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 in creased tyrosine phosphorylation of the cytoplasmic focal adhesion pro tein-tyrosine kinase (PTK) and also the stimulation of the mitogen-act ivated protein kinase ERK2, Focal adhesion kinase (FAK) associates wit h integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr- 397 and Tyr-925 promotes the binding of Src family PTKs and Grb2, resp ectively, 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 transien t transfection, FAK overexpression enhanced FN-stimulated activation o f ERK2 similar to 4-fold, This was blocked by co-expression of the dom inant negative Asn-17 mutant Ras, indicating that FN stimulation of ER K2 was Ras-dependent. FN-stimulated c-Src PTK activity was enhanced by wild type FAR expression, whereas FN-stimulated activation of ERK2 wa s blocked by expression of the c-Src binding site Phe-397 mutant of FA R, Expression of the Grb2 binding site Phe-925 mutant of FAK enhanced activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK did n ot, Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enha nced p130(Cas) association with FAR, She tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation, FN-induced Grb2-Shc associa tion is another pathway leading to activation of ERK2 via Ras, The inh ibitory effects of Tyr-397 FAK expression show that FAR-mediated assoc iation and activation of c-Src is essential for maximal signaling to E RK2. Moreover, multiple signaling pathways are activated upon the form ation of an FAK-c-Src complex, and several of these can lead to Ras de pendent ERK2 mitogen-activated protein kinase activation.