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* 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.
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.
). 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 (
) 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(
) 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 (
), 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 (
), 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.
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 (
). 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).
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 (
), 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.
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 (
). 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 (
). 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 (
). 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.
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.
In mammalian cells, PI 3-kinase has been implicated in signal transduction pathways triggered by a variety of cell surface receptors (
). 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.
) 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
) 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 (
). 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 (
) 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.
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.