Focal adhesion kinase overexpression enhances Ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src

Cell adhesion to extracellular matrix proteins such as fibronectin (FN) triggers a number of intracellular signaling events including the increased tyrosine phosphorylation of the cytoplasmic focal adhesion protein-tyrosine kinase (PTK) and also the stimulation of the mitogen-activated protein kinase ERK2, Focal adhesion kinase (FAK) associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family PTKs and Grb2, respectively, To investigate the mechanisms by which FAR, c-Src, and Grb2 function in FN stimulated signaling events to ERK2, we expressed wild type and mutant forms of FAK in human 293 epithelial cells by transient transfection, FAK overexpression enhanced FN-stimulated activation of ERK2 similar to 4-fold, This was blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 was Ras-dependent. FN-stimulated c-Src PTK activity was enhanced by wild type FAR expression, whereas FN-stimulated activation of ERK2 was blocked by expression of the c-Src binding site Phe-397 mutant of FAR, Expression of the Grb2 binding site Phe-925 mutant of FAK enhanced activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK did not, Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhanced p130(Cas) association with FAR, She tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation, FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras, The inhibitory effects of Tyr-397 FAK expression show that FAR-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of an FAK-c-Src complex, and several of these can lead to Ras dependent ERK2 mitogen-activated protein kinase activation.