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* This research was supported by National Science Council, Taiwan, Grant NSC88-2311-B005-027 (to H.-C. C.) and grants from the National Institutes of Health (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. § These authors contributed equally to this work. ‖ Is an established investigator of the American Heart Association.
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.
). p85 contains an SH3 domain and two SH2 domains. This kinase phosphorylates the D-3 position of the inositide ring of phosphatidylinositol and its derivatives, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (
) in NIH 3T3 cells. The significance of this interaction in cellular processes is unknown.
In this report, we have found that a FAK mutant capable of binding to Src, but not to PI3K, failed to stimulate cell migration and that inhibition of PI3K decreases cell migration. Taken together, these results strongly implicate a role for PI3K in the facilitation of FAK-promoted cell migration and suggest that the binding of Src and p130Cas to FAK may not be sufficient for this process.
It is known that PI3K plays an important role in growth factor-induced cell migration (
). To examine the putative role of PI3K in FAK-promoted cell migration, CHO cells overexpressing WT FAK and control cells (Neo) were subjected to cell migration assays in the presence of specific PI3K inhibitors (Fig.1). Treatment of WT cells with either PI3K inhibitor resulted in an inhibition of cell migration in a dose-dependent manner. Furthermore, in the presence of either 100 nm wortmannin or 75 μm LY294002, the majority (∼80%) of cell migration promoted by FAK overexpression in WT cells was inhibited, with little effect on control cell migration. These results demonstrate that PI3K plays a role downstream of FAK in promoting CHO cell migration on fibronectin. However, from these results, it was not clear whether FAK-promoted cell migration is dependent on PI3K, either through direct binding at Tyr-397 of FAK or through another FAK-promoted signaling pathway.
To determine whether the direct binding of PI3K to FAK is a prerequisite for its function in FAK-promoted cell migration, a FAK mutant deficient only in PI3K binding was generated by a substitution of Asp-395 with Ala (see below). Songyang et al. (
) suggested that residues both N- and C-terminal to the phosphotyrosine may contribute to the specific binding of the SH2 domain to its cognate phosphotyrosine and showed that the optimal binding sequence for the p85 N-terminal SH2 domain is EEDpYVEM. An examination of FAK sequences revealed that sequences flanking Tyr-397 (TDDpYAEI) conformed well with the binding motif for the p85 N-terminal SH2 domain. Based on these observations, two Asp residues upstream of Tyr-397 were mutated either singly or in combination to generate a PI3K binding-defective FAK mutant. These FAK mutants were transiently expressed in HEK 293 cells, and their ability to bind the SH2 domains of Src and p85 was examinedin vitro (Fig. 2A). Similar to the mutation in Tyr-397, the mutation converting Asp-396 (or in combination with Asp-395) to Ala abolished FAK binding to the SH2 domains of Src and p85. Interestingly, a mutation in Asp-395 inhibited FAK binding to the SH2 domain of p85 but not Src. To examine if the FAK D395A mutant also selectively binds to Src but not p85 in intact cells, HEK 293 cells were transiently co-transfected with expression plasmids encoding HA epitope-tagged FAK or its mutants, c-Src, and p85. Two days after transfection, cells were lysed, and the association of FAK with Src or p85 was detected by co-immunoprecipitation (Fig. 2B). Consistent with the results from in vitro binding experiments, the mutation in Asp-396 abolished FAK association with both Src and p85, and importantly, the mutation in Asp-395 only prevented FAK association with p85 but not Src.
To examine the effects of FAK mutations in Asp-395 and Asp-396 on its tyrosine phosphorylation and in vitro autophosphorylation, HA epitope-tagged WT FAK or its mutants were transiently expressed in HEK 293 cells, immunoprecipitated with anti-HA, and subjected to immunoblotting with anti-PY or to an in vitro kinase assay (Fig. 3A). The tyrosine phosphorylation and in vitro autophosphorylation of FAK mutants containing a substitution of Asp-396 with Ala were as low as those of the Y397F mutant, indicating that Asp-396 is essential for the phosphorylation event upon Tyr-397. This also reflects the inability of the FAK D396A mutant to bind the SH2 domains of Src and p85. In contrast, the tyrosine phosphorylation and in vitroautophosphorylation of FAK D395A mutant remained comparable with the WT. These results indicated that the inability of the D395A mutant to bind PI3K was most likely not because of an overall conformational change. To exclude the possibility that the mutation in Asp-395 causes a subtle change in protein conformation and thereby affects the binding of FAK to other proteins, the capacity of FAK D395A to bind to the SH3 domain of p130Cas was examined in vitro (Fig.3B). The result clearly showed that the ability of FAK to bind the SH3 domain of p130Cas was not affected by mutating Asp-395. Together, these data suggest that the inability of FAK D395A to bind p85 is indeed because of the preference of the p85 SH2 domain for an aspartic acid at residue 395 in FAK.
To determine whether PI3K binding is required for FAK-promoted cell migration, stable CHO cell lines overexpressing FAK D395A were established. The expression of FAK D395A was examined by immunoblotting with anti-FAK or KT3 (Fig.4A). The expression levels of ectopic WT FAK and D395A mutant in CHO cells were comparable and severalfold higher than that of endogenous FAK. To examine the association of endogenous PI3K with WT and mutant FAK in CHO cells, ectopically expressed FAK proteins were immunoprecipitated by mAb KT3, and the immunocomplexes were subjected to an in vitro PI3K activity assay (Fig. 4B). Approximately 10% of total cellular PI3K activity were found to associate with ectopic WT FAK (data not shown). As expected, the levels of PI3K activity associated with WT FAK and P712/715A mutant were similar. Surprisingly, approximately 15% of PI3K activity that WT FAKs had were co-precipitated with Y397F and D395A mutants. The cell migration assays indicated a marked increase (∼2.5-fold) in cell migration for WT cells compared with both Y397F and control (Neo) cells, which were similar in their rates of migration. Interestingly, both D395A clones failed to promote cell migration (Fig. 4C). These results together suggest that the residual binding of PI3K to FAK Y397F or D395A mutant in CHO cells is not sufficient for promoting migration. Moreover, adhesion assays indicated that all clones had similar adhesive strength to fibronectin (data not shown), indicating that the decrease in migration observed in D395A clones was not because of an alteration in cell adhesion.
We have previously demonstrated that phosphorylation of Tyr-397 in FAK is required for binding the SH2 domains of Src and PI3K (
). Thus, it is unclear whether the inability of FAK Y397F to promote cell migration is because of its inability to bind Src and/or PI3K. To clarify this, the association of FAK D395A with c-Src in CHO cells was examined. Both WT FAK and FAK D395A but not FAK Y397F co-precipitated with c-Src in CHO cells (Fig. 5A). We have previously demonstrated that the association of p130Caswith FAK and FAK-promoted phosphorylation of p130Cas by Src are critical for FAK-promoted migration (
). We examined the phosphorylation state of p130Cas in FAK D395A cells as an indicator of a FAK/Src-depending cell migration pathway as well as an indirect indicator of FAK/p130Cas association. Consistent with prior experiments (
). FAK D395A was able to promote p130Cas phosphorylation at an estimated 2-fold level above WT FAK, indicating that the FAK/Src specific pathway is still functioning in the D395A mutant. Taken together, these results strongly suggest that PI3K binding to FAK is required for FAK to promote cell migration and that FAK:Src association alone may be insufficient for this cellular function.
In this report, we examined the role of PI3K in FAK-promoted cell migration. First we showed that two PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Our results are consistent with other work showing the importance of PI3K in increased cell motility stimulated by growth factors such as PDGF and hepatocyte growth factor (
). However, these data do not indicate whether PI3K association with FAK is required for increased cell migration and cannot rule out the involvement of PI3K in a FAK-independent migration pathway. To address this, we next established stable cell lines overexpressing FAK and a mutant defective in PI3K binding. Using this system, we showed that the PI3K binding-deficient mutant failed to promote cell migration toward fibronectin. These results strongly suggest that FAK:PI3K association is required for FAK-promoted cell migration.
We have previously identified that the autophosphorylation site Tyr-397 is the primary site for PI3K binding (
). We show here that the introduction of a mutation in Asp-395 completely abolishes FAK association with PI3K SH2 domain but not Src SH2 domain in vitro. The D395A mutant transiently expressed in HEK 293 cells also loses its ability to bind to intact p85 but not Src. However, in CHO cells some residual binding of PI3K to Y397F and D395A mutants can be detected, indicating that other mechanisms such as SH3 interaction may also be involved. This result is consistent with the previous report that the SH3 domain of p85 could bind to a proline-rich region (amino acids 875–884) of FAK in vitro (
) and suggest that an SH3 interaction besides SH2 interaction might also participate in FAK:PI3K interactions in response to certain stimuli in different cell types. Furthermore, together with our observation that FAK D395A mutant retained its tyrosine phosphorylation and in vitrophosphorylation to the same level as WT FAK, it appears that Asp-395 makes a major contribution for specific binding of the SH2 domains of PI3K to FAK. This finding also supports the notion that residues N-terminal of phosphotyrosine are critical for specific binding to certain SH2 domain-containing proteins.
Previous results from our laboratory showed that a FAK mutant (P712/715A) deficient in p130Cas binding failed to promote cell migration (
). In this report, we showed that FAK D395A deficient in PI3K binding also failed to increase cell migration. Although a residual binding of PI3K to D395A mutant was detected in CHO cells, it was apparently not sufficient for promoting cell migration. In fact, an analogous situation has also been described that a residual binding of p130Cas to FAK P712/715A mutant possibly mediated by a second proline-rich region (amino acids 875–884) on FAK does not allow for FAK-promoted migration (
). These results together suggest that thresholds of signals transmitted independently through the associations of p130Cas and PI3K with FAK have to be reached for enhanced cell migration. Lack of either one will not turn on an “intracellular machinery” for the induction of cell migration. Another possible interpretation is that the inability of FAK D395A to promote cell migration is because of a defect in p130Cas binding. This defect may result from a subtle conformation change by the D395A substitution, or a mutually dependent binding of PI3K and p130Cas to FAK. The latter possibility was excluded by our observation that the D395A mutant retained its ability to bind the SH3 domain of p130Casin vitro and promote p130Cas phosphorylation in vivo.
The tyrosine phosphorylation of p130Cas and its subsequent association with the adapter protein Crk have been shown to play an important role in promoting cell migration (
). Here, we have shown that expression of FAK D395A increased p130Cas tyrosine phosphorylation at a roughly 2-fold level above WT FAK in CHO cells. Although the reason for this elevation in p130Cas phosphorylation is unclear, this result supports our conclusion that p130Cas binding and phosphorylation is necessary but not sufficient for FAK-promoted cell migration. Furthermore, several studies have shown that Src is important for phosphorylation of p130Cas (
). Because FAK D395A still binds to Src, our results also suggest that FAK:Src association alone is not sufficient for the enhancement of cell migration. However, we cannot exclude the necessity of Src in FAK-promoted cell migration because of its function, at least, in phosphorylating p130Cas. Therefore, it is likely that FAK-promoted cell migration depends on the coordinate regulation of a number of these signaling events involving PI3K, p130Cas, and Src.
We have previously shown that FAK:PI3K association is enhanced by cell adhesion to ECM proteins (
). In this report, we have shown that this association is likely to be involved in cell migration upon cell adhesion to ECM proteins. However, it is not clear if this interaction is also involved in PDGF-stimulated cell migration. Furthermore, we do not know if FAK generally participates in growth factor-induced cell motility using a mechanism similar to that in ECM protein-induced cell migration. It is known that the tyrosine phosphorylation of FAK is stimulated by a number of growth factors (
), it is possible that phosphorylated FAK recruits the same set of intracellular signaling molecules to facilitate cell migration. Answers to these questions will be interesting, and experiments are now in progress to test these possibilities.
We thank Dr. L. C. Cantley for the cDNA encoding the p85 subunit of PI3K and Dr. D. Shalloway for the expression vector encoding c-Src. We also thank Dr. D. C. Han for the construction of pGEX-Cas.SH3 and P.-C. Chan for the critical reading of this manuscript.