p190RhoGEF contributes to aberrant FAK^−/− MEF morphology, RhoA activity, and FA formation. (A) Stable p190RhoGEF knockdown. FAK^−/− MEFs were infected with lentiviral Scr or p190RhoGEF shRNA and sorted for GFP expression, and lysates were analyzed by p190RhoGEF, Pyk2, actin, and GFP blotting. (B) Phase-contrast images show that FAK^−/− p190RhoGEF shRNA MEFs exhibit more of a fibroblast phenotype than FAK^−/− Scr shRNA control MEFs. Reduced numbers of FAs connected to F-actin after FN replating (60 min) upon p190RhoGEF knockdown as determined by anti-paxillin and Alexa Fluor 350 phalloidin costaining. Bars, 30 μm. (C) Decreased FAK^−/− FA formation upon p190RhoGEF knockdown. Box-and-whisker plots of paxillin-positive–stained points within FAK^−/− Scr shRNA or FAK^−/− p190RhoGEF shRNA MEFs plated on FN for 60 min (n = 15 cells per point). (D) p190RhoGEF knockdown prevents deregulated FAK^−/− MEF RhoA activation. GTP-bound RhoA was determined by GST-RBD pulldown assays from lysates of suspended and FN-replated FAK^−/− Scr shRNA and FAK^−/− p190RhoGEF shRNA MEFs followed by blotting for total RhoA levels. (E) Loss of p190RhoGEF inhibits motility. 10-μg/ml-FN–stimulated FAK^+/+, FAK^−/− Scr, and FAK^−/− p190RhoGEF shRNA MEF Boyden chamber motility over 4 h. Means ± SD were determined from three experiments and are a percentage of FAK^−/− control (set to 100). (F) p190RhoGEF contributes to FAK^−/− MEF proliferation. Cell growth assays over 5 d show that FAK^−/− p190RhoGEF shRNA MEFs grow slower than FAK^−/− Scr shRNA MEF controls and that Ad-Pyk2 overexpression does not function to enhance FAK^−/− p190RhoGEF shRNA MEF cell proliferation. Cells were infected 24 h before initiating growth analyses. Values are means ± SD from two experiments. (G) Pyk2 overexpression weakly activates RhoA in FAK^−/− p190RhoGEF shRNA MEFs. Cells were preinfected with Ad-Pyk2 WT, subjected to FN replating, and analyzed for Rho-GTP binding followed by blotting for total RhoA and Myc-Pyk2 levels.