Tsichlis for antibodies to FAK, Src, and Akt. This work was supported by grants CA17007 (to R.O. of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent Dihydrofolic acid activation of extracellular signalCregulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior. (Santa Cruz, CA), and the anti-hemagglutinin (HA) antibody (12CA5) was purchased from (Indianapolis, IN). The polyclonal antiserum to the COOH-terminal of FAK was a gift from J.-L. Guan (Cornell University, Ithaca, NY). The antibody to Src (phosphotyrosine 416) was a gift from A. Laudano (University of New Hampshire, Durham, NH). The polyclonal antiserum to Akt was provided by P. Tsichlis (Fox Chase Cancer Center, Philadelphia, PA). The antiCintegrin 1 subunit antibody (130) was previously characterized (Marcantonio and Hynes, 1988). Cell Culture Rat1 lines expressing N17Rac1 or N19RhoA driven by a tetracycline- repressible promoter were established as described (Qiu et al., 1995 for 10 min at 4C, and protein concentration was then determined using a Pierce Micro Rabbit polyclonal to HMBOX1 BCA protein assay kit (for 10 min at 4C, and protein concentration was determined as described above. Immunoprecipitation was performed with 500 g of lysate, 10 g of 12CA5, and protein A-Sepharose. The immunoprecipitates were then washed and subjected to an in vitro kinase reaction (using myelin basic protein as substrate) as described (King et al., 1997). The specific activity of Erk2 was determined by quantitating Dihydrofolic acid the 32P incorporated into myelin basic protein with a PhosphorImager and dividing it by the level of protein in the precipitate. Akt In Vitro Kinase Assay Cells were washed twice in PBS and lysed in Akt lysis buffer (20 mM Tris, pH 7.5, 150 mM sodium chloride, 10% glycerol, 1% NP-40, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 500 g/ml leupeptin, 2 mM aprotinin, and 1 mM PMSF). Akt was Dihydrofolic acid immunoprecipitated from 250 to 500 g of lysate with a polyclonal antibody to Akt or the HA tag antibody (12CA5) and subjected to an in vitro kinase reaction using histone H2B as an exogenous substrate as described (King et al., 1997). Adhesion and Spreading Assays Serum-starved cells (500,000 cells/ml) suspended in 1 ml of DME as described above were plated on fibronectin-coated/BSA-blocked 35-mm wells. To initiate adhesion, the cells were centrifuged for 30 s at 50 and allowed to adhere (10 min) or spread (20 min) at 37C. Adherent cells were washed twice with PBS and then fixed with 4% PFA in PBS for 15 min at room temperature. The fixed cells were then rinsed twice with water, stained with 0.1% crystal violet in water for 30 min at room temperature, and rinsed twice with water. Adhesion was quantitated by adding 10% acetic acid to the crystal violetCstained well and examining the solution in a spectrophotometer at 600 nm. Background staining was determined by staining cells that adhere to a BSA-coated well in the absence of fibronectin. Spreading was quantitated by photographing the cells that adhere to fibronectin-coated wells after 20 min and counting the cells that had spread. Determination of F-Actin Content F-actin content was examined essentially as described (Southwick et al., 1989). In brief, serum-starved cells (800,000 cells/ml) suspended in 1 ml of DME were plated on fibronectin- or polylysine-coated/BSA-blocked 35-mm wells for 15 or 60 min at 37C. The cells were then washed twice in PBS and fixed with 4% PFA in PBS for 10 min at room temperature. Next, the cells were permeabilized with.