p190RhoGEF-stimulated RhoA activity and FA formation are dependent on FAK binding. (A) GFP-p190RhoGEF or GFP-GEFH1 overexpression in normal MEFs. (A) FAK^+/+ MEFs were infected with a lentiviral expression vector for GFP or GFP fusions of p190RhoGEF WT, p190RhoGEF Y1003A (catalytically inactive), p190RhoGEF Δ1292-1301 (FAK-binding mutant), GEFH1 WT, or GEFH1 Y393A (catalytically inactive) and sorted for GFP expression. Shown is the flow cytometry profile of GFP expression (open peaks) compared with FAK^+/+ autofluorescence (shaded peaks). (B) GFP-p190RhoGEF–mediated RhoA activation is dependent on FAK binding. GTP-bound RhoA was determined by GST-RBD pulldown assays from lysates of suspended and FN-replated FAK^+/+-expressing GFP or the indicated GFP-190RhoGEF constructs followed by blotting for RhoA expression. (C) FAK^+/+ p190RhoGEF WT MEFs show enhanced FA formation and actin fibers similar to FAK^−/− MEFs. Cells were plated onto 1-μg/ml-FN–coated coverslips for 60 min and costained with antibodies to vinculin and Alexa Fluor 350 phalloidin for F-actin. Bar, 30 μm. (D) p190RhoGEF and GEFH1 promote FA formation in MEFs. Box-and-whisker plots of paxillin-positive–stained points within MEFs stably overexpressing the indicated GFP-p190RhoGEF or GFP-GEFH1 constructs upon FN plating for 60 min (n = 15 cells per point). (E) GEF-mediated increased FA formation is associated with decreased cell motility. MEFs overexpressing the indicated GFP-p190RhoGEF or GFP-GEFH1 constructs were evaluated for 10-μg/ml-FN–stimulated Boyden chamber motility over 4 h. Means ± SD were determined from two experiments and are a percentage of FAK^+/+ GFP MEFs (set to 100). Asterisks indicate that the difference between FAK^+/+ GFP and FAK^+/+ GFP–p190RhoGEF Δ1292-1301 or FAK^+/+ GFP–p190RhoGEF Y1003A is significant (P < 0.05).