Characterization of Tyrosine Phosphorylation of Paxillin in Vitro by Focal Adhesion Kinase

The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion. The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion. Cell adhesion to an extracellular matrix plays a fundamental role in regulating cellular behaviors such as migration, proliferation, and differentiation(1Hynes R.O. Lander A.D. Cell. 1992; 68: 303-322Abstract Full Text PDF PubMed Scopus (762) Google Scholar, 2Damsky C.H. Werb Z. Curr. Opin. Cell Biol. 1992; 4: 772-781Crossref PubMed Scopus (487) Google Scholar). The organization of cell adhesion sites is directed by a family of transmembrane receptors known as integrins(3Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). The binding of integrins to extracellular matrix ligands catalyzes the recruitment of multiple cytoskeletal associated proteins to the cytoplasmic face of cell attachment sites (focal adhesions) and thereby organizes the actin cytoskeleton(4Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Annu. Rev. Cell Biol. 1988; 4: 487-525Crossref PubMed Scopus (1701) Google Scholar, 5Turner C.E. Burridge K. Curr. Opin. Cell Biol. 1991; 3: 849-853Crossref PubMed Scopus (190) Google Scholar). The molecular mechanisms by which cell adhesion triggers various cellular events have not yet been defined. However, evidence is emerging that implicates the tyrosine phosphorylation of certain focal adhesion proteins in at least one aspect of integrin-meditated signaling, that of cytoskeleton reorganization. In particular, the binding of integrins to extracellular matrix ligands results in an increase in tyrosine-phosphorylated forms of several focal adhesion proteins(6Schaller M.D. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 7Bockholt S.M. Burridge K. J. Biol. Chem. 1993; 268: 14565-14567Abstract Full Text PDF PubMed Google Scholar). Two focal adhesion proteins that demonstrate a high stoichiometry of tyrosine phosphorylation upon integrin activation are the nonreceptor tyrosine kinase, focal adhesion kinase (FAK)1 1The abbreviations used are: FAKfocal adhesion kinaseGSTglutathione S-transferasePCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresis. (8Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar, 9Hanks S.K. Calalb M.B. Harper M.C. Patel S.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (730) Google Scholar, 10Kornberg L. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar, 11Schaller 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 (1293) Google Scholar, 12Guan J.-L. Trevithick J.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar), and the 68-kDa protein, paxillin(8Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar, 13Turner C.E. BioEssays. 1994; 16: 47-52Crossref PubMed Scopus (149) Google Scholar). The tyrosine phosphorylation of these two proteins has been suggested to be involved in both the formation of focal adhesions and the assembly of actin stress fibers(8Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar). focal adhesion kinase glutathione S-transferase polymerase chain reaction polyacrylamide gel electrophoresis. The coordinate phosphorylation of paxillin and FAK on tyrosine residues has also been observed in other systems. Cells transformed by Rous sarcoma virus (14Glenney J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (360) Google Scholar) and tissues undergoing embryogenesis (15Turner C.E. J. Cell Biol. 1991; 115: 201-207Crossref PubMed Scopus (97) Google Scholar, 16Turner C.E. Schaller M.D. Parsons J.T. J. Cell Sci. 1993; 105: 637-645Crossref PubMed Google Scholar) have been shown to contain enriched populations of tyrosine-phosphorylated forms of paxillin and FAK. Additionally, tyrosine-phosphorylated paxillin and FAK have been detected in cultured cells that have been treated with a variety of stimuli, including lysophosphatidic acid (17Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar), angiotensin II(18Turner C.E. Pietras K.M. Taylor D.S. Molloy C.J. J. Cell Sci. 1995; 108: 333-342Crossref PubMed Google Scholar), sphingosine(19Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 27610-27617Abstract Full Text PDF PubMed Google Scholar), low doses of platelet-derived growth factor(20Rankin S. Rozengurt E. J. Biol. Chem. 1994; 269: 704-710Abstract Full Text PDF PubMed Google Scholar), and the neuropeptides bombesin, endothelin, and vasopressin(21Zachary I. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1992; 267: 19031-19034Abstract Full Text PDF PubMed Google Scholar, 22Zachary I. Sinnett-Smith J. Turner C.E. Rozengurt E. J. Biol. Chem. 1993; 268: 22060-22065Abstract Full Text PDF PubMed Google Scholar). The multiple observations of concomitant phosphorylation of FAK and paxillin suggest that tyrosine phosphorylation of paxillin may be a direct consequence of FAK activity. Additional support for this hypothesis is provided by the finding that purified paxillin can be phosphorylated in vitro by a FAK immunoprecipitate(16Turner C.E. Schaller M.D. Parsons J.T. J. Cell Sci. 1993; 105: 637-645Crossref PubMed Google Scholar). The paxillin cDNA has been recently cloned(23Salgia R. Li J-L. Lo S.H. Brunkhorst B. Kansas G.S. Sobhany E.S. Sun Y. Pisick E. Hallek M. Ernst T. Tantravahi R. Chen L.B. Griffin J.D. J. Biol. Chem. 1995; 270: 5039-5047Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 24Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar), and a region of the paxillin molecule has been identified that supports the binding of both vinculin and FAK(24Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar). An analysis of the paxillin amino acid sequence reveals the presence of additional domains, which are thought to function in protein-protein interactions. Among these domains are a proline-rich, putative SH3 binding motif(25Musacchio A. Wilmanns M. Saraste M. Prog. Biophys. Mol. Biol. 1994; 61: 283-297Crossref PubMed Scopus (144) Google Scholar, 26Pawson T. Gish G.D. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (796) Google Scholar, 27Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1438) Google Scholar), four LIM domains (28Schmeichel K.L. Beckerle M.C. Cell. 1994; 79: 211-219Abstract Full Text PDF PubMed Scopus (408) Google Scholar), and multiple tyrosine residues, which are likely to represent sites of phosphorylation, given that these residues lie within good consensus sequences for binding to proteins that contain SH2 domains (29Songyang 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 (2384) Google Scholar). In the present study, we have determined that one of the tyrosine residues that potentially comprises part of an SH2 binding domain for the adapter protein, Crk, is a primary site of paxillin phosphorylation by FAK in vitro. Selected regions of paxillin cDNA were amplified from paxillin clone 10 (24Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar) by polymerase chain reaction (PCR) using oligonucleotides containing 5’-BamHI or 3’-EcoRI restriction sites. The PCR products were subcloned into BamHI/EcoRI-digested pGEX-2T (Pharmacia Biotech Inc.). Verification that paxillin was subcloned into pGEX-2T in the correct reading frame was provided by dideoxy chain termination sequencing (Sequenase 2, U. S. Biochemical Corp.). The GST-paxillin fusion proteins were expressed and purified using glutathione (GSH)-Sepharose as described elsewhere(30Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). For use in kinase assays, fusion proteins were eluted from GSH-Sepharose beads with 20 mM GSH in 50 mM Tris-HCl buffer (pH 8.0), then dialyzed overnight into kinase buffer (10 mM HEPES, 3 mM MnCl2, pH 7.3). 14-16-day-old embryonic chicken gizzards (a source of smooth muscle focal adhesion proteins) were homogenized in 10 volumes of buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Triton X-100, 0.1% deoxycholate, 10 εg/ml leupeptin, and 50 εM sodium orthovanadate (lysis buffer). Lysates were centrifuged at 100,000 × g to remove insoluble material. FAK was immunoprecipitated by incubating the lysates for 90 min at 4°C with anti-FAK monoclonal antibody 2A7 (gift from Dr. J. T. Parsons, University of Virginia), followed by the addition of rabbit, anti-mouse IgG complexed to protein A-Sepharose. The immune complexes were pelleted, washed extensively with lysis buffer, washed once with 10 mM HEPES, 3 mM MnCl2, pH 7.3 (kinase buffer), then resuspended into 20 εl of kinase buffer. Approximately 5-20 εg of GST-paxillin fusion protein (previously dialyzed into kinase buffer) were added to the FAK immunoprecipitate with 10 εCi of γ-32PATP. In some kinase assays, 100 εM tyrphostin (Life Technologies, Inc.) was also added. Following a 20-min incubation at room temperature, kinase reactions were terminated by boiling the samples in SDS-PAGE sample buffer. Samples were resolved on 10% acrylamide gels by SDS-PAGE (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) and then visualized by autoradiography. Protein amounts were determined by using a Bio-Rad protein assay kit, as well as by Coomassie staining of SDS-PAGE gels. To perform kinase assays with native paxillin, anti-paxillin monoclonal antibody 165 (32Turner C.E. Glenney J.R. Burridge K. J. Cell Biol. 1990; 111: 1059-1068Crossref PubMed Scopus (529) Google Scholar) and anti-FAK monoclonal antibody 2A7 were added simultaneously to gizzard lysates. Following a 90-min incubation at 4°C, paxillin and FAK were coprecipitated by the addition of rabbit anti-mouse saturated protein A-Sepharose. The precipitates were washed and then resuspended in kinase buffer containing 10 εCi of γ-32PATP. As described above, kinase reactions proceeded for 20 min at room temperature, and the phosphorylated samples were resolved by electrophoresis. Phenylalanine substitutions for tyrosine residues were introduced into the paxillin sequence using a PCR method described by Landt et al.(33Landt D. Grunert H. Hahn U. Gene (Amst.). 1990; 96: 125-128Crossref PubMed Scopus (639) Google Scholar). Mutated paxillin cDNA was subcloned into pGEX-2T as described above. The correct sequence of the mutated GST-paxillin fusion protein was verified by sequencing. GST, GST-paxillin 54-313, and GST-paxillin 54-313/Y118F, each coupled to GSH-Sepharose beads(30Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar), were incubated with embryonic chicken gizzard lysates for 90 min at 4°C. Samples were pelleted and washed extensively with lysis buffer. Samples were resolved by electrophoresis on 10% acrylamide gels and transferred to nitrocellulose using standard procedures(34Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44924) Google Scholar). FAK and FRNK, an alternately spliced FAK isoform(6Schaller M.D. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar), were detected by incubating nitrocellulose blots with anti-FAK polyclonal (gift from Dr. Parsons, University of Virginia), followed by incubation with fusion proteins were eluted from SDS-PAGE and to to standard K. T. Protein University Scholar). were in the first by electrophoresis in pH buffer K. T. Protein University Scholar) and then resolved by in acid K. T. Protein University Scholar). acid of was not to the paxillin cDNA amino acid residues was amplified by PCR using paxillin clone 10 (24Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar) as a PCR 5’-BamHI and 3’-EcoRI restriction sites to into the restriction sites of the a which a In residue of the paxillin sequence was mutated to from the is at the first residue within the of tyrosine 118 to phenylalanine was as described above. of NIH 3T3 on were using the method of et J. J-L. Trevithick J.E. C.A. Hynes R.O. J. Cell Biol. 1989; PubMed Scopus Google Scholar). the cells were and for as C.E. Glenney J.R. Burridge K. J. Cell Biol. 1990; 111: 1059-1068Crossref PubMed Scopus (529) Google Scholar). and of transfected paxillin were detected using a polyclonal C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar). Cells were with to stress Following of the of paxillin and the identification of FAK binding to a paxillin C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar), we have using of cDNA an additional chicken cDNA sequence amino at the amino used in this is on a protein of amino The additional cDNA sequence has been in An acid sequence was for paxillin R. Li J-L. Lo S.H. Brunkhorst B. Kansas G.S. Sobhany E.S. Sun Y. Pisick E. Hallek M. Ernst T. Tantravahi R. Chen L.B. Griffin J.D. J. Biol. Chem. 1995; 270: 5039-5047Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) of this a GST-paxillin fusion protein containing paxillin sequence from amino was shown to support the binding of FAK (24Turner C.E. Miller J.T. J. Cell Sci. 1994; 107: 1583-1591Crossref PubMed Google Scholar) and a in fusion protein, GST-paxillin was used in in vitro kinase assays with FAK of the GST-paxillin fusion protein not was that the phosphorylation of the fusion protein was specific to the paxillin of immunoprecipitated FAK fusion protein was also observed and The phosphorylation of FAK likely represents an M.D. J.D. J.D. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). of phosphorylated GST-paxillin from SDS-PAGE gels and acid that the phosphorylation of the fusion protein was to tyrosine residues not Additionally, the phosphorylation of both paxillin and FAK was by the of a tyrosine kinase A. C. A. J. Chem. 1989; PubMed Scopus Google Scholar), in the assay buffer not has been shown to FAK in vitro and in L.H. Turner C.E. Burridge K. Mol. Biol. Cell. 1994; PubMed Scopus Google phosphorylation of GST-paxillin and of GST-paxillin FAK was immunoprecipitated from embryonic chicken gizzard lysates with monoclonal antibody the fusion proteins (previously dialyzed into kinase buffer) were added with 10 εCi of γ-32PATP. reactions proceeded for 20 min at room and were terminated by boiling in SDS-PAGE sample buffer. were resolved by electrophoresis on 10% acrylamide gels. The protein samples were added to the FAK GST-paxillin fusion protein added and and of GST-paxillin and of results from kinase assays with assays with of GST-paxillin that the primary site of phosphorylation within amino To the of paxillin phosphorylation by a of GST-paxillin fusion proteins was and used in in vitro kinase assays with FAK 2, and The fusion protein, GST-paxillin 54-313, four tyrosine residues, Tyr-118, and In vitro kinase assays using of this fusion protein that phosphorylation of the fusion protein by the FAK immunoprecipitate within a region of the paxillin molecule amino finding Tyr-118 as the likely site of phosphorylation by the FAK To that tyrosine 118 was the primary site of phosphorylation by the FAK was used to tyrosine residues within the fusion protein to Mutated fusion proteins were then used in in vitro kinase assays with one additional of the mutated fusion proteins paxillin sequence from amino of the fusion protein used in The of paxillin sequence from amino acid to was fusion protein to the as IgG chain on SDS-PAGE gels. to the fusion protein by Coomassie staining and thereby that amounts of mutated fusion proteins were added to each kinase GST-paxillin of the tyrosine residues that were present in of phosphorylation of was observed with to and In vitro kinase assays with mutated fusion proteins that the phosphorylation of paxillin by the FAK immunoprecipitate was by a phenylalanine substitution at tyrosine 118 In in the of phosphorylation was observed in of the other mutated indicating that the phosphorylation of GST-paxillin by FAK on a tyrosine is that the in phosphorylation of the Y118F mutant was to a in the of the paxillin molecule the Y118F mutant was shown by analysis to bind both FAK and FRNK, a of M.D. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar), equally as well as the wild type fusion protein of FAK by GST-paxillin fusion GST-paxillin and GST-paxillin containing a phenylalanine substitution at tyrosine 118 were incubated with embryonic chicken gizzard lysates for 90 min at 4°C. and proteins were with and resolved by sample of the embryonic chicken gizzard was also Following proteins were transferred to and both FAK and FRNK, a of M.D. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar), were detected by using anti-FAK polyclonal The of molecular are on the The of paxillin used in the represents of the paxillin sequence of paxillin as a protein in an insoluble a of the paxillin sequence was from the fusion proteins used in assays, was to the phosphorylation of the fusion protein by the FAK immunoprecipitate the in vitro phosphorylation of native paxillin by FAK. To this both GST-paxillin and native paxillin were phosphorylated in vitro by the FAK The phosphorylated proteins were from SDS-PAGE and to As shown in multiple were observed in the of both native paxillin and GST-paxillin fusion protein. that and suggested that phosphorylation at a site (Tyr-118), is likely that multiple at least in the of the fusion protein, from in the of a of the two that the of were that the phosphorylation of native paxillin at the as the fusion protein. in the of are likely to the of sites in the native paxillin the GST-paxillin fusion protein. these may the phosphorylation of paxillin at a which was in the native The analysis two one of the in both the tyrosine Tyr-118, as a principle site of phosphorylation in both native paxillin and the GST-paxillin fusion protein. were in the of native paxillin as with the fusion protein, that of the primary sites of paxillin phosphorylation by the FAK immunoprecipitate were present within fusion protein To the that tyrosine residues not in GST-paxillin be for phosphorylation by we in vitro kinase assays with several additional fusion GST-paxillin GST-paxillin GST-paxillin GST-paxillin and GST-paxillin phosphorylation of of these fusion proteins by FAK was observed not hypothesis that Tyr-118 is the primary site of phosphorylation by FAK this was the identification of Tyr-118 as a substrate for phosphorylation by FAK was by a indicating that Tyr-118 of paxillin is phosphorylated in cultured cells that M.D. Parsons J.T. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). In this however, was also as a phosphorylation In with this we have not detected phosphorylation of paxillin on the used in in vitro kinase In of the that focal adhesion formation is with the tyrosine phosphorylation of paxillin and K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar), we were in the phosphorylation of paxillin on tyrosine 118 was for the of paxillin to focal paxillin cDNA and paxillin cDNA containing a phenylalanine substitution at tyrosine 118 were transfected into NIH 3T3 cells cDNA chicken paxillin sequence for amino Similar to paxillin the Y118F mutant paxillin was detected in focal adhesions by the antibody indicating that phosphorylation at tyrosine 118 was not for the recruitment of paxillin to adhesion sites. is also that the proline-rich, putative SH3 binding domain of paxillin not to be for the amino for this domain were not within the transfected cDNA is of have shown an paxillin and the SH3 domain of Z. Taylor Turner C.E. C. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), a tyrosine kinase that has been identified in focal Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). is a protein containing binding sites for several focal adhesion is likely that the transfected paxillin are to focal adhesions with vinculin FAK or the paxillin LIM the phosphorylation of paxillin on Tyr-118 is not essential for the of paxillin to focal adhesion however, the for phosphorylation of Tyr-118 in the assembly of focal adhesions to be given that paxillin is present in NIH 3T3 cells. function of paxillin phosphorylation on Tyr-118 may the recruitment of other to focal adhesion sites. residues to sites for proteins containing SH2 domains and thereby the formation of has been that the amino a residue a for binding to SH2 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 (2384) Google Scholar). An of the amino acid sequence tyrosine 118 of paxillin suggests that phosphorylation at this site is likely to be for the known paxillin and the SH2 domain of the protein, R.B. Fajardo J.E. C. Shoelson S.E. Z. Cantley L.C. Hanafusa H. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar). hypothesis is by a an paxillin phosphorylated on Tyr-118 and the SH2 domain of M.D. Parsons J.T. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). In we have identified a specific tyrosine residue within paxillin that is a primary target for phosphorylation by FAK and have that the phosphorylation of this residue is not essential for the of paxillin to focal adhesion sites. Additional be to events that may lie of paxillin phosphorylation by FAK. are to Dr. J. Parsons of for the of FAK. also Dr. for reading of this