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.

The focal adhesion kinase (FAK) has been Implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To gain insight into FAK function, we examined the potential interaction of FAK with intracellular signaline molecules contini the Src homology 2 domains. We report here the stable association of FAK with phosphatidylnositol 3-kinase (PI 3-kinase; EC 2.7.1.137) in NIH 3T3 monse flbroblasts. Evidence from several laboratories has shown that a cytoplasmic protein tyrosine kinase called focal adhesion kinase (FAK) is involved in the signal transduction pathways initiated by integrins on the cell surface (reviewed in refs. 5 and 6). FAK becomes rapidly phosphorylated following cell attachment to fibronectin-coated surfaces or integrin clustering by antibodies (7-13). Integrin clustering on the cell surface also stimulated the tyrosine kinase activity of FAK in both fibroblasts and platelets (10,14). Mutant analysis of the heterologous chicken integrin ,31 subunit expressed in NIH 3T3 mouse cells showed that the cytoplasmic sequences of (31 are necessary for inducing tyrosine phosphorylation of FAK (7). FAK has been localized to focal contacts where integrin aggregates after binding to ECM ligands, consistent with its role in initiating the signaling cascade by integrins (9,(11)(12)(13) Sequence analyses of cDNAs derived from several species revealed that FAK is highly conserved, suggesting that it has a critical role in cell regulation (11,13,18 Hildebrand et al. (20). The corresponding recombinant proteins were expressed by a baculovirus system in Spodopterafrugiperda (Sf21) insect cells, and lysates Abbreviations: anti-pY, anti-phosphotyrosine; ECM, extracellular matrix; FAK, focal adhesion kinase; GST, glutathione S-transferase; PI 3-kinase, phosphatidylinositol 3-kinase; SH2, Src homology 2. *To whom reprint requests should be addressed. 10148The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

We have previously shown that overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promoted their migration on fibronectin. This effect was dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. In this study, we have examined the role of PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant failed to promote cell migration although its association with Src was retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130Cas to FAK may not be sufficient for this event.