Tyrosine Phosphorylation of Crk-associated Substrates by Focal Adhesion Kinase

Abstract

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125FAK) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins. Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125FAK) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130Cas and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins. Integrin-ligand binding induces various biological and biochemical signals in addition to cell adhesion (1Ruoslahti E. J. Clin. Invest. 1991; 87: 1-5Crossref PubMed Scopus (1494) Google Scholar, 2Ruoslahti E. Reed J.C. Cell. 1994; 77: 477-478Abstract Full Text PDF PubMed Scopus (971) Google Scholar, 3Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9050) Google Scholar, 4Juliano R.L. Haskill S. J. Cell. Biol. 1993; 120: 577-585Crossref PubMed Scopus (1548) Google Scholar). We and others showed the costimulatory effect of the ligation of β1 integrin in CD3-dependent T cell proliferation, indicating that integrin-ligand binding can promote cell proliferation (5Matsuyama T. Yamada A. Kay J. Yamada K.M. Akiyama S.K. Schlossman S.F. Morimoto C. J. Exp. Med. 1989; 170: 1133-1148Crossref PubMed Scopus (251) Google Scholar, 6Shimizu Y. Van Seventer G.A. Horgan K.J. Shaw S. J. Immunol. 1990; 145: 59-67PubMed Google Scholar). To reveal the mechanism of the integrin-mediated biological signals, numbers of laboratories have studied the role of protein tyrosine phosphorylation in various cell types (7Guan J.L. Trevithick J.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (475) Google Scholar, 8Kornberg L.J. Earp H.S. Turner C.E. Prockop C. Juliano R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8392-8396Crossref PubMed Scopus (634) Google Scholar, 9Lipfert L. Haimovich B. Schaller M.D. Cobb B.S. Parsons J.T. Brugge J.S. J. 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DNA Cell Biol. 1993; 12: 823-830Crossref PubMed Scopus (91) Google Scholar). FAK, 1The abbreviations used are: FAK, focal adhesion kinase; Cas, Crk-associated substrate; SH2, Src homology 2; SH3, Src homology 3; HA, hemagglutinin; FN, fibronectin; PLL, poly-l-lysine; CT, COOH-terminal domain; JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase. a 125-kDa cytoplasmic tyrosine kinase, is localized to focal adhesions, which are the sites where cells attach to the substrata via integrin-extracellular matrix binding (14Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Ann. Rev. Cell Biol. 1988; 4: 487-525Crossref PubMed Scopus (1706) Google Scholar). FAK is tyrosine phosphorylated following integrin stimulation in various cells (9Lipfert L. Haimovich B. Schaller M.D. Cobb B.S. Parsons J.T. Brugge J.S. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (633) Google Scholar, 15Guan J.-L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (728) Google Scholar, 16Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1185) Google Scholar, 17Nojima Y. Tachibana K. Sato T. Schlossman S.F. Morimoto C. Cell. Immunol. 1995; 161: 8-13Crossref PubMed Scopus (49) Google Scholar). FAK autophosphorylates Tyr-397, which is a binding site for the Src family kinases when phosphorylated (18Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (486) Google Scholar, 19Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1124) Google Scholar, 20Xing Z. Chen H.-C. Nowlen J.K. Taylor S.J. Shalloway D. Guan J. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (287) Google Scholar, 21Eide B.L. Turck C.W. Escobedo J.A. Mol. Cell. Biol. 1995; 15: 2819-2827Crossref PubMed Scopus (164) Google Scholar). However, besides autophosphorylation, no other substrate of FAK has been revealed. A 68-kDa cytoskeletal protein, paxillin, is tyrosine phosphorylated upon integrin stimulation (16Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1185) Google Scholar) and binds to FAK (22Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar, 23Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (505) Google Scholar, 24Tachibana K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar). Schaller and Parsons (23Schaller M.D. Parsons J.T. Mol. Cell. Biol. 1995; 15: 2635-2645Crossref PubMed Scopus (505) Google Scholar) reported FAK-dependent tyrosine phosphorylation of paxillin, although it is not clear whether FAK itself or a FAK-associated tyrosine kinase phosphorylates paxillin. Crk-associated substrates (Cas) form a protein family of at least three distinct proteins. p130Cas is a 120–130-kDa protein that was first identified as a highly tyrosine-phosphorylated protein in both v-Src- and v-Crk-transformed fibroblasts (25Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (287) Google Scholar, 26Birge R.B. Fajardo J.E. Mayer B.J. Hanafusa H. J. Biol. Chem. 1992; 267: 10588-10595Abstract Full Text PDF PubMed Google Scholar, 27Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (595) Google Scholar). Subsequently, a 105–110-kDa p130Cas-related protein (Cas-L or HEF1) was identified as a 105-kDa protein that is tyrosine phosphorylated upon the ligation of β1 integrins in T lymphocytes (28Minegishi M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar, 29Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar). Furthermore, an 83-kDa Cas-related protein, Efs (or Sin), was reported as the binding proteins for the Src homology 3 (SH3) domains of Fyn and Src (30Ishino M. Ohba T. Sasaki H. Sasaki T. Oncogene. 1995; 11: 2331-2338PubMed Google Scholar, 31Alexandropoulos K. Baltimore D. Genes & Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar). All Cas proteins contain one SH3 domain in the NH2-terminal region, and they all contain multiple putative binding sites for Src homology 2 (SH2) domains, including 8–15 binding sites for the Crk SH2 domain. All three Cas proteins also contain a YDYVHL sequence, which is a potential binding site for the Src SH2 domain (31Alexandropoulos K. Baltimore D. Genes & Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 32Nakamoto 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). We and others reported that both p130Casand Cas-L were tyrosine phosphorylated upon the ligation of integrins and that tyrosine-phosphorylated Cas proteins bind to the SH2 domain-containing proteins Crk, Nck, and SHP-2 (28Minegishi M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar, 33Nojima Y. Morino N. Mimura T. Hamasaki H. Furuya K. 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 (294) Google Scholar, 34Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Furthermore, Cas proteins bind directly to FAK (28Minegishi M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar, 29Law S.F. Estojak J. Wang B. Mysliwiec T. Kruh G. Golemis E.A. Mol. Cell. Biol. 1996; 16: 3327-3337Crossref PubMed Scopus (221) Google Scholar, 35Polte T.R. Hanks S.K. Proc. Natl Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (391) Google Scholar, 36Harte 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 (324) Google Scholar). These findings suggest the role of Cas proteins in integrin-mediated signaling, especially in the recruitment of Crk and the other signaling molecules containing SH2 domains. In this study, we show that FAK phosphorylates the YDYVHL motif within Cas proteins and initiates tyrosine phosphorylation of Cas by pleural tyrosine kinases. We also demonstrate the putative involvement of FAK in integrin-mediated tyrosine phosphorylation of Cas proteins. Cos-1 cells and 293T cells were obtained from ATCC and Dr. David Baltimore (MIT, Cambridge, MA), respectively. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 20 μmGentamicin (Life Technologies, were Cos-1 cells by the (28Minegishi M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar) and 293T cells by the R. J.A. K. in and Scholar). 3 of Cas and of FAK were cells cultured in a 293T cells were from the substrata by in Dulbecco's modified Eagle's cultured for in a with (Life Technologies, and with serum Y. Tachibana K. Sato T. Schlossman S.F. Morimoto C. Cell. Immunol. 1995; 161: 8-13Crossref PubMed Scopus (49) Google Scholar). FAK was by the addition of a sequence, to the of the of the FAK K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar). p130Cas and Cas-L were by the addition of a sequence, to the of p130Cas and Cas-L R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (595) Google Scholar, M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). were by as K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar). FAK and Cas-L were and p130Cas was sites were by the of p130Cas residues or by Cas-L residues Fyn was obtained from Dr. of was from Crk obtained from Dr. J. Mayer Cells were in as K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar). The proteins were with and as (28Minegishi M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar). The protein of the Fyn SH2 domain was from a from Dr. and was used for and were by and was with a and or with as K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google Scholar). was protein containing Cas-L residues K. H. K. and C. in was with protein by with in at for the kinase domain and were by the of the In kinase was by the of kinase domain and in the in kinase and at for p130Cas and are tyrosine phosphorylated upon the ligation of β1 Cas proteins bind to FAK, it was that Cas proteins are tyrosine phosphorylated by activated FAK following β1 integrin-ligand To whether this was the we FAK and p130Cas or Cas-L in Cos-1 cells and tyrosine phosphorylation of Cas proteins by and in both p130Casand Cas-L were tyrosine phosphorylated by the of FAK, p130Cas Cas-L was tyrosine phosphorylated FAK A kinase of FAK not tyrosine phosphorylation of Cas indicating that this Cas phosphorylation was the kinase of the other tyrosine phosphorylation of Cas proteins was when with the although this phosphorylation was with phosphorylation by is an site that a binding site for the SH2 domains of Src family tyrosine kinases, and the the recruitment of Src family kinases to that FAK directly phosphorylates Cas although FAK-associated tyrosine kinases also in FAK-dependent Cas Furthermore, of which does not bind to Cas, in Cas indicating that the binding of FAK to Cas is not for FAK-dependent tyrosine phosphorylation of Cas proteins when FAK is However, when the of FAK is the binding of FAK to Cas a in tyrosine phosphorylation of Cas and the role of binding in the FAK-dependent tyrosine phosphorylation of Cas proteins. We to which Cas sites are tyrosine phosphorylated by the FAK by Cas The p130Cas and Cas-L in were in the or of FAK in Cos-1 cells and were as in and were phosphorylated by FAK, p130Cas was FAK-dependent phosphorylation of Cas-L and were both with that of that contain multiple residues that contain tyrosine residues including the conserved YDYVHL Cas-L residues containing Cas-L residues to COOH-terminal containing the YDYVHL suggest that at least Cas domains, and a domain containing the YDYVHL sequence, are tyrosine phosphorylated when FAK is To the phosphorylation site we a Cas-L with a site the and the YDYVHL was with FAK, by and by in was by a COOH-terminal which was by and NH2-terminal which were by In Cas-L was not by The was highly tyrosine phosphorylated by FAK, were also tyrosine We also a in p130Cas and in 2 COOH-terminal were from by and were by These were tyrosine phosphorylated by FAK or by an NH2-terminal was also tyrosine phosphorylated and was by not These further that at least sites in Cas protein are tyrosine phosphorylated by FAK Furthermore, the of tyrosine-phosphorylated to phosphorylated was when with with that when with the involvement of FAK in the phosphorylation of the and the involvement of a kinase in the phosphorylation of the A p130Cas with a of the YDYVHL to was and used to the phosphorylation in 2 the FAK-dependent tyrosine phosphorylation of was with phosphorylation of Cas from Furthermore, of the in a phosphorylation in These that a tyrosine within the p130Cas YDYVHL sequence is the site of tyrosine phosphorylation by FAK and that a tyrosine kinase with phosphorylates Cas tyrosine A was a Cas-L with the of YDYVHL to although was tyrosine phosphorylated by not To further the phosphorylation of Cas by FAK, we phosphorylation of Cas-L kinase domain protein as a kinase and proteins as substrates. in Cas-L and Cas-L residues were tyrosine phosphorylated by the in kinase Cas-L residues were not The tyrosine residues localized in Cas-L are within the YDYVHL These that the domain of FAK can phosphorylate tyrosine residues within the and the YDYVHL sequence of was tyrosine phosphorylated by p130Cas was the involvement of the Cas YDYVHL sequence in tyrosine phosphorylation of the Cas To the of the tyrosine-phosphorylated YDYVHL sequence, we and a containing the YDYVHL sequence was tyrosine phosphorylated in by not in the NH2-terminal of were phosphorylated the of indicating the involvement of the phosphorylated YDYVHL sequence in the tyrosine phosphorylation of strongly that the Cas-L are phosphorylated by a tyrosine kinase that is recruited to the phosphorylated YDYVHL The Cas YDYVHL sequence was reported as a binding site for Src family kinases (31Alexandropoulos K. Baltimore D. Genes & Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 32Nakamoto 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). To whether tyrosine phosphorylation of the YDYVHL sequence by FAK in the recruitment of Src family kinases, we an Src family tyrosine kinase, with Cas-L or in the or of in cells and the of Fyn with Cas-L proteins. Cas-L does not contain a sequence that is a binding site for the Fyn SH3 domain 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). was used to the of Fyn by with in was with Cas-L when with FAK Fyn was not FAK Fyn was not with with FAK These strongly suggest that Fyn was recruited to the tyrosine-phosphorylated YDYVHL sequence of To that Fyn binds to the YDYVHL sequence via SH2 we used the SH2 domain to Cas proteins. in p130Cas was by the SH2 domain when with FAK, p130Cas was not FAK In was by the SH2 domain. A was obtained Cas-L and not These strongly suggest that Src family tyrosine kinases are recruited to Cas-L in a tyrosine Cas proteins are tyrosine phosphorylated by FAK in and in although it Cas proteins are tyrosine phosphorylated by FAK following integrin To the involvement of FAK in integrin-mediated Cas Cas proteins were in 293T Cells were from the substrata and to with or and were Cas proteins were by and in both p130Casand Cas-L showed tyrosine phosphorylation following the of cells with the Cas-L was strongly phosphorylated by stimulation we to the phosphorylation site in Cas-L following integrin the phosphorylation site by FAK was to within the YDYVHL sequence in Cas integrin-mediated tyrosine phosphorylation of was with that of in was tyrosine phosphorylated following integrin stimulation with indicating that the Cas-L YDYVHL is tyrosine phosphorylated upon the ligation of To further the integrin-mediated phosphorylation sites in we of and The COOH-terminal was tyrosine phosphorylated following the from was not phosphorylated following indicating that a tyrosine within the Cas-L YDYVHL is the integrin-mediated phosphorylation site The NH2-terminal of were tyrosine A was by the of FAK in Cos-1 cells However, when with was highly tyrosine and the NH2-terminal from both and were and to was the were strongly tyrosine phosphorylated the COOH-terminal These strongly suggest that FAK phosphorylates Cas-L upon integrin To further the involvement of FAK in the integrin-mediated Cas a FAK that the FAK COOH-terminal domain was with and the effect integrin-mediated Cas-L phosphorylation was binds to Cas-L (28Minegishi M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Scholar) and is localized to focal K. Sato T. D'Avirro N. Morimoto C. J. Exp. Med. 1995; 182: 1089-1099Crossref PubMed Scopus (239) Google although does not contain a kinase domain or in the of inhibited integrin-mediated tyrosine phosphorylation of strongly the involvement of FAK in the integrin-mediated tyrosine phosphorylation of Cas proteins. adhesion kinase has been to a role in integrin-mediated protein tyrosine although no for phosphorylation by FAK has been and Cas, are the candidates for FAK We have that Cas-L is tyrosine phosphorylated following integrin stimulation in cells Y. Rothstein D.M. Sugita K. Schlossman S.F. Morimoto C. J. Exp. Med. 1992; 175: 1045-1053Crossref PubMed Scopus (142) Google Scholar, M. Tachibana T. Sato T. Iwata S. Nojima Y. Morimoto C. J. Exp. Med. 1996; 184: 1365-1375Crossref PubMed Scopus (147) Google Cas-L is not tyrosine phosphorylated upon integrin stimulation in which are the cells for T. Y. K. H. and C. FAK is in FAK is in this we that FAK is essential for the integrin-mediated tyrosine phosphorylation of Cas-L in cells and that FAK directly phosphorylate However, Src family tyrosine kinases, Fyn and can bind to autophosphorylated FAK (18Cobb B.S. Schaller M.D. Leu T.-H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (486) Google Scholar, 21Eide B.L. Turck C.W. Escobedo J.A. Mol. Cell. Biol. 1995; 15: 2819-2827Crossref PubMed Scopus (164) Google it is to FAK kinase from FAK-associated Src family kinase In this study, we have that FAK directly phosphorylates p130Cas and Cas-L by the which does not bind to the Src SH2 domain. that FAK from Src cells can phosphorylate p130Cas in T. Mol. Cell. Biol. PubMed Scopus Google although it is to the FAK-associated kinase, as in this of We that the phosphorylation site by FAK is within the Cas YDYVHL sequence, and we further that an Src family kinase, binds to the tyrosine-phosphorylated YDYVHL FAK has a substrate and the substrate of FAK is We have a FAK Cas which is also a binding site for the Fyn SH2 domain. These findings the FAK substrate and the binding for the SH2 domains of Src family tyrosine kinases. Furthermore, we have tyrosine phosphorylation of Cas by a tyrosine kinase recruited to phosphorylated FAK also can phosphorylate the Cas at least in although phosphorylation by FAK is with phosphorylation by a kinase recruited to phosphorylated Cas and FAK in We have also that the sequence is phosphorylated upon stimulation in 293T in which FAK is activated and phosphorylated upon stimulation not this integrin-mediated phosphorylation of Cas-L is inhibited by the of further the involvement of FAK in the integrin-mediated Cas was also reported that of the FAK COOH-terminal domain tyrosine phosphorylation of and and the of focal A. Parsons J.T. Nature. 1996; PubMed Scopus Google Scholar, Romer L.H. Mol. Biol. Cell. 1996; PubMed Scopus Google Scholar). for integrin-mediated tyrosine phosphorylation of Cas is as integrin-mediated cell adhesion FAK phosphorylates the and an Src family kinase(s) is recruited to the phosphorylation and it phosphorylates Cas are binding sites for the Crk SH2 domain. These that a Cas phosphorylation by FAK and Src family kinases is essential for integrin-mediated recruitment of Crk to Cas. that of Crk induces of c-Jun NH2-terminal kinase protein kinase via a S. T. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. T. Y. S. M. K. T. T. K. S. M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). was reported that a tyrosine kinase S. H. R. E. B. J. Nature. 1995; PubMed Scopus Google Scholar, H. K. M. H. K. Sasaki T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google is in the of G. S. J. Science. 1996; PubMed Scopus Google Scholar). These findings suggest that FAK in the of tyrosine phosphorylation of Cas proteins and the recruitment of Crk and In to it was reported that FAK not in integrin-mediated tyrosine phosphorylation of the of FAK cells K. Hirai H. S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, K. Mimura T. Morino N. Furuya H. T. S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. 1996; PubMed Scopus Google Scholar). However, we that a tyrosine-phosphorylated protein, which binds to and Cas, is in FAK cells not phosphorylation of this protein was upon integrin We identified that this protein is from However, it is highly that this protein is a kinase, as S. H. R. E. B. J. Nature. 1995; PubMed Scopus Google Scholar, H. K. M. H. K. Sasaki T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google binds to and protein for FAK in the integrin-mediated protein tyrosine phosphorylation in FAK was also reported that integrin-mediated is in Src cells T. Mol. Cell. Biol. PubMed Scopus Google Scholar, K. Hirai H. S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: PubMed Google Scholar, K. Mimura T. Morino N. Furuya H. T. S. Morimoto C. Yazaki Y. Hirai H. Nojima Y. 1996; PubMed Scopus Google Scholar). are recruitment to phosphorylated p130Cas and FAK essential for the phosphorylation of p130Cas it to p130Cas tyrosine phosphorylation upon integrin stimulation in Src in FAK Calalb M.B. T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: PubMed Google Scholar) and others S.K. T.R. PubMed Scopus Google Scholar) showed that FAK kinase that essential for FAK of integrins, phosphorylates the β1 integrin cytoplasmic A. C. K. L. Oncogene. 1989; 4: Google Scholar). we have that p130Cas and Cas-L are tyrosine phosphorylated in a we proteins. The p130Cas and Cas-L is that a sequence of the YDYVHL sequence, Cas-L does not contain a sequence is a binding site for the Src SH3 domain 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 and is to in tyrosine phosphorylation of p130Cas 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). However, we that tyrosine phosphorylation of p130Cas upon integrin stimulation in 293T cells or by FAK in cells is that of These findings suggest that the Src SH3 is not essential for the recruitment of Src family kinases to Cas proteins or for the Cas phosphorylation by Src family kinases upon integrin phosphorylation as the YDYVHL sequence and the are conserved p130Cas and that tyrosine phosphorylation of p130Cas by a mechanism from that of We Dr. David Dr. J. Dr. and Dr. for and Dr. Dr. A. Dr. G. and Dr. Yamada for We also D'Avirro and for and for and