Interaction of Focal Adhesion Kinase with Cytoskeletal Protein Talin

The interaction of cells with extracellular matrix proteins plays a critical role in a variety of biological processes. Recent studies suggest that cell-matrix interactions mediated by integrins can transduce biochemical signals to the cell interior that regulate cell proliferation and differentiation. These studies have placed the focal adhesion kinase (FAK), an intracellular protein tyrosine kinase, in a central position in integrin-initiated signal transduction pathways (Zachary, I., and Rozengurt, E.(1992) Cell 71, 891-894; Medline Schaller, M., and Parsons, J. T. (1993) Trends Cell Biol. 3, 258-262). Here, we report data suggesting a possible association of FAK with the cytoskeletal protein talin in NIH 3T3 cells. We have identified a 48-amino acid sequence in the carboxyl-terminal domain of FAK necessary for talin binding in vitro. Furthermore, we have correlated the ability of integrin to induce FAK phosphorylation with its ability to bind talin using a mutant integrin lacking the carboxyl-terminal 13 amino acids. These studies suggest talin may be a mediator for FAK activation in signaling initiated by integrins and may provide an explanation for the dependence on the integrity of actin-cytoskeleton of multiple intracellular signaling pathways converging to FAK activation and autophosphorylation. The interaction of cells with extracellular matrix proteins plays a critical role in a variety of biological processes. Recent studies suggest that cell-matrix interactions mediated by integrins can transduce biochemical signals to the cell interior that regulate cell proliferation and differentiation. These studies have placed the focal adhesion kinase (FAK), an intracellular protein tyrosine kinase, in a central position in integrin-initiated signal transduction pathways (Zachary, I., and Rozengurt, E.(1992) Cell 71, 891-894; Medline Schaller, M., and Parsons, J. T. (1993) Trends Cell Biol. 3, 258-262). Here, we report data suggesting a possible association of FAK with the cytoskeletal protein talin in NIH 3T3 cells. We have identified a 48-amino acid sequence in the carboxyl-terminal domain of FAK necessary for talin binding in vitro. Furthermore, we have correlated the ability of integrin to induce FAK phosphorylation with its ability to bind talin using a mutant integrin lacking the carboxyl-terminal 13 amino acids. These studies suggest talin may be a mediator for FAK activation in signaling initiated by integrins and may provide an explanation for the dependence on the integrity of actin-cytoskeleton of multiple intracellular signaling pathways converging to FAK activation and autophosphorylation. Focal adhesion kinase (FAK)1 1The abbreviations used are: FAKfocal adhesion kinasePDGFplatelet-derived growth factorGSTglutathione S-transferase. 1The abbreviations used are: FAKfocal adhesion kinasePDGFplatelet-derived growth factorGSTglutathione S-transferase. is a cytoplasmic tyrosine kinase localized in focal contacts(1Zachary I. Rozengurt E. Cell. 1992; 71: 891-894Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 2Schaller M. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 3Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Crossref PubMed Scopus (1533) Google Scholar). Consistent with its subcellular localization, FAK has been implicated in signal transduction pathways initiated by integrin-mediated cell adhesion to extracellular matrix proteins. FAK becomes rapidly phosphorylated in response to cell attachment to fibronectin-coated surfaces or integrin clustering by antibodies(4Guan J.-L. Trevithick L.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar, 5Kornberg L. Earp H.S. Turner C. Prokop C. Juliano R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8392-8396Crossref PubMed Scopus (632) Google Scholar, 6Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1180) Google Scholar, 7Guan J.-L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (724) Google Scholar, 8Hanks S. Calalb M. Harper M. Patel S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (728) Google Scholar, 9Kornberg L.J. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar, 10Schaller M. Borgman C. Cobb B. Reynolds A. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1290) Google Scholar). Integrin clustering on the cell surface also stimulates the tyrosine kinase activity of FAK in both fibroblasts and platelets(7Guan J.-L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (724) Google Scholar, 11Lipfert L. Haimovich B. Schaller M. Cobb B. Parsons J.T. Brugge J.S. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (629) Google Scholar). In addition, transformation of fibroblasts by v-Src results in an elevated tyrosine phosphorylation of FAK, associated with increased kinase activity(7Guan J.-L. Shalloway D. Nature. 1992; 358: 690-692Crossref PubMed Scopus (724) Google Scholar, 12Kanner S. Reynolds A. Richard V. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (397) Google Scholar). FAK phosphorylation is also stimulated by several neuropeptides, including bombesin, vasopressin, and endothelin(13Leeb-Lundberg L. Song X.-H. J. Biol. Chem. 1991; 266: 7746-7749Abstract Full Text PDF PubMed Google Scholar, 14Zachary I. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1992; 267: 19031-19034Abstract Full Text PDF PubMed Google Scholar), which bind to cell surface receptors coupled to G-proteins(13Leeb-Lundberg L. Song X.-H. J. Biol. Chem. 1991; 266: 7746-7749Abstract Full Text PDF PubMed Google Scholar, 15Zachary I. Gill J. Lehmann W. Sinnett-Smith J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4577-4581Crossref PubMed Scopus (119) Google Scholar, 16Zachary I. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1991; 266: 24126-24133Abstract Full Text PDF PubMed Google Scholar, 17Huckle W.R. Prokop C.A. Dy R.C. Herman B. Earp S. Mol. Cell. Biol. 1990; 10: 6290-6298Crossref PubMed Scopus (87) Google Scholar, 18Force T. Kyriakis J.M. Avruch J. Bonventre J.V. J. Biol. Chem. 1991; 266: 6650-6656Abstract Full Text PDF PubMed Google Scholar). Finally, tyrosine phosphorylation of FAK was recently shown to be modulated by platelet-derived growth factor (PDGF) (19Rankin S. Rozengurt E. J. Biol. Chem. 1994; 269: 704-710Abstract Full Text PDF PubMed Google Scholar) and lysophosphatidic acid, a platelet-derived phospholipid that elicits a wide variety of cellular responses in diverse cell types by acting on its receptor coupled to G-proteins(20Moolenaar W.H. Adv. Cancer Res. 1991; 57: 87-102Crossref PubMed Scopus (37) Google Scholar, 21van der Bend R.L. Brunner J. Jalink K. van Corven E.J. Moolenaar W.H. van Blitterswijk W.J. EMBO J. 1992; 11: 2495-2501Crossref PubMed Scopus (178) Google Scholar). Therefore, FAK activation and phosphorylation are likely to be a point of convergence of multiple intracellular signaling pathways(1Zachary I. Rozengurt E. Cell. 1992; 71: 891-894Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 2Schaller M. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar). focal adhesion kinase platelet-derived growth factor glutathione S-transferase. focal adhesion kinase platelet-derived growth factor glutathione S-transferase. The mechanism of FAK activation in response to various stimuli is poorly understood(2Schaller M. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar). Inactivation of protein kinase C by a number of methods does not block increase in FAK phosphorylation stimulated by neuropeptides, although treatment of cells with protein kinase C activators lead to increased tyrosine phosphorylation of FAK(22Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar). In contrast, inactivation of p21rho by ADP-ribosylation with botulinum C3 exoenzyme blocks the tyrosine phosphorylation of FAK induced by lysophosphatidic acid (23Kumagai N. Morii N. Fujisawa K. Nemoto Y. Narumiya S. J. Biol. Chem. 1993; 268: 24535-24538Abstract Full Text PDF PubMed Google Scholar) or bombesin and endothelin(24Rankin S. Morii N. Narumiya S. Rozengurt E. FEBS Lett. 1994; 354: 315-319Crossref PubMed Scopus (131) Google Scholar). It is unclear, however, if p21rho is involved in FAK activation by integrins. Studies using cytochalasin D, which selectively disrupts the networks of actin filaments, show that the integrity of the actin cytoskeleton is required for increased phosphorylation of FAK in response to a variety of extracellular stimuli(6Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1180) Google Scholar, 22Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar). Furthermore, integrins have been shown to interact with the actin cytoskeleton via their direct binding to two cytoskeletal proteins talin and α-actinin(25Horwitz A. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (824) Google Scholar, 26Otey C.A. Pavalko F.M. Burridge K. J. Cell Biol. 1990; 111: 721-729Crossref PubMed Scopus (650) Google Scholar). Therefore, we hypothesized that some of the cytoskeletal proteins colocalized with FAK and integrins in the focal contacts may be involved in mediating FAK activation by integrins. Here, we report data suggesting a possible association of FAK with talin in NIH 3T3 cells and the identification of a 48-amino acid sequence in the carboxyl-terminal domain of FAK necessary for talin binding in vitro. Furthermore, we have correlated the ability of integrin to induce FAK phosphorylation with its ability to bind talin using a mutant integrin lacking the carboxyl-terminal 13 amino acids. Protein A-Sepharose 4B, glutathione-agarose beads, Ponceau S, and monoclonal antibody against talin were purchased from Sigma. Rabbit antiserum against PDGF receptor was from UBI. Antisera against talin and vinculin were kind gifts of Dr. R. O. Hynes (Massachusetts Institute of Technology), and monoclonal antibody (12CA5) against an epitope of the hemagglutinin (HA1) protein of the influenza virus was a kind gift of Dr. Q. Yu (Boston University Medical School). Rabbit anti-FAK serum was prepared as previously described(27Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). NIH 3T3 cells or insect Spodoptera frugiperda (Sf21) cells were maintained as previously described(27Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). Cell lysates were prepared in 1% Nonidet P-40 lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory unit/ml aprotinin, and 20 μg/ml leupeptin) as previously described(27Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). Lysates from NIH 3T3 cells (500 μg) or Sf21 cells (100 μg) were immunoprecipitated by incubation with 3 μl of various antisera for 1 h at 4°C. Immune complexes were collected on protein A-Sepharose and washed four times in lysis buffer. They were then boiled for 3 min in SDS-sample buffer and resolved on SDS-polyacrylamide gel electrophoresis. Western blotting was performed with monoclonal anti-talin antibody (1:1000 dilution) or 12CA5 (1:1000 dilution) using the Amersham electrochemiluminescence system, as previously described(27Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). The various FAK mutants have been previously described(28Hildebrand J.D. Schaller M.D. Parsons J.T. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (358) Google Scholar). Two complementary synthetic oligonucleotides encoding an epitope derived from the influenza virus hemagglutinin sequence (YPYDVPDYA, which is recognized by the monoclonal antibody 12CA5) were ligated to the 5′-end of the FAK coding sequence, replacing 29 amino acid residues. Epitope-tagged FAK or its mutants were cloned into expression vector pBlueBac2 (Invitrogen). These were then transfected into Sf21 cells along with the linear genomic DNA of baculoviruses, and the recombinant viruses were harvested and used to infect Sf21 for recombinant protein production, as previously described(27Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). The cDNAs encoding chicken integrin β1 and the Δ4 mutant have been previously described(29Marcantonio E.E. Guan J-L. Trevithick J. Hynes R.O. Cell Regul. 1990; 1: 597-604Crossref PubMed Scopus (116) Google Scholar). cDNA fragments containing the cytoplasmic domains of the wild type or mutant chicken integrin β1 were excised by digestions with BclI and PvuII and inserted into the cloning site of pGEX-3X (Pharmacia Biotech Inc.) after digestions with BamHI and SmaI. The resulting plasmids were used to generate in-frame fusion proteins GST-cytoβ1wt and GST-cytoβ1Δ4, respectively, as previously described(27Xing Z. Chen H.-C. Nowlen J.K. Taylor S. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar). For talin-FAK binding assays, cellular talin from 500 μg of NIH 3T3 cell lysates were immobilized on protein A beads by immunoprecipitation with polyclonal anti-talin serum. Talin-bound beads were further incubated with 100 μg of recombinant virus-infected Sf21 cell lysates in 1% Nonidet P-40 lysis buffer for 2 h at 4°C. The bound recombinant FAK or mutants were eluted in SDS sample buffer, resolved on SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with 12CA5. The membranes were then washed in stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol) at 55°C for 30 min and reblotted with anti-talin antibody to show that similar amounts of talin were present in the binding assays. Aliquots of lysates (15 μg) containing recombinant proteins were also analyzed directly by Western blotting with 12CA5 to show that similar amounts of recombinant FAK and mutants were used in the binding assays. In some experiments, SDS was added to the cell lysates (final concentration, 2%) prepared in 1% Nonidet P-40 lysis buffer. The lysates with SDS were then boiled for 5 min to disrupt potential associations of talin with other proteins. The samples were cooled and diluted (10-fold) in 1% Nonidet P-40 lysis buffer and used for immunoprecipitation with anti-talin serum or normal rabbit serum to prepare for immobilized talin in binding assays. For talin-integrin binding assays, about 5 μg of bacterially expressed GST fusion proteins were immobolized on glutathione beads and then incubated with 1 mg of NIH 3T3 cell lysates in 1% Nonidet P-40 lysis buffer overnight at 4°C. The bound complexes were eluted in SDS sample buffer and analyzed by Western blotting with anti-talin antibody. The membranes were then stained with Ponceau S to show that similar amounts of fusion proteins were present in the binding assays. To study the role of FAK in integrin signaling, we searched for FAK-interacting proteins that may mediate FAK activation in response to integrin aggregation in cell adhesion. both integrins and FAK are localized in focal we were in potential interactions of FAK with other cytoskeletal proteins localized in focal To the potential association of FAK with of two proteins was performed using lysates prepared from NIH 3T3 cells. shown in talin is present in the anti-FAK which with the talin as by anti-talin serum of the that about of cellular talin was associated with that the talin was also in anti-talin which the of talin Hynes R.O. Nature. 1990; PubMed Scopus Google Scholar). was not in of anti-FAK serum or antiserum against PDGF receptor the of talin association with Consistent with on their interaction in A. Duggan K. R. C. Buck C. J. Cell Biol. PubMed Scopus Google Scholar), talin was also in data the possible association of FAK with talin in To the of FAK necessary for association with recombinant FAK and a of its mutants were in insect cells by expression These mutants have been previously J.D. Schaller M.D. Parsons J.T. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (358) Google Scholar) and are as to of amino of amino of amino and of amino To the and of the recombinant an encoding an epitope derived from the influenza virus hemagglutinin sequence (YPYDVPDYA, which is recognized by the monoclonal antibody 12CA5) was to the amino of FAK or its replacing 29 amino acid residues. The recombinant FAK and mutants were expressed in insect Sf21 cells as FAK was expressed in Sf21 cells not and the recombinant proteins can be by immunoprecipitation with anti-FAK serum by Western blotting with 12CA5 of the recombinant proteins by in kinase that recombinant FAK and mutants kinase not that in were for recombinant proteins. kinase activity was for as not of mutant is likely can bind to talin 3 and association of talin and recombinant of lysates from insect cells were analyzed by Western blotting using 12CA5 to that similar amounts of were used in binding for cellular talin was immobilized on protein A-Sepharose beads by anti-talin immunoprecipitation and then incubated with various recombinant FAK proteins. The bound proteins were eluted in SDS sample buffer and analyzed by Western with 12CA5. the was and using anti-talin monoclonal antibody to that similar amounts of talin were present in samples in the binding used are as in 2 In binding were performed using cell lysates prepared from Sf21 cells with various recombinant viruses encoding FAK or its of sample was analyzed by Western blotting with 12CA5 to show that a similar of recombinant proteins was expressed in insect cells The samples were incubated with cellular talin immobilized on protein A-Sepharose beads by immunoprecipitation with polyclonal anti-talin serum. the bound proteins were resolved on SDS-polyacrylamide gel and by Western blotting with 12CA5. shown in recombinant proteins bound to Western blotting of the membranes that similar amounts of talin were present in samples of the recombinant proteins bound to beads normal rabbit serum was used for talin immunoprecipitation not These data that amino of FAK in were necessary for binding talin immobilized on of recombinant FAK to beads be to its direct binding to talin or mediated by other proteins that are associated with For vinculin has been shown to be associated with talin A. Duggan K. R. C. Buck C. J. Cell Biol. PubMed Scopus Google Scholar) and is present in anti-talin To two cell lysates were boiled in SDS to disrupt associations by anti-talin serum shown in vinculin be in beads prepared from the lysates 1 and potential proteins as and not be in anti-talin the treatment not It is also that other proteins were associated with talin after in SDS of the cell recombinant FAK bound to beads prepared from boiled lysates as as that from lysates and The recombinant FAK not bind to beads normal rabbit serum was used for talin immunoprecipitation 3 and results suggest that FAK likely to talin directly in and to a of amino on data from and have implicated that the carboxyl-terminal amino in the cytoplasmic domain of integrin β1 is for intracellular signaling, resulting in tyrosine phosphorylation of J.-L. Trevithick L.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). To if the ability of integrin to FAK is correlated with its binding to two integrin β1 cytoplasmic GST fusion proteins were in including an cytoplasmic domain and a mutant The mutant 13 amino from the of integrin β1 cytoplasmic domain and was to induce FAK tyrosine J.-L. Trevithick L.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar). The GST fusion proteins were immobilized on glutathione-agarose beads and further incubated with NIH 3T3 cell the bound protein complexes were eluted in SDS sample buffer and analyzed by Western with shown in talin was by GST-cytoβ1wt not GST or Ponceau S of the membranes that similar amounts of the fusion proteins were present in samples These results integrin binding to talin and its ability to induce FAK which is with the that talin-FAK interaction plays a role in integrin-mediated The results suggest a possible association of FAK with a cytoskeletal protein association was in by and in by binding of recombinant FAK to immobilized cellular for direct association of talin to FAK was using talin from NIH 3T3 lysates in in binding These results suggest a possible mechanism for FAK activation and tyrosine phosphorylation by integrins the cytoplasmic domain of integrin β1 with talin and The binding of talin to the FAK is as as that to the wild type FAK that the possible association of talin with FAK is of FAK activation and is with the that talin is an mediator of FAK activation by integrins not a of FAK The potential association of FAK with talin may also to that of actin-cytoskeleton with cytochalasin block FAK phosphorylation in several K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1180) Google Scholar, 22Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar). FAK activation and tyrosine phosphorylation have been implicated as a point of convergence in the of encoding tyrosine and with receptors coupled to I. Rozengurt E. Cell. 1992; 71: 891-894Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 2Schaller M. Parsons J.T. Trends Cell Biol. 1993; 3: 258-262Abstract Full Text PDF PubMed Scopus (156) Google Scholar). The of diverse pathways are at the integrity of actin-cytoskeleton to a role in FAK responses to of and the cytochalasin block FAK phosphorylation in of K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1180) Google Scholar, 22Sinnett-Smith J. Zachary I. Valverde A.M. Rozengurt E. J. Biol. Chem. 1993; 268: 14261-14268Abstract Full Text PDF PubMed Google Scholar). Therefore, the actin cytoskeleton may be involved in diverse which is then to FAK by a of chicken integrin β1 mutants expressed in NIH 3T3 and J.M. Mol. Biol. Cell. PubMed Scopus Google Scholar) have recently shown in the correlated of FAK, and as induced by with The of proteins with integrin the of amino in the cytoplasmic domain of β1 These are with present suggesting a role for talin in FAK activation by integrins. talin not bind the GST fusion protein containing the cytoplasmic domain of integrin mutant Δ4 mutant as used by and which amino that its binding to the GST fusion protein containing the cytoplasmic domain of β1 integrin studies also that the mutant was localized to focal contacts E.E. Guan J-L. Trevithick J. Hynes R.O. Cell Regul. 1990; 1: 597-604Crossref PubMed Scopus (116) Google Scholar) induced FAK J.-L. Trevithick L.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar). A. Buck C. Duggan K. L. Google Scholar) have identified that amino of the cytoplasmic domain of β1 integrin bind to talin in vitro. sequence to amino and as and J.M. Mol. Biol. Cell. PubMed Scopus Google Scholar), amino from both talin binding site in of amino may the to association in may for the of the Δ4 mutant to FAK in J.-L. Trevithick L.E. Hynes R.O. Cell Regul. 1991; 2: 951-964Crossref PubMed Scopus (473) Google Scholar). We have identified amino in the carboxyl-terminal domain of FAK in necessary for talin These amino have also been previously to be necessary for FAK to focal J.D. Schaller M.D. Parsons J.T. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (358) Google Scholar). Therefore, talin may be of the FAK binding proteins for its to the focal interaction with talin may not be for FAK to focal contacts was not localized to focal J.D. Schaller M.D. Parsons J.T. J. Cell Biol. 1993; 123: 993-1005Crossref PubMed Scopus (358) Google Scholar), although bind talin in It is possible that interaction of FAK with protein in complexes in is necessary for its in the focal contacts for its activation and have recently shown that integrin clustering by a antibody on the beads induced of and FAK not other cytoskeletal proteins. It is unclear, however, which of FAK is for its association with FAK has also been shown to bind to cytoskeletal C.E. J.T. J. Cell Sci. 1994; PubMed Google Scholar). can interact with talin via vinculin is that FAK with talin via its binding to which does not bind J. Schaller, M. and Parsons, J. T. Mol. Biol. in with talin are in to the binding on FAK for various cytoskeletal proteins and to of interactions to the activation and tyrosine of We are to Dr. R. O. Hynes for antisera against talin and Dr. Q. Yu for monoclonal antibody and for critical of the and