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Phosphorylation of Tyrosine 397 in Focal Adhesion Kinase Is Required for Binding Phosphatidylinositol 3-Kinase*

  • Hong-Chen Chen
    Affiliations
    Cancer Biology Laboratories, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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  • Paul A. Appeddu
    Footnotes
    Affiliations
    Cancer Biology Laboratories, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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  • Hiroko Isoda
    Affiliations
    Cancer Biology Laboratories, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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  • Jun-Lin Guan
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Cancer Biology Laboratories, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
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  • Author Footnotes
    * This research was supported by National Institutes of Health Grants GM48050 and GM52890 (to J.-L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    Present address: Center for Extracellular Matrix Biology, Texas A & M University, Houston, TX 77030.
Open AccessPublished:October 18, 1996DOI:https://doi.org/10.1074/jbc.271.42.26329
      We have shown previously that cell adhesion or platelet-derived growth factor (PDGF) promotes the in vivo association of focal adhesion kinase (FAK) with phosphatidylinositol (PI) 3-kinase. In vitro experiments indicated that this interaction was mediated by the p85 subunit of PI 3-kinase and dependent on the tyrosine phosphorylation of FAK. Here we report data suggesting that the major autophosphorylation site of FAK (Tyr-397) is the binding site for the SH2 domains of p85 in vitro and is also required for the association of FAK with PI 3-kinase in vivo. We also show that Tyr-397 is responsible for the increased FAK:PI 3-kinase association upon PDGF stimulation, implying that no additional site of FAK was involved in its binding to PI 3-kinase after PDGF stimulation. Finally, we present evidence that the interaction of PI 3-kinase with Tyr-397 of FAK stimulates its activity. Together, these results suggest that FAK activation and autophosphorylation at Tyr-397 may lead to its association with PI 3-kinase through the SH2 domains of p85, which can subsequently activate PI 3-kinase during cell adhesion.

      INTRODUCTION

      Focal adhesion kinase (FAK)
      The abbreviations used are: FAK
      focal adhesion kinase
      PI
      phosphatidylinositol
      GST
      glutathione S-transferase
      PDGF
      platelet-derived growth factor
      HA
      hemagglutinin
      kd
      kinase-defective.
      is a cytoplasmic tyrosine kinase involved in integrin-mediated signal transduction pathways (
      • Clark E.A.
      • Brugge J.S.
      ,
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ,
      • Juliano R.L.
      • Haskill S.
      ,
      • Zachary I.
      • Rozengurt E.
      ). In adherent cells, FAK colocalizes with integrins in focal contacts. FAK activation and tyrosine phosphorylation have been shown in a variety of cell types to be dependent on integrins binding to their extracellular ligands (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ). Furthermore, FAK-deficient mouse embryos generated by FAK gene knockout exhibit a general deficiency in mesoderm which is very similar to what is seen in fibronectin-deficient mice (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      ,
      • Furuta Y.
      • Ilic D.
      • Kanazawa S.
      • Takeda N.
      • Yamamoto T.
      • Aizawa S.
      ,
      • George E.L.
      • Georges-Labouesse E.N.
      • Patel-King R.S.
      • Rayburn H.
      • Hynes R.O.
      ). These results complement the observations in cell culture systems which suggest a unique role of FAK in signaling pathways initiated by integrin binding to fibronectin.
      Recent studies have suggested that complexes of FAK with other cellular proteins may play important roles in signal transduction by integrins (
      • Clark E.A.
      • Brugge J.S.
      ). In fibroblasts, integrin engagement promotes the association of FAK with both c-Src and the adaptor protein Grb2 (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ) and results in activation of mitogen-activated protein kinase (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ,
      • Chen Q.
      • Kinch M.S.
      • Lin T.H.
      • Burridge K.
      • Juliano R.L.
      ,
      • Zhu X.
      • Assoian R.K.
      ). Tyrosine residue 397 has been identified as the major site of FAK autophosphorylation (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.X.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Chan P.-Y.
      • Kanner S.B.
      • Whitney G.
      • Aruffo A.
      ) and the binding site for the SH2 domain of Src family kinases (
      • Cobb B.S.
      • Schaller M.D.
      • Leu T.-H.
      • Parsons J.T.
      ,
      • Xing Z.
      • Chen H.-C.
      • Nowlen J.K.
      • Taylor S.
      • Shalloway D.
      • Guan J.-L.
      ). Tyr-925 of FAK, which is phosphorylated by Src in vitro, has been identified as the binding site for the SH2 domain of Grb2 (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ). Based on these results, it has been proposed that the interaction of FAK with Src and Grb2 can link integrin-initiated signals to the Ras/mitogen-activated protein kinase pathway (
      • Clark E.A.
      • Brugge J.S.
      ,
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ).
      Studies from both our laboratory (
      • Chen H.-C.
      • Guan J.-L.
      ) and that of Guinebault et al. (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ) have shown that FAK can also bind to phosphatidylinositol (PI) 3-kinase upon ligand engagement of integrins. PI 3-kinase phosphorylates at the D-3 position of the inositol ring of phosphatidylinositides to produce PI(
      • Juliano R.L.
      • Haskill S.
      )P, PI(3,4)P2, and PI(3,4,5)P3, which are potential second messengers that can activate protein kinase Cζ (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ) or δ, ϵ, and η (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ), and the related c-Akt protein kinase (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ). PI 3-kinase is a heterodimer composed of a 110-kDa catalytic p110 subunit and an 85-kDa regulatory p85 subunit. The latter contains an SH3 domain and two SH2 domains (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ). Binding of PI 3-kinase to FAK upon cell adhesion is mediated by the p85 subunit (
      • Chen H.-C.
      • Guan J.-L.
      ,
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ). Guinebault et al. (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ) have demonstrated that a GST fusion protein containing the p85 SH3 domain can bind to FAK as well as a synthetic peptide derived from a proline-rich region of FAK, indicating a role of the SH3 domain of p85 in FAK association with PI 3-kinase. However, several lines of evidence suggest that the p85 SH2 domains may also be responsible for the association. First, cell adhesion stimulates FAK association with PI 3-kinase in vivo (
      • Chen H.-C.
      • Guan J.-L.
      ) as well as its binding to the NH2-terminal SH2 domain of p85 in vitro (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ), concomitant with FAK activation and autophosphorylation. Second, autophosphorylation of recombinant FAK in vitro increases its binding to PI 3-kinase (
      • Chen H.-C.
      • Guan J.-L.
      ). Finally, FAK contains three tyrosines (Tyr-180, Tyr-652, and Tyr-950) in the YXXM motif for optimal binding to p85 SH2 domains (
      • Songyang 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.
      ), although it is not clear whether any of these sites is phosphorylated in addition to Tyr-397.
      In this paper, we have investigated further the nature of the interaction between FAK and PI 3-kinase. Our results suggest that the major autophosphorylation site of FAK (Tyr-397) is the binding site for the SH2 domains of p85 and is responsible for the in vivo association of FAK with PI 3-kinase, even though it is not in the typical YXXM motif for binding p85 SH2 domains. We have also shown that Tyr-397 is responsible for the increased FAK:PI 3-kinase association upon platelet-derived growth factor (PDGF) stimulation (
      • Chen H.-C.
      • Guan J.-L.
      ), implying that no additional site of FAK was involved in its binding to PI 3-kinase after PDGF stimulation. Lastly, we present evidence that interaction of PI 3-kinase with Tyr-397 of FAK stimulates its activity. Together, these results suggest that FAK activation and autophosphorylation at Tyr-397 may lead to its association with PI 3-kinase through the SH2 domains of p85, which can subsequently activate PI 3-kinase during cell adhesion.

      RESULTS

      To determine the potential p85 binding sites on FAK, we first performed a series of in vitro binding assays using recombinant FAK and its mutants produced in insect Sf21 cells via baculovirus expression systems. The wild type FAK and mutants kd, ΔN, ΔC1, ΔC2, and ΔC3 were as described previously (
      • Chen H.-C.
      • Appeddu P.A.
      • Parsons J.T.
      • Hildebrand J.D.
      • Schaller M.D.
      • Guan J.-L.
      ). The mutant Y397F was obtained by using similar methods, as described under “Experimental Procedures.” All recombinant FAK proteins were tagged with an epitope derived from the influenza virus HA sequence (YPYDVPDYA, HA epitope), which allows detection of the recombinant proteins by the monoclonal antibody 12CA5 (26; see Fig. 1A).
      Figure thumbnail gr1
      Fig. 1In vitro association of recombinant FAK with immobilized GST-p85 and its SH2 domains. Panel A, aliquots of Sf21 cell lysates containing recombinant FAK or its mutants were analyzed by Western blotting using 12CA5 to verify that similar amounts of input were used in binding assays for all samples. Panels B-D, GST fusion protein GST-p85 (panel B), GST-p85·NSH2 (panel C), or GST-p85·CSH2 (panel D) was immobilized on glutathione-agarose beads and then incubated with Sf21 cell lysates containing recombinant FAK proteins. The bound proteins were eluted in SDS sample buffer and analyzed by Western blotting with 12CA5.
      In vitro binding assays were performed using cell lysates prepared from Sf21 cells infected with various recombinant viruses encoding HA epitope-tagged FAK or its mutants. An aliquot of each sample was analyzed by Western blotting with 12CA5 to show that similar amounts of recombinant proteins were used in the binding assays (Fig. 1A). The remaining samples were incubated with immobilized GST fusion proteins containing p85 (GST-p85) or its NH2- or COOH-terminal SH2 domains (GST-p85·NSH2 or GST-p85·CSH2). After washing, the bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by Western blotting with 12CA5. As shown in Fig. 1, wild type FAK and all four FAK deletion mutants (ΔN, ΔC1, ΔC2, and ΔC3) bound GST-p85 (panel B) as well as GST-p85·NSH2 and GST-p85·CSH2 (panels C and D, respectively). In contrast, the FAK mutant kd or Y397F mutant did not bind GST fusion proteins containing p85 or its two individual SH2 domains (panels B-D). The Y397F mutant bound the SH2 domain of Grb2 and had tyrosine kinase activity comparable to that of wild type FAK (data not shown), rendering it unlikely that the point mutation of Tyr-397 to Phe caused an overall conformational change leading to the inability of Y397F mutant to interact with p85. None of the recombinant FAK proteins bound to GST alone (data not shown). Taken together, these results suggested that FAK could associate with p85 by binding to either of its NH2- or COOH-terminal SH2 domain. They also showed that kinase activity and Tyr-397 were required for FAK binding to p85 domains, suggesting that autophosphorylated Tyr-397 bound to these SH2 domains.
      FAK contains three tyrosines (Tyr-180, Tyr-652, and Tyr-950) in the YXXM motif described for optimal binding to p85 SH2 domains (
      • Songyang 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.
      ), although it is not clear whether any of these sites is phosphorylated in addition to Tyr-397. Results shown in Fig. 1 indicated that neither Tyr-180 nor Tyr-950 was responsible for FAK binding to p85 because FAK mutants ΔN (lacking Tyr-180) and ΔC2 (lacking Tyr-950) bound to GST-p85 as effectively as wild type FAK. To test whether Y652 mediates FAK binding to p85, a FAK mutant Y652F (Tyr-652 mutated to Phe) was generated, transiently expressed in 293 cells, and used for in vitro binding assays shown in Fig. 2. Panel A shows that similar expression levels of the HA epitope-tagged wild type FAK, Y397F, and Y652F mutants were obtained from 293 cells. Panel B shows that wild type FAK bound GST-p85 but Y397F mutant did not, as expected. The Y652F mutant bound to GST-p85 as efficiently as the wild type FAK, suggesting that Tyr-652 was not involved in FAK binding to p85. Therefore, none of the three tyrosine residues of FAK in YXXM motif was responsible for FAK binding to p85 in vitro.
      Figure thumbnail gr2
      Fig. 2In vitro association of GST-p85 with FAK and mutants expressed in 293 cells. Lysates were prepared from 293 cells that had been transfected with expression plasmids encoding the HA epitope-tagged FAK (wt), Y397F or Y652F mutant, or pKH3 vector alone (control), as indicated. An aliquot from each sample was analyzed by Western blotting using 12CA5 to verify similar levels of expression of FAK and its mutants (panel A). The remaining portion of each sample was incubated with immobilized GST-p85. After washing, the bound proteins were resolved on SDS-polyacrylamide gel electrophoresis and detected by Western blotting with 12CA5 (panel B).
      To examine the specificity of the interaction of p85 with phosphorylated Tyr-397 of FAK, a synthetic phosphopeptide pY397 (12-mer containing phosphorylated Tyr-397 and its flanking sequences) was tested for its ability to inhibit the binding of recombinant FAK to GST-p85. As shown in Fig. 3A, the phosphopeptide pY397 inhibited FAK binding to GST-p85 or GST-p85·NSH2. It had no effect on FAK binding to GST-Grb2·SH2, which is mediated by Tyr-925 (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ). These results confirmed that the phosphorylated Tyr-397 of FAK was responsible for its interaction with p85 via binding to the SH2 domains of p85.
      Figure thumbnail gr3
      Fig. 3Inhibition of FAK-p85 binding by a phosphopeptide containing Tyr-397 of FAK. Panel A, immobilized GST fusion protein GST-p85·NSH2, GST-p85, GST-Src·SH2, or GST-Grb2·SH2 was preincubated with (+) or without (−) 100 μM pY397, as indicated. The protein was then used for binding assays for association with recombinant FAK in Sf21 cell lysates as described in the legend except that 100 μM pY397 was present (+) or absent (−) in the incubations. Panel B, similar peptide competition experiments were performed for FAK binding to GST-p85 (top panel) and GST-Src·SH2 (bottom panel) as described in panel A except that increasing concentrations of the phosphopeptide pY397 (0-200 μM) were used as indicated. A peptide (200 μM) corresponding to the COOH-terminal 15 residues of FAK served as a negative control.
      Phosphorylation of Tyr-397 in FAK has been shown to be responsible for its association with Src through its SH2 domain (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.X.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Eide B.L.
      • Turck C.W.
      • Escobedo J.A.
      ). Indeed, the phosphopeptide pY397 also inhibited FAK binding to GST fusion protein containing the Src SH2 domain (GST-Src·SH2; Fig. 3A). Fig. 3B shows that pY397 inhibited the binding of recombinant FAK to GST-p85 or GST-Src·SH2 in a dose-dependent manner. The IC50 to inhibit FAK:GST-p85 and FAK:GST-Src·SH2 associations were approximately 10 and 3 μM, respectively. A control peptide corresponding to the carboxyl 15 residues of FAK did not inhibit FAK binding to either GST-p85 or GST-Src·SH2 even at 200 μM.
      From our in vitro data to this point, it appeared that phosphorylated Tyr-397 in FAK was responsible for FAK binding to PI 3-kinase through the SH2 domains of p85. To test if this was also the case in vivo, we established NIH 3T3 cell lines, which stably expressed HA epitope-tagged wild type FAK and three mutants (kd, Y397F, and Y652F), as described under “Experimental Procedures.” Association of PI 3-kinase with the transfected FAK and its mutants was assessed by immunoprecipitation with 12CA5 followed by PI 3-kinase activity assays, as described previously (
      • Chen H.-C.
      • Guan J.-L.
      ). Fig. 4A shows that similar amounts of HA epitope-tagged FAK and its mutants were present in all immunoprecipitates with the exception of cells transfected with pKH3 alone. Fig. 4B shows that PI 3-kinase was associated with wild type FAK and the Y652F mutant but not Y397F or kd mutants. Fig. 4C shows that PDGF increased the quantity of PI 3-kinase activity associated with wild type FAK and the Y652F mutant, consistent with our previous observation that PDGF could specifically stimulate the interaction of FAK with PI 3-kinase in NIH 3T3 cells (
      • Chen H.-C.
      • Guan J.-L.
      ). However, no PI 3-kinase activity was detected in association with either the kd or Y397F mutant, even after PDGF stimulation. Therefore, phosphorylation of Tyr-397 in FAK was also required for its association with PI 3-kinase in vivo in response to both cell adhesion and PDGF stimulation. Furthermore, these results indicated that other regions of FAK besides Tyr-397 were not involved in the increased FAK:PI 3-kinase association in response to PDGF.
      Figure thumbnail gr4
      Fig. 4In vivo association of PI 3-kinase with FAK and its mutants. Lysates were prepared from NIH 3T3 cells that stably expressed HA epitope-tagged FAK (wt), kd, Y397F or Y652F mutant, or control cells transfected with pKH3 vector alone (control), as indicated. They were immunoprecipitated by 12CA5 and divided into two portions, as described under “Experimental Procedures.” Panel A, one aliquot was analyzed by Western blotting using 12CA5 to verify that similar amounts of HA epitope-tagged FAK and its mutants were present in the immune complexes. Panel B, the other part was assayed for the associated PI 3-kinase activity, as described under “Experimental Procedures.” The locations of the origin (Ori) and phosphatidylinositol 3-phosphate (PIP) are indicated on the right. Panel C, various NIH 3T3 cell clones were serum-starved and treated with (+) or without (−) 25 μg/ml PDGF for 10 min, as indicated. The cells were then lysed, and HA epitope-tagged FAK proteins were immunoprecipitated using 12CA5. The immune complexes were assayed for PI 3-kinase activities as described under “Experimental Procedures.” The location of phosphatidylinositol 3-phosphate is indicated on the right.
      To examine the potential functional consequences of FAK association with PI 3-kinase, we tested if the binding of the phosphopeptide pY397 to p85 could affect PI 3-kinase activity. Cellular PI 3-kinase was immunoprecipitated from NIH 3T3 cells using polyclonal anti-p85 and assayed for its activity in the presence or absence of pY397 or the control peptides. Fig. 5 shows that PI 3-kinase activity was increased 2-3-fold in the presence of pY397 but was not affected by the control peptide. These results suggested that association of FAK with PI 3-kinase might result in the activation of PI 3-kinase.
      Figure thumbnail gr5
      Fig. 5Effect of phosphopeptide pY397 on PI 3-kinase activity. Cellular PI 3-kinase was immunoprecipitated by rabbit anti-p85 serum from NIH 3T3 cells. The immunoprecipitates were divided into four equal portions and assayed for PI 3-kinase activities in the presence (+) or absence (−) of the phosphopeptide pY397 or the control peptide as described under “Experimental Procedures.” The locations of the origin (Ori) and phosphatidylinositol 3-phosphate (PIP) are indicated on the right.

      DISCUSSION

      In mammalian cells, PI 3-kinase has been implicated in signal transduction pathways triggered by a variety of cell surface receptors (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Grazizni A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ,
      • Bjorge J.D.
      • Chan T.
      • Antczak M.
      • Kung H.
      • Fujita D.J.
      ,
      • Giorgetti S.
      • Ballotti R.
      • Kowalski-Chauvel A.
      • Tartare S.
      • Van Obberghen E.
      ). For example, PI 3-kinase interacts with activated growth factor receptor tyrosine kinases upon ligand binding (
      • Valis M.
      • Kazlauskas A.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp-Lorenzino L.
      • Tan J.C.
      • Besmer P.
      ). This association has been demonstrated to be mediated by binding of autophosphorylated tyrosine residues in the receptor tyrosine kinase to the SH2 domains of p85 subunit of PI 3-kinase (
      • Escobedo J.A.
      • Kaplan D.R.
      • Kavanaugh W.M.
      • Turck C.W.
      • Williams L.T.
      ,
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ,
      • Lev S.
      • Givol D.
      • Yarden Y.
      ,
      • Reedijk M.
      • Liu X.
      • van der Geer P.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ). Recent studies have also shown that PI 3-kinase associates with activated FAK in response to integrin binding to its ligands (
      • Chen H.-C.
      • Guan J.-L.
      ,
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ). However, less is known about the nature of FAK:PI 3-kinase interactions or the binding sites on FAK. In this paper we demonstrated that FAK kinase activity and the major FAK autophosphorylation site Tyr-397 were required for FAK association with PI 3-kinase. A mutation converting Tyr-397 to Phe abolished recombinant FAK binding to p85 or its SH2 domains in vitro. Consistent with this, a phosphopeptide corresponding to Tyr-397 and its flanking sequences (pY397) inhibited the binding of FAK to p85. Furthermore, the Tyr-397 → Phe mutation also prevented FAK association with PI 3-kinase in vivo in response to either cell adhesion or PDGF stimulation. Finally, we observed that the binding of the phosphopeptide pY397 to p85 resulted in the activation of PI 3-kinase by 2-3-fold (Fig. 5). Together, these results suggested that FAK activation and autophosphorylation at Tyr-397 might lead to its association with PI 3-kinase through the SH2 domains of p85, which could subsequently activate PI 3-kinase during cell adhesion.
      Guinebault et al. (
      • Guinebault C.
      • Payrastre B.
      • Racaud-Sultan C.
      • Mazarguil H.
      • Breton M.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      ) have observed that the SH3 domain of p85 could bind to a proline-rich sequence in FAK (amino acids 875-880) using in vitro binding experiments, suggesting that the SH3 domain of p85 may play a role in FAK:PI 3-kinase association. Similarly, we also observed that the GST fusion protein containing the p85 SH3 domain was able to bind to recombinant FAK in vitro (data not shown). However, this proline-rich sequence appeared not to be necessary for FAK binding to p85 itself because the ΔC2 mutant (lacking amino acids 875-880) bound to GST-p85 as effectively as the wild type FAK (Fig. 1). Indeed, our results here strongly suggested that the primary FAK binding sites in PI 3-kinase were the SH2 domains of p85. Nevertheless, our results did not rule out the possibility that the p85 SH3 domain binding to the proline-rich sequences of FAK might also participate in FAK:PI 3-kinase association in response to certain stimuli in different cell types.
      Our identification of Tyr-397 in FAK as its primary binding site for p85 SH2 domains was surprising because Tyr-397 was not in the YXXM motif for optimal binding to p85 SH2 domains reported previously (
      • Songyang 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.
      ). Although there are three tyrosine residues in FAK within the YXXM motif, our results here clearly ruled out their involvement in FAK binding to p85 (Fig. 1, Fig. 2). Furthermore, there are several precedents where tyrosine residues not in the YXXM motif have been reported to bind to the SH2 domains of p85. These include YVXV in the hepatocyte growth factor receptor (
      • Ponzetto C.
      • Bardeli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ), YVNA in the vascular endothelial growth factor receptor (
      • Cunningham S.A.
      • Waxham M.N.
      • Arrate P.M.
      • Brock T.A.
      ), and YVAC in the erythropoietin receptor (
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ). One possible explanation for these exceptions is provided by recent results showing that the residues amino-terminal to the phosphotyrosine may also contribute to specific binding of phosphotyrosine to SH2 domains (
      • Songyang Z.
      • Margolis B.
      • Chaudhuri M.
      • Shoelson S.E.
      • Cantley L.C.
      ). Using a degenerate library of peptides in which residues both NH2- and COOH-terminal to the phosphotyrosine are varied, it has been found that the optimal motif for binding the p85 NH2-terminal SH2 domain is EDDpYVEM and that the preference for Met at the +3 position is not as strong as that when a peptide library varying only residues COOH-terminal to the phosphotyrosine is used (
      • Songyang 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.
      ). An examination of FAK sequences revealed that sequences flanking Tyr-397 (TDDpYAEI) conformed well with the EDDpYVEM motif for binding the p85 NH2-terminal SH2 domain. Two aspartic acid and one glutamic acid residue were identical to those in the motif at the −2, −1, and +2 positions, respectively. Furthermore the general pattern of pY-hydrophobic-hydrophilic-hydrophobic residues for binding p85 SH2 domains (
      • Songyang 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.
      ) was also maintained in Tyr397-Ala-Glu-Ile of FAK.
      Tyr-397 in FAK has previously been identified as the binding site for Src through its SH2 domain (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.X.
      • Vines R.R.
      • Parsons J.T.
      ,
      • Eide B.L.
      • Turck C.W.
      • Escobedo J.A.
      ). Furthermore, significantly more FAK:Src association has been detected in v-Src transformed NIH 3T3 cells than that in normal NIH 3T3 cells (
      • Xing Z.
      • Chen H.-C.
      • Nowlen J.K.
      • Taylor S.
      • Shalloway D.
      • Guan J.-L.
      ). However, we did not detect any increases in FAK:PI 3-kinase association in v-Src-transformed NIH 3T3 cells compared with normal NIH 3T3 cells (data not shown), which suggested that PI 3-kinase binding to FAK was likely to be independent of the Src:FAK interaction. In addition, the identification of Tyr-397 for both PI 3-kinase and Src binding suggested the interesting possibility that FAK might exist in different cellular pools complexed with either Src or PI 3-kinase. In v-Src-transformed CEF cells, at least 80% of FAK is complexed with v-Src (
      • Cobb B.S.
      • Schaller M.D.
      • Leu T.-H.
      • Parsons J.T.
      ). Experiments are in progress to determine the fraction of FAK associated with c-Src or PI 3-kinase in NIH 3T3 cells under various conditions.
      Recent studies have suggested that FAK is involved in an integrin-triggered signaling pathway leading to cell migration. Ilic et al. (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      ) have shown that embryonic cells from FAK-deficient mice exhibited a decreased migration on fibronectin, which was suggested to be responsible for a defect in mesodermal migration resulting in an embryonic lethal phenotype of the FAK-deficient mice. We have found that overexpression of FAK in Chinese hamster ovary cells caused a significant increase in cell migration on fibronectin and that autophosphorylation of FAK at Tyr-397 and its subsequent association with Src and Fyn were correlated with this increased migration (
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ). Identification of Tyr-397 as the binding site for p85 here raised the possibility that FAK:PI 3-kinase as well as FAK:Src bindings may be important for downstream signaling events leading to cell migration.
      Association of FAK with p85 may lead to activation of PI 3-kinase. We found that incubation of a phosphopeptide containing Tyr-397 and its flanking residues with PI 3-kinase increased its activity by 2-3-fold in vitro (Fig. 5). This was comparable to the level of activation of PI 3-kinase by phosphopeptides derived from receptor tyrosine kinases which are believed to activate PI 3-kinase in vivo (
      • Backer J.M.
      • Myers M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis D.
      • Skolnik E.Y.
      • Schlessinger J.
      • Write M.F.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chaudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ). These results and our previous observation that FAK could phosphorylate p85 in vitro (
      • Chen H.-C.
      • Guan J.-L.
      ) suggested that PI 3-kinase may be an important downstream effector of FAK in integrin signaling leading to cell migration. In preliminary experiments, we have found that wortmannin, a specific inhibitor of PI 3-kinase, could reduce cell migration on fibronectin of Chinese hamster ovary cells overexpressing FAK (data not shown). Identification of the PI 3-kinase binding site on FAK should allow us to dissect the relative contributions of PI 3-kinase and other FAK-binding proteins to downstream signaling events leading to cell migration.

      Acknowledgments

      We are grateful to Dr. I. Macara for expression vector pKH3; Dr. L. C. Cantley for plasmid pGEX-p85; Dr. T. Pawson for plasmids pGEX-p85·NSH2, pGEX-p85·CSH2, and pGEX-Grb2·SH2; Drs. Bibbins and Varmus for plasmid pGEX-Src·SH2; Jared Cohen for construction of the Y397F mutant; Korena Kosco for construction of pKH3-FAK; and Laurie Warner for technical help. We thank Dr. Steve Taylor and Michael Dolenga for a critical reading of the manuscript and helpful comments.

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