Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion. FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of PIX. INTRODUCTION Integrin 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 (2000) exhibited that p190RhoGAP is usually tyrosine-phosphorylated in a Src-dependent manner, and tyrosine phosphorylation of p190RhoGAP by FAK has been shown in vitro by Holinstat (2006) . Thus, Src and FAK inactivate RhoA activity via p190RhoGAP after integrin-mediated adhesion. This relaxes cytoskeletal tension, allowing the formation of membrane extensions. The Rho family GTPases cycle between inactive GDP-bound and active GTP-bound forms and their activation is (+)-CBI-CDPI2 usually mediated by guanine nucleotide exchange factors (GEFs; Hall, 2005 ). FAK interactions with Rac-activation mechanisms that may positively mobilize cell spreading are still incompletely understood and may proceed along multiple pathways. Thus, Hsia and coworkers showed that viral Src transformation does not fully restore Rac-dependent invasive behavior in FAK null cells. In fact, the transient accumulation of FAK at the lamellipodia of FAK-expressing fibroblasts is usually (+)-CBI-CDPI2 associated with the formation of a signaling complex with Src, p130CAS, and Dock180 and elevation of both Rac and JNK activity (Hsia (2004) also showed that Rac activation induced by PIX was increased after phosphorylation on S525 and T526 by PAK. In addition to PAK, PIX has been shown to bind to the ArfGAP family IL22RA2 protein paxillin kinase linker (PKL; Turner as a glutathione at 4C for 4 min. GST-PBD, 50 g, immobilized on glutathione-Sepharose beads was added to 500 g of protein from cell lysates and incubated at 4C with rotation for 60 min. The beads were then washed three times with lysis buffer and boiled in Laemmli sample buffer (Laemmli BL21 (Stratagene, La Jolla, CA). The PIX-GST fusion protein was purified from bacterial lysates, immobilized on glutathione-Sepharose beads, and then released with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione; Sigma). Cells were transfected with either full-length FAK or the truncated carboxy-terminus FAK mutant Dter that lacks the kinase domain name. Both FAK constructs were EGFP-tagged. 48 h after transfection protein lysates were obtained using modified RIPA buffer (0.1% DOC, 0.1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 2 mM sodium vanadate, 20 g/ml leupeptin, 20 g/ml aprotinin in PBS). EGFP-FAK or EGFP-Dter were immunoprecipitated with anti-GFP (+)-CBI-CDPI2 antibody from 500 g of protein lysates for 2 h and captured on protein A-Sepharose by rotation for 1 h at 4C. After four washes in lysis buffer EGFP-FAK or EGFP-Dter immunoprecipitates were resuspended in kinase buffer (50 mM Tris, pH 7.4, 5 mM MnCl2, and 5 mM MgCl2). Approximately 2 g of purified PIX-GST and 20 M of ATP (final concentration) were added to the beads as substrate to a total volume of 60 l and incubated at 37C for 30 min on (+)-CBI-CDPI2 a shaker to (+)-CBI-CDPI2 keep the beads in suspension. In other experiments, aliquots of GST- wtFAK411-686 or GST-FAK411-686K454R (1, 3, or 6 g) were added to 2 g of purified GST-PIX or 4 g of purified GST in kinase buffer with or without 20 M of ATP (final concentration) in a total volume of 60 l and incubated at 37C for 30 min. The kinase reactions were stopped by the addition of sample buffer and analyzed using.