We have investigated a role for the amino-terminal FERM-like domain of the focal adhesion kinase (FAK) as a negative regulator of its own activity and phosphorylation state. Deletion of the first 375 amino acids from the amino terminus of FAK increases its catalytic activity in vitro, its phosphorylation when expressed in mammalian cells, and the phosphorylation of a FAK substrate, paxillin. Deletion mutants are phosphorylated in suspension, suggesting that they are no longer regulated by adhesion. The amino terminus of FAK can interact with the kinase domain of FAK in vitro and in vivo, suggesting that it might act as an autoinhibitor of FAK activity. The amino terminus of FAK can act in trans to inhibit FAK phosphorylation when expressed in mammalian cells or to directly inhibit FAK activity in vitro. Expression of the amino terminus of FAK inhibits cell cycle progression in CHO cells, consistent with its inhibition of FAK phosphorylation and function in trans. A glutathione S-transferase fusion protein containing the cytoplasmic tail of the ␤1 integrin stimulates FAK activity in vitro, suggesting that FAK could be regulated by molecular interactions with the amino terminus. Based on these and previous data, we propose a working model for activation of FAK in cell adhesion.Focal adhesion kinase (FAK) is a 125-kDa tyrosine kinase whose activity and phosphorylation status are regulated by cell adhesion to the extracellular matrix through integrins (25,28,40). FAK is maintained in a dephosphorylated and inactive state when cells are held in suspension. Upon attachment of cells to the extracellular matrix, FAK becomes localized to focal adhesions and is activated. Such regulation positions FAK as a key enzyme in transmitting information from integrins to the various signaling pathways that coordinate events that modulate or are dependent on cell adhesion. Indeed, FAK has been implicated as an important regulator of a variety of developmental and signaling pathways, including those that regulate cellular events such as cell cycle progression, cell survival, and cell migration (20,23,24,28,40).Previous studies suggest that a number of factors contribute to the activation of FAK by cell adhesion. FAK's ability to localize to focal adhesions appears to be critical in promoting its phosphorylation. Most FAK mutants that contain deletions or point mutations in their carboxy-terminal domains which abolish their ability to be recruited into focal adhesions no longer become strongly phosphorylated (9, 32). Autophosphorylation of FAK at Y397 is critical for many FAK-dependent functions, including phosphorylation of FAK on other residues (2, 3). Phosphorylation of Y397 creates a binding site for several SH2 domain-containing molecules, including phosphatidylinositol 3-kinase, Grb7, and Src (5, 16, 26, 39). Mutation of this residue to phenylalanine blocks phosphorylation of FAK on residues within its kinase domain and carboxy terminus (2). Clustering of chimeric molecules containing the extracellular and transmembrane domains...

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr 256 . We found that phosphorylation of Tyr 256 affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr 256 of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr 256 plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.

Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-L-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK.

Focal adhesion kinase (FAK) is an important mediator of signal transduction pathways initiated by integrins in cell migration, survival and cell cycle regulation. The ability of FAK to mediate integrin signaling in the regulation of cell cycle progression depends on the phosphorylation of Tyr397, which implies a functional significance for the formation of FAK signaling complexes with Src, phosphatidylinositol-3-kinase (PI3K) and Grb7. We have previously described a FAK mutant, D395A, that selectively disrupts FAK binding to PI3K, but allows FAK association with Src. Using this mutation in a mislocalized FAK mutant background, we show here that formation of a FAK/PI3K complex is not sufficient for cell cycle progression but the formation of a FAK/Src complex plays an essential role. We also show that mutation of D395 to A disrupted FAK association with Grb7. This suggests that a FAK/Grb7 complex is not involved in the cell cycle regulation either, which is supported by direct analysis of cells expressing a dominant negative Grb7 construct. Finally, we provide evidence that the Src-dependent association of FAK with Grb2 and p130Cas are both required for the regulation of cell cycle progression by FAK. Together, these studies identify important FAK downstream signaling pathways in cell cycle regulation. ß