No abstract

A rapid increase in the tyrosine phosphorylation of the nonreceptor tyrosine kinase FAK 1 (1, 2), which localizes to focal adhesion plaques (3), has been identified as a prominent early event in cells stimulated by diverse signaling molecules that regulate cell proliferation, migration, and apoptosis (4, 5 (14,27). Phosphorylation at Tyr-925 within the focal adhesion targeting (FAT) domain creates a binding site for the Src homology 2 domain of the adapter protein Grb2-SOS (Ras exchange factor) complex and provides a possible mechanism of activation of the Ras/Raf/MEK/ERK pathway (36). The FAT domain binds paxillin and talin, which are responsible for targeting FAK to the focal adhesions and for promoting downstream signaling (36 -38). The importance of FAK-mediated signal transduction is underscored by experiments showing that this tyrosine kinase is implicated in embryonic development (39) and in the control of cell migration (27, 40 -42), proliferation (40, 43), and apoptosis (44, 45). It is increasingly recognized that overexpression of FAK is linked to the invasive properties of cancer cells (46,47).In addition to being phosphorylated at multiple tyrosines in response to external stimuli, FAK is also phosphorylated at serine residues (48, 49). Recently, Ser-722 and Ser-910, in the COOH-terminal, noncatalytic region of FAK (termed FAK-related nonkinase (FRNK)), have been identified as prominent phosphorylation sites (49). Despite the importance of FAK in signal transduction, virtually nothing is known about the regulation of these phosphorylation events. In particular, none of the previous studies demonstrated that the phosphorylation of any of these serine residues of FAK can be regulated in re-

A rapid increase in the tyrosine phosphorylation of the nonreceptor tyrosine kinase FAK 1 (1, 2), which localizes to focal adhesion plaques, has been identified as a prominent early event in cells stimulated by diverse signaling molecules that regulate cell proliferation, migration, and apoptosis (3-5). In particular, FAK is activated and tyrosine-phosphorylated in response to integrin clustering induced by cell adhesion or antibody cross-linking (1, 2, 6 -9). In addition, FAK is rapidly tyrosine-phosphorylated in cells stimulated by mitogenic neuropeptide agonists including bombesin (10 -16) and bioactive lipids including LPA (17-19) that act via heptahelical GPCRs, polypeptide growth factors (20 -24), bacterial toxins (25,26), and activated variants of pp60 Src (27,28). The importance of FAK-mediated signal transduction is underscored by experiments implicating this tyrosine kinase in embryonic development (29) and in the control of cell migration (30 -33), proliferation (30, 34), and apoptosis (35,36). In addition, there is increasing evidence linking overexpression of FAK to the invasive properties of cancer cells (37,38). It is increasingly recognized that the tyrosine phosphorylation and activation of FAK is an important point of convergence in the action of integrins, GPCR agonists, growth factors, and oncogenes (5, 39, 40). However, the molecular pathways leading to FAK tyrosine phosphorylation in response to multiple extracellular factors remain incompletely understood.Plating suspended cells onto fibronectin-coated dishes, a paradigm of integrin receptor activation (41), induces adhesion-dependent phosphorylation of FAK at multiple sites including tyrosines 397, 576, 577, 861, and 925 (32, 42-45). Tyr-397, the only apparent autophosphorylation site (46 -51), has emerged as a critical residue in FAK-mediated signaling (5). The autophosphorylation of FAK at Tyr-397 creates a high affinity binding site for the SH2 domain of Src family kinases including Src, Yes, and Fyn and leads to the formation of a signaling complex between FAK and Src family kinases (46, 47, 49 -52). A model has recently been proposed that envisages reciprocal catalytic activation of FAK and Src family kinases in response to adhesiondependent signals. Src family kinases associated with FAK are thought to phosphorylate FAK at additional sites including Tyr-576 and Tyr-577 that are located in the activation loop of the kinase catalytic domain of FAK (32, 42, 53), thereby promoting maximal FAK catalytic activation. Because phenylalanine mutation of Tyr-576 and Tyr-577 reduced adhesion-mediated FAK autophosphorylation (at Tyr-397), it has been proposed that activation loop phosphorylation of FAK by Src stimulates intermolecular phosphorylation at Tyr-397, thereby leading to signal amplification at sites of integrin-mediated cell adhesion (32). Therefore, in this model Src family kinases are thought to play a major role leading to autophosphorylation of FAK at Tyr-397 as part of an adhesion-dependent signaling response. Recently, we demons...

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAKSrc complex formation. Bombesin stimulated FAK/Src association through a Ca 2؉ and phosphatidylinositol 3-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr 577 induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.

No abstract