cas is a specific substrate for PTP-PEST in vitro and in intact cells (11Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar). Similar results have also been obtained in studies performed on fibroblasts derived from PTP-PEST−/− mice (12Cote J.F. Charest A. Wagner J. Tremblay M.L. Biochemistry. 1998; 37: 13128-13137Crossref PubMed Scopus (92) Google Scholar). This high degree of selectivity of PTP-PEST for p130Cas is derived from two distinct high affinity interactions. The catalytic domain of PTP-PEST itself displays intrinsic specificity for tyrosine-phosphorylated p130Cas(11Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar), but this specificity is greatly enhanced by an interaction between the SH3 domain of p130Cas and a proline-rich sequence surrounding Pro-337 in the phosphatase (13Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar). These data strongly suggest that a major role of PTP-PEST is to regulate the level of tyrosine phosphorylation of p130Cas within the cell.p130Cas was initially identified as a major tyrosine-phosphorylated protein in cells transformed by the oncogenes v-crk (14Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 15Mayer B.J. Hamaguchi M. Hanafusa H. Nature. 1988; 332: 272-275Crossref PubMed Scopus (516) Google Scholar) and v-src (16Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 17Reynolds A.B. Kanner S.B. Wang H.C. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar). In addition, p130Cas is rapidly phosphorylated following mitogenic stimulation by a wide variety of agonists (18Leeb-Lundberg L.M.F. Song X.H. Mathis S.A. J. Biol. Chem. 1994; 269: 24328-24334Abstract Full Text PDF PubMed Google Scholar, 19Rankin S. Rozengurt E. J. Biol. Chem. 1994; 269: 704-710Abstract Full Text PDF PubMed Google Scholar, 20Ribon V. Saltiel A.R. J. Biol. Chem. 1996; 271: 7375-7380Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 21Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar, 22Zachary I. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1992; 267: 19031-19034Abstract Full Text PDF PubMed Google Scholar), during B-cell activation via antigen receptor ligation (23Manie S.N. Beck A.R. Astier A. Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 24Ingham R.J. Krebs D.L. Barbazuk S.M. Turck C.W. Hirai H. Matsuda M. Gold M.R. J. Biol. Chem. 1996; 271: 32306-32314Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and following cell attachment to fibronectin (25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 26Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 27Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 28Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 29Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Although these observations implicate tyrosine phosphorylation of p130Cas in the control of a variety of cellular processes, the precise role of p130Cas remains largely obscure. The structure of p130Cas suggests that it is involved in the formation of multiprotein complexes via several types of protein-protein interaction. Thus, p130Cas contains an SH3 domain, which has been shown to interact with proline-rich sequences in several proteins (13Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar, 25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 30Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 31Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 32Ohba T. Ishino M. Aoto H. Sasaki T. Biochem. J. 1998; 330: 1249-1254Crossref PubMed Scopus (62) Google Scholar), a proline-rich segment, representing a potential SH3 domain binding sequence, a central domain with multiple copies of the motif YXXP which, when tyrosine-phosphorylated, represent potential binding sites for a variety of SH2 domains (33Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar), and a C-terminal segment containing high affinity binding sites for the SH2 and SH3 domains of Src (34Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar).In order to investigate further the cellular function of PTP-PEST, we have generated Rat1 fibroblast lines that overexpress the wild-type phosphatase protein. These cells display a specific defect in p130Cas phosphorylation following cell attachment to fibronectin and exhibit a considerably reduced rate of migration. The data suggest that regulation of the tyrosine phosphorylation status of p130Cas by PTP-PEST is a critical control element of cell motility.DISCUSSIONThe results described in this paper suggest that PTP-PEST is a regulator of integrin-mediated movement of fibroblasts across the extracellular matrix via its regulation of the tyrosine phosphorylation state of p130Cas. Although previous reports have suggested a potential involvement of p130Cas in cell motility (50Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (588) Google Scholar, 51Cary L.A. Han D.C. Polte T.R. Hanks S.K. Guan J.L. J. Cell Biol. 1998; 140: 211-221Crossref PubMed Scopus (416) Google Scholar, 52Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar), our data are the first to reveal a requirement for tyrosine phosphorylation of p130Cas in this process. The precise role of p130Cas in controlling cell movement is not clear. However, data recently obtained from mouse embryos lacking the p130Cas protein suggest that it is involved in organization of the actin cytoskeleton in fibroblasts and cardiocytes (53Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar).Integrin-mediated cell movement across the extracellular matrix is a highly dynamic process requiring continual assembly and disassembly of focal contacts, together with extensive remodeling of the actin cytoskeleton (54Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (903) Google Scholar, 55Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar, 56Keely P. Parise L. Juliano R. Trends Cell Biol. 1998; 8: 101-106Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 57Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). Many of the proteins found in focal contacts, including p130Cas, are highly tyrosine-phosphorylated; in fact, focal contacts represent one of the major sites of tyrosine phosphorylation within the cell, as assessed by immunofluorescence analysis using anti-Tyr(P) antibodies (58Maher P.A. Pasquale E.B. Wang J.Y. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6576-6580Crossref PubMed Scopus (155) Google Scholar) (see Fig. 3). Furthermore, at least two protein tyrosine kinases, p125FAK and Src, are localized to focal contacts (59Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1286) Google Scholar, 60Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar), and both kinases have been implicated in the control of cell movement (52Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar, 61Fincham V.J. Frame M.C. EMBO J. 1998; 17: 81-92Crossref PubMed Scopus (264) Google Scholar, 62Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar, 63Gilmore A.P. Romer L.H. Mol. Biol. Cell. 1996; 7: 1209-1224Crossref PubMed Scopus (461) Google Scholar). These observations have led to the suggestion that tyrosine phosphorylation plays a significant role in controlling the assembly and disassembly of focal adhesion complexes, thereby regulating cell motility. However, the control of cell movement is a reversible, dynamic process suggesting that its regulation by tyrosine phosphorylation must involve both kinases and phosphatases. Our results suggest that the protein tyrosine phosphatase PTP-PEST regulates cell motility by modulating specifically the extent of tyrosine phosphorylation of p130Cas.The observation that p130Cas is localized at the leading edge of migrating fibroblasts suggests a model for the observed effects of PTP-PEST expression on cell movement. By limiting the accumulation of tyrosine-phosphorylated p130Cas, the ability of p130Cas to interact with appropriate SH2 domain-containing binding partners is impaired in cells overexpressing PTP-PEST. It appears likely that some of these interactions are essential for targeting tyrosine-phosphorylated p130Cas to the correct cytoskeletal location. Thus, it has been shown that the SH2 domain of v-Crk is required for the v-Crk-induced translocation of tyrosine-phosphorylated p130Cas to a detergent-insoluble fraction (48Nievers M.G. Birge R.B. Greulich H. Verkleij A.J. Hanafusa H. van Bergen en Henegouwen P.M. J. Cell Sci. 1997; 110: 389-399PubMed Google Scholar). Also, in p130Cas-overexpressing COS cells, fibronectin-induced relocation of p130Cas to focal adhesion complexes requires both the SH3 domain of p130Cas (which can bind directly to the focal adhesion kinase p125FAK (25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar,30Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 49Polte T.R. Hanks S.K. J. Biol. Chem. 1997; 272: 5501-5509Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)) and a C-terminal segment which contains binding sites for the SH2 and SH3 domains of Src (34Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 64Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (135) Google Scholar). Our observation that cells overexpressing PTP-PEST display a defect in the formation of complexes between p130Cas and Crk following fibronectin stimulation (Fig. 5 B) suggests that such complexes are involved in regulating cell movement. Indeed, recent evidence obtained using COS cells transfected with mutant forms of Crk and p130Cassuggested an involvement of p130Cas-Crk complexes in COS cell migration on vitronectin-coated membranes (50Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (588) Google Scholar).Recently, p130Cas−/− fibroblasts have been reported to display a defect in their response to an activated, transforming variant of the Src tyrosine kinase; they fail to form colonies in soft agar, a defect that can be rescued by re-expression of p130Cas (53Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar). These results suggest that the involvement of p130Cas in regulating the organization of actin cytoskeleton structures may also influence the response of cells to transforming oncogenes, a suggestion that is supported by several earlier observations implicating a direct regulatory role for p130Cas in cellular transformation (65Auvinen M. Paasinen-Sohns A. Hirai H. Andersson L.C. Holtta E. Mol. Cell. Biol. 1995; 15: 6513-6525Crossref PubMed Scopus (75) Google Scholar, 66Mayer B.J. Hanafusa H. J. Virol. 1990; 64: 3581-3589Crossref PubMed Google Scholar, 67Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar). These data further suggest that PTP-PEST may play a key role in suppressing cellular transformation through its ability to dephosphorylate p130Cas. Indeed, such a role is supported by our observation that overexpression of PTP-PEST dramatically influences the location of p130Cas within v-Crk-transformed cells, suggesting that PTP-PEST potentially influences the response of cells to a variety of transforming oncogenes.The results in this paper represent the first description of the physiological role of PTP-PEST. The data suggest that PTP-PEST is involved in controlling the rate of cell movement via the dephosphorylation of p130Cas. Our results also provide new insight into the function of p130Cas in integrin-mediated processes. Thus, it appears that tyrosine phosphorylation of p130Cas is largely dispensable for many of the cellular responses to fibronectin, including cell attachment, cell spreading, and MAP kinase activation. However, tyrosine phosphorylation of p130Cas is required for the efficient movement of cells across the extracellular matrix and therefore is likely to represent an important mechanism for regulating the dynamic process of cell migration in vivo. PTP-PEST is a ubiquitously expressed mammalian, cytosolic protein tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.1The abbreviations used are: PTP, protein tyrosine phosphatase; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein.that was cloned in our laboratory (1Yang Q. Co D. Sommercorn J. Tonks N.K. J. Biol. Chem. 1993; 268 (Correction (1993) J. Biol. Chem.268, 17650): 6622-6628Abstract Full Text PDF PubMed Google Scholar) and by others (2Charest A. Wagner J. Shen S.H. Tremblay M.L. Biochem. J. 1995; 308: 425-432Crossref PubMed Scopus (62) Google Scholar, 3den Hertog J. Pals C.E. Jonk L.J. Kruijer W. Biochem. Biophys. Res. Commun. 1992; 184: 1241-1249Crossref PubMed Scopus (26) Google Scholar, 4Takekawa M. Itoh F. Hinoda Y. Arimura Y. Toyota M. Sekiya M. Adachi M. Imai K. Yachi A. Biochem. Biophys. Res. Commun. 1992; 189: 1223-1230Crossref PubMed Scopus (34) Google Scholar). Several signaling proteins have been shown to be capable of binding to PTP-PEST, including Shc (5Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar, 6Charest A. Wagner J. Jacob S. McGlade C.J. Tremblay M.L. J. Biol. Chem. 1996; 271: 8424-8429Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), paxillin (7Shen Y. Schneider G. Cloutier J.F. Veillette A. Schaller M.D. J. Biol. Chem. 1998; 273: 6474-6481Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), Grb2 (8Charest A. Wagner J. Kwan M. Tremblay M.L. Oncogene. 1997; 14: 1643-1651Crossref PubMed Scopus (48) Google Scholar), and Csk (9Davidson D. Cloutier J.F. Gregorieff A. Veillette A. J. Biol. Chem. 1997; 272: 23455-23462Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), but the physiological significance of these interactions and their relevance to the function of PTP-PEST is unclear. In order to investigate the physiological substrate specificity of members of the PTP family, we developed a novel method involving expression of substrate-trapping mutant forms of PTPs (10Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (677) Google Scholar, 11Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar). These mutants retain a high affinity for substrates but are catalytically impaired. Therefore, they form complexes with appropriate target substrates that are amenable to isolation and characterization. We have used this method to identify substrates of PTP-PEST and thus gain insight into its physiological function. We have shown that the adaptor protein p130Cas is a specific substrate for PTP-PEST in vitro and in intact cells (11Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar). Similar results have also been obtained in studies performed on fibroblasts derived from PTP-PEST−/− mice (12Cote J.F. Charest A. Wagner J. Tremblay M.L. Biochemistry. 1998; 37: 13128-13137Crossref PubMed Scopus (92) Google Scholar). This high degree of selectivity of PTP-PEST for p130Cas is derived from two distinct high affinity interactions. The catalytic domain of PTP-PEST itself displays intrinsic specificity for tyrosine-phosphorylated p130Cas(11Garton A.J. Flint A.J. Tonks N.K. Mol. Cell. Biol. 1996; 16: 6408-6418Crossref PubMed Scopus (231) Google Scholar), but this specificity is greatly enhanced by an interaction between the SH3 domain of p130Cas and a proline-rich sequence surrounding Pro-337 in the phosphatase (13Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar). These data strongly suggest that a major role of PTP-PEST is to regulate the level of tyrosine phosphorylation of p130Cas within the cell. p130Cas was initially identified as a major tyrosine-phosphorylated protein in cells transformed by the oncogenes v-crk (14Mayer B.J. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2638-2642Crossref PubMed Scopus (105) Google Scholar, 15Mayer B.J. Hamaguchi M. Hanafusa H. Nature. 1988; 332: 272-275Crossref PubMed Scopus (516) Google Scholar) and v-src (16Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (396) Google Scholar, 17Reynolds A.B. Kanner S.B. Wang H.C. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar). In addition, p130Cas is rapidly phosphorylated following mitogenic stimulation by a wide variety of agonists (18Leeb-Lundberg L.M.F. Song X.H. Mathis S.A. J. Biol. Chem. 1994; 269: 24328-24334Abstract Full Text PDF PubMed Google Scholar, 19Rankin S. Rozengurt E. J. Biol. Chem. 1994; 269: 704-710Abstract Full Text PDF PubMed Google Scholar, 20Ribon V. Saltiel A.R. J. Biol. Chem. 1996; 271: 7375-7380Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 21Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar, 22Zachary I. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1992; 267: 19031-19034Abstract Full Text PDF PubMed Google Scholar), during B-cell activation via antigen receptor ligation (23Manie S.N. Beck A.R. Astier A. Law S.F. Canty T. Hirai H. Druker B.J. Avraham H. Haghayeghi N. Sattler M. Salgia R. Griffin J.D. Golemis E.A. Freedman A.S. J. Biol. Chem. 1997; 272: 4230-4236Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 24Ingham R.J. Krebs D.L. Barbazuk S.M. Turck C.W. Hirai H. Matsuda M. Gold M.R. J. Biol. Chem. 1996; 271: 32306-32314Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and following cell attachment to fibronectin (25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 26Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 27Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 28Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 29Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar). Although these observations implicate tyrosine phosphorylation of p130Cas in the control of a variety of cellular processes, the precise role of p130Cas remains largely obscure. The structure of p130Cas suggests that it is involved in the formation of multiprotein complexes via several types of protein-protein interaction. Thus, p130Cas contains an SH3 domain, which has been shown to interact with proline-rich sequences in several proteins (13Garton A.J. Burnham M.R. Bouton A.H. Tonks N.K. Oncogene. 1997; 15: 877-885Crossref PubMed Scopus (144) Google Scholar, 25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 30Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 31Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 32Ohba T. Ishino M. Aoto H. Sasaki T. Biochem. J. 1998; 330: 1249-1254Crossref PubMed Scopus (62) Google Scholar), a proline-rich segment, representing a potential SH3 domain binding sequence, a central domain with multiple copies of the motif YXXP which, when tyrosine-phosphorylated, represent potential binding sites for a variety of SH2 domains (33Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar), and a C-terminal segment containing high affinity binding sites for the SH2 and SH3 domains of Src (34Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In order to investigate further the cellular function of PTP-PEST, we have generated Rat1 fibroblast lines that overexpress the wild-type phosphatase protein. These cells display a specific defect in p130Cas phosphorylation following cell attachment to fibronectin and exhibit a considerably reduced rate of migration. The data suggest that regulation of the tyrosine phosphorylation status of p130Cas by PTP-PEST is a critical control element of cell motility. DISCUSSIONThe results described in this paper suggest that PTP-PEST is a regulator of integrin-mediated movement of fibroblasts across the extracellular matrix via its regulation of the tyrosine phosphorylation state of p130Cas. Although previous reports have suggested a potential involvement of p130Cas in cell motility (50Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (588) Google Scholar, 51Cary L.A. Han D.C. Polte T.R. Hanks S.K. Guan J.L. J. Cell Biol. 1998; 140: 211-221Crossref PubMed Scopus (416) Google Scholar, 52Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar), our data are the first to reveal a requirement for tyrosine phosphorylation of p130Cas in this process. The precise role of p130Cas in controlling cell movement is not clear. However, data recently obtained from mouse embryos lacking the p130Cas protein suggest that it is involved in organization of the actin cytoskeleton in fibroblasts and cardiocytes (53Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar).Integrin-mediated cell movement across the extracellular matrix is a highly dynamic process requiring continual assembly and disassembly of focal contacts, together with extensive remodeling of the actin cytoskeleton (54Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (903) Google Scholar, 55Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar, 56Keely P. Parise L. Juliano R. Trends Cell Biol. 1998; 8: 101-106Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 57Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). Many of the proteins found in focal contacts, including p130Cas, are highly tyrosine-phosphorylated; in fact, focal contacts represent one of the major sites of tyrosine phosphorylation within the cell, as assessed by immunofluorescence analysis using anti-Tyr(P) antibodies (58Maher P.A. Pasquale E.B. Wang J.Y. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6576-6580Crossref PubMed Scopus (155) Google Scholar) (see Fig. 3). Furthermore, at least two protein tyrosine kinases, p125FAK and Src, are localized to focal contacts (59Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1286) Google Scholar, 60Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar), and both kinases have been implicated in the control of cell movement (52Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar, 61Fincham V.J. Frame M.C. EMBO J. 1998; 17: 81-92Crossref PubMed Scopus (264) Google Scholar, 62Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar, 63Gilmore A.P. Romer L.H. Mol. Biol. Cell. 1996; 7: 1209-1224Crossref PubMed Scopus (461) Google Scholar). These observations have led to the suggestion that tyrosine phosphorylation plays a significant role in controlling the assembly and disassembly of focal adhesion complexes, thereby regulating cell motility. However, the control of cell movement is a reversible, dynamic process suggesting that its regulation by tyrosine phosphorylation must involve both kinases and phosphatases. Our results suggest that the protein tyrosine phosphatase PTP-PEST regulates cell motility by modulating specifically the extent of tyrosine phosphorylation of p130Cas.The observation that p130Cas is localized at the leading edge of migrating fibroblasts suggests a model for the observed effects of PTP-PEST expression on cell movement. By limiting the accumulation of tyrosine-phosphorylated p130Cas, the ability of p130Cas to interact with appropriate SH2 domain-containing binding partners is impaired in cells overexpressing PTP-PEST. It appears likely that some of these interactions are essential for targeting tyrosine-phosphorylated p130Cas to the correct cytoskeletal location. Thus, it has been shown that the SH2 domain of v-Crk is required for the v-Crk-induced translocation of tyrosine-phosphorylated p130Cas to a detergent-insoluble fraction (48Nievers M.G. Birge R.B. Greulich H. Verkleij A.J. Hanafusa H. van Bergen en Henegouwen P.M. J. Cell Sci. 1997; 110: 389-399PubMed Google Scholar). Also, in p130Cas-overexpressing COS cells, fibronectin-induced relocation of p130Cas to focal adhesion complexes requires both the SH3 domain of p130Cas (which can bind directly to the focal adhesion kinase p125FAK (25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar,30Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 49Polte T.R. Hanks S.K. J. Biol. Chem. 1997; 272: 5501-5509Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)) and a C-terminal segment which contains binding sites for the SH2 and SH3 domains of Src (34Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 64Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (135) Google Scholar). Our observation that cells overexpressing PTP-PEST display a defect in the formation of complexes between p130Cas and Crk following fibronectin stimulation (Fig. 5 B) suggests that such complexes are involved in regulating cell movement. Indeed, recent evidence obtained using COS cells transfected with mutant forms of Crk and p130Cassuggested an involvement of p130Cas-Crk complexes in COS cell migration on vitronectin-coated membranes (50Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (588) Google Scholar).Recently, p130Cas−/− fibroblasts have been reported to display a defect in their response to an activated, transforming variant of the Src tyrosine kinase; they fail to form colonies in soft agar, a defect that can be rescued by re-expression of p130Cas (53Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar). These results suggest that the involvement of p130Cas in regulating the organization of actin cytoskeleton structures may also influence the response of cells to transforming oncogenes, a suggestion that is supported by several earlier observations implicating a direct regulatory role for p130Cas in cellular transformation (65Auvinen M. Paasinen-Sohns A. Hirai H. Andersson L.C. Holtta E. Mol. Cell. Biol. 1995; 15: 6513-6525Crossref PubMed Scopus (75) Google Scholar, 66Mayer B.J. Hanafusa H. J. Virol. 1990; 64: 3581-3589Crossref PubMed Google Scholar, 67Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar). These data further suggest that PTP-PEST may play a key role in suppressing cellular transformation through its ability to dephosphorylate p130Cas. Indeed, such a role is supported by our observation that overexpression of PTP-PEST dramatically influences the location of p130Cas within v-Crk-transformed cells, suggesting that PTP-PEST potentially influences the response of cells to a variety of transforming oncogenes.The results in this paper represent the first description of the physiological role of PTP-PEST. The data suggest that PTP-PEST is involved in controlling the rate of cell movement via the dephosphorylation of p130Cas. Our results also provide new insight into the function of p130Cas in integrin-mediated processes. Thus, it appears that tyrosine phosphorylation of p130Cas is largely dispensable for many of the cellular responses to fibronectin, including cell attachment, cell spreading, and MAP kinase activation. However, tyrosine phosphorylation of p130Cas is required for the efficient movement of cells across the extracellular matrix and therefore is likely to represent an important mechanism for regulating the dynamic process of cell migration in vivo. The results described in this paper suggest that PTP-PEST is a regulator of integrin-mediated movement of fibroblasts across the extracellular matrix via its regulation of the tyrosine phosphorylation state of p130Cas. Although previous reports have suggested a potential involvement of p130Cas in cell motility (50Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (588) Google Scholar, 51Cary L.A. Han D.C. Polte T.R. Hanks S.K. Guan J.L. J. Cell Biol. 1998; 140: 211-221Crossref PubMed Scopus (416) Google Scholar, 52Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar), our data are the first to reveal a requirement for tyrosine phosphorylation of p130Cas in this process. The precise role of p130Cas in controlling cell movement is not clear. However, data recently obtained from mouse embryos lacking the p130Cas protein suggest that it is involved in organization of the actin cytoskeleton in fibroblasts and cardiocytes (53Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar). Integrin-mediated cell movement across the extracellular matrix is a highly dynamic process requiring continual assembly and disassembly of focal contacts, together with extensive remodeling of the actin cytoskeleton (54Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (903) Google Scholar, 55Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar, 56Keely P. Parise L. Juliano R. Trends Cell Biol. 1998; 8: 101-106Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 57Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1461) Google Scholar). Many of the proteins found in focal contacts, including p130Cas, are highly tyrosine-phosphorylated; in fact, focal contacts represent one of the major sites of tyrosine phosphorylation within the cell, as assessed by immunofluorescence analysis using anti-Tyr(P) antibodies (58Maher P.A. Pasquale E.B. Wang J.Y. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6576-6580Crossref PubMed Scopus (155) Google Scholar) (see Fig. 3). Furthermore, at least two protein tyrosine kinases, p125FAK and Src, are localized to focal contacts (59Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1286) Google Scholar, 60Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar), and both kinases have been implicated in the control of cell movement (52Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar, 61Fincham V.J. Frame M.C. EMBO J. 1998; 17: 81-92Crossref PubMed Scopus (264) Google Scholar, 62Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1580) Google Scholar, 63Gilmore A.P. Romer L.H. Mol. Biol. Cell. 1996; 7: 1209-1224Crossref PubMed Scopus (461) Google Scholar). These observations have led to the suggestion that tyrosine phosphorylation plays a significant role in controlling the assembly and disassembly of focal adhesion complexes, thereby regulating cell motility. However, the control of cell movement is a reversible, dynamic process suggesting that its regulation by tyrosine phosphorylation must involve both kinases and phosphatases. Our results suggest that the protein tyrosine phosphatase PTP-PEST regulates cell motility by modulating specifically the extent of tyrosine phosphorylation of p130Cas. The observation that p130Cas is localized at the leading edge of migrating fibroblasts suggests a model for the observed effects of PTP-PEST expression on cell movement. By limiting the accumulation of tyrosine-phosphorylated p130Cas, the ability of p130Cas to interact with appropriate SH2 domain-containing binding partners is impaired in cells overexpressing PTP-PEST. It appears likely that some of these interactions are essential for targeting tyrosine-phosphorylated p130Cas to the correct cytoskeletal location. Thus, it has been shown that the SH2 domain of v-Crk is required for the v-Crk-induced translocation of tyrosine-phosphorylated p130Cas to a detergent-insoluble fraction (48Nievers M.G. Birge R.B. Greulich H. Verkleij A.J. Hanafusa H. van Bergen en Henegouwen P.M. J. Cell Sci. 1997; 110: 389-399PubMed Google Scholar). Also, in p130Cas-overexpressing COS cells, fibronectin-induced relocation of p130Cas to focal adhesion complexes requires both the SH3 domain of p130Cas (which can bind directly to the focal adhesion kinase p125FAK (25Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar,30Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (386) Google Scholar, 49Polte T.R. Hanks S.K. J. Biol. Chem. 1997; 272: 5501-5509Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)) and a C-terminal segment which contains binding sites for the SH2 and SH3 domains of Src (34Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 64Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (135) Google Scholar). Our observation that cells overexpressing PTP-PEST display a defect in the formation of complexes between p130Cas and Crk following fibronectin stimulation (Fig. 5 B) suggests that such complexes are involved in regulating cell movement. Indeed, recent evidence obtained using COS cells transfected with mutant forms of Crk and p130Cassuggested an involvement of p130Cas-Crk complexes in COS cell migration on vitronectin-coated membranes (50Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (588) Google Scholar). Recently, p130Cas−/− fibroblasts have been reported to display a defect in their response to an activated, transforming variant of the Src tyrosine kinase; they fail to form colonies in soft agar, a defect that can be rescued by re-expression of p130Cas (53Honda H. Oda H. Nakamoto T. Honda Z. Sakai R. Suzuki T. Saito T. Nakamura K. Nakao K. Ishikawa T. Katsuki M. Yazaki Y. Hirai H. Nat. Genet. 1998; 19: 361-365Crossref PubMed Scopus (306) Google Scholar). These results suggest that the involvement of p130Cas in regulating the organization of actin cytoskeleton structures may also influence the response of cells to transforming oncogenes, a suggestion that is supported by several earlier observations implicating a direct regulatory role for p130Cas in cellular transformation (65Auvinen M. Paasinen-Sohns A. Hirai H. Andersson L.C. Holtta E. Mol. Cell. Biol. 1995; 15: 6513-6525Crossref PubMed Scopus (75) Google Scholar, 66Mayer B.J. Hanafusa H. J. Virol. 1990; 64: 3581-3589Crossref PubMed Google Scholar, 67Kanner S.B. Reynolds A.B. Wang H.C. Vines R.R. Parsons J.T. EMBO J. 1991; 10: 1689-1698Crossref PubMed Scopus (157) Google Scholar). These data further suggest that PTP-PEST may play a key role in suppressing cellular transformation through its ability to dephosphorylate p130Cas. Indeed, such a role is supported by our observation that overexpression of PTP-PEST dramatically influences the location of p130Cas within v-Crk-transformed cells, suggesting that PTP-PEST potentially influences the response of cells to a variety of transforming oncogenes. The results in this paper represent the first description of the physiological role of PTP-PEST. The data suggest that PTP-PEST is involved in controlling the rate of cell movement via the dephosphorylation of p130Cas. Our results also provide new insight into the function of p130Cas in integrin-mediated processes. Thus, it appears that tyrosine phosphorylation of p130Cas is largely dispensable for many of the cellular responses to fibronectin, including cell attachment, cell spreading, and MAP kinase activation. However, tyrosine phosphorylation of p130Cas is required for the efficient movement of cells across the extracellular matrix and therefore is likely to represent an important mechanism for regulating the dynamic process of cell migration in vivo. We thank Kim Pennino for assistance in characterizing the G104 monoclonal anti-phosphotyrosine antibody used in this work; Amy Bouton (University of Virginia) for p130Cas antibodies, Rat1 fibroblast and 3Y1v-Crk fibroblast cells; and Scott Lowe (Cold Spring Harbor Laboratory) for providing the pWZL(hygro) vector and Bosc23 cell line.