1995;6:637C647. to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation. Cell adhesion to extracellular matrix (ECM) proteins can generate transmembrane signals important for cell survival and can promote directed cell migration events. In a variety of cell types, integrin stimulation by ECM proteins such as fibronectin (FN) leads to changes in intracellular protein tyrosine phosphorylation events. In fibroblasts, the focal adhesion kinase (FAK), a nonreceptor protein-tyrosine kinase (PTK), colocalizes with integrin receptors at sites of cell attachment to ECM proteins. FAK may associate directly with integrin cytoplasmic domains (44) or may cocluster with integrin receptors through interactions with other integrin-associated proteins (4, 8, 22). FAK tyrosine phosphorylation is stimulated by cell binding Dianemycin to ECM proteins (for a review, see reference 50), by overexpression of the integrin cytoplasmic domains (52) and also by other growth factor or serum mitogens (for a review, see reference 24). Since integrin receptors lack catalytic activity, FAK association and activation may be important for integrin-mediated signal transduction events (for a review, see reference 20). This hypothesis is supported by gene knockout results in which both the FN- and FAK-null mice die as a result of LATS1 similar developmental gastrulation defects (15, 25). In addition to integrin stimulation of FAK, ECM protein binding to cells can lead to changes in the tyrosine phosphorylation of a number of different signaling proteins, including Dianemycin p130Cas, Shc, and Cbl, as well Dianemycin as structural proteins such as paxillin and tensin. Integrin stimulation can also promote increases in intracellular calcium levels (51), protein kinase C activity (32, 56), and phosphatidylinositol (PI) 3-kinase activity (7, 28). One downstream target for integrin-initiated signaling events is the activation of the extracellular signal-regulated kinase 2/mitogen-activated protein (ERK2/MAP) kinase pathway (9, 38, 39, 47, 59). Although integrin-initiated signaling to ERK2 is dependent on the integrity of the actin cytoskeleton and involves the activation of both the Rho and the Ras families of small GTPase Dianemycin proteins (12, 40), the integrin signaling pathways upstream of Ras have not been clearly defined. Attempts to delineate the molecular mechanisms of integrin-stimulated signaling to ERK2 have yielded potentially conflicting results. In NIH 3T3 fibroblasts, Grb2 transiently binds to a motif surrounding FAK Tyr-925 after FN stimulation (47), with the binding of Src-family PTKs to the motif surrounding the FAK autophosphorylation site (Tyr-397) being important for Src-mediated phosphorylation of FAK Tyr-925 in vivo (48). Direct Grb2 binding to FAK and.