FAK Potentiates Rac1 Activation and Localization to Matrix Adhesion Sites: A Role for βPIX

Abstract

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosinephosphorylated ␤PIX and thereby increased its binding to Rac1. In addition, ␤PIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of ␤PIX. INTRODUCTIONIntegrin receptors are activated and clustered at sites of extracellular matrix (ECM) binding, leading to the tyrosine phosphorylation of a number of downstream signaling proteins including FAK (Hanks et al., 1992;Schaller et al., 1992;Romer et al., 2006). Autophosphorylation of FAK at Tyr-397 creates a binding site for Src. After binding to FAK, Src phosphorylates FAK on several other tyrosine residues, including Tyr-925 and Tyr-576/577 to achieve full FAK activation and scaffolding potential Schaller 2001). are in the FAK kinase activation loop, and phosphorylation on these sites enhances catalytic activity (Ruest et al., 2000). Phosphorylation on Tyr-925 induces the recruitment of Grb2 and promotes the activation of the Ras/Raf/MEK/ERK pathway (Schlaepfer et al., 1997). In addition to Src, phosphorylation on FAK Tyr-397 also induces the recruitment of Shc and p130CAS to focal adhesions (Schlaepfer et al., 1997;Takahashi et al., 1999). Src and FAK also directly mediate the tyrosine phosphorylation of p130CAS and paxillin, leading in turn to the recruitment of Crk and Nck and the assembly of multiphosphocomponent signaling complexes at focal adhesions (Schaller and Parsons 1995;Schlaepfer et al., 1999;Turner, 2000;Romer et al., 2006).FAK's role in cell spreading has been investigated since reports that FAK null fibroblasts from knockout mice exhibited similar plating efficiency but poor spreading when compared with normal controls (Ilic et al., 1995). Reexpression of FAK in the FAK null cells restores their ability to spread on fibronectin (Owen et al., 1999;Sieg et al., 1999), whereas overexpression of either the dominant negative FAK protein FRNK, or the FAK-inactivating phosphatases PTEN or Shp-2, results in delayed or impaired cell spreading (Richardson and Parsons 1996;Gu et al., 1998;Yu et al., 1998). FAK was once though...

Tyrosine 397 phosphorylation is critical for FAK-promoted Rac1 activation and invasive properties in oral squamous cell carcinoma cells

“…Studies indicated that FAK can upregulate Rac1 activity through the activation of PI3K. 11,29 In addition, FAK can phosphorylate RhoGDI and cause the dissociation of RhoGDI from Rac1, thus promoting Rac1 activation.…”

“…9,10 Importantly, their activities can be modulated by FAK signaling. 11,12 Rac1 is a member of Rho GTPases that cycle between an inactive GDP-bound form and an active GTP-bound form. In response to extracellular signals, the active Rac1-GTP can interact with different effectors and activate multiple intracellular signals that regulate actin cytoskeletal reorganization, vesicle trafficking, and cell migration.…”

The Rac1 hypervariable region in targeting and signaling

“…Rac1 binds to Crk, CD2AP and PACSIN2 via their SH3 domains and also binds to the SH3 domain of β-PIX, which is one of the FA-targeting proteins for activated Rac1. 31,34 Some of the proteins that bind the Rac1 C-terminus promote its downstream signaling. CD2AP and Nedd4 co-localize with Rac1 at cell-cell contacts in epithelial cells and appear to stimulate Rac1-induced cell-cell adhesion.…”

Host epithelial geometry regulates breast cancer cell invasiveness

“…Furthermore, the phosphorylation and activation of FAK has been found to be enhanced in high-stress regions of mammary epithelial tissues (36). To determine whether FAK regulates the response of tumor cells to host tissue contractility, we expressed a dominant-negative mutant that lacks the kinase domain (FAKDter) (44) (Fig. S7D).…”