Pyk2 signaling activates RhoA and promotes p190RhoGEF expression. (A) Pyk2 knockdown restores normal RhoA regulation during FN adhesion of FAK^−/− MEFs. RhoA activation analyses were performed using the indicated serum-starved MEFs held in suspension for 30 min and then replated on 10 μg/ml FN for the indicated times. GTP-bound RhoA was determined by GST-RBD pulldown and blotting for RhoA levels in lysates. (B) Quantitation of FN-associated RhoA-GTP binding. RhoA values are means ± SD of three independent experiments and are plotted relative to RhoA-GTP levels in suspended FAK^−/− vector shRNA MEFs. (C) Pyk2 kinase activity and Y402 phosphorylation are required to promote Rho activation. FAK^−/− Pyk2 shRNA MEFs were preinfected with Ad-TA control, Myc-Pyk2 WT, Myc-Pyk2 F402, or Myc-Pyk2 A457 (kinase inactive), subjected to FN replating, and analyzed for Rho-GTP binding as in A. (D) Quantitation of FN-stimulated RhoA-GTP binding from C. Values are means ± SD of two independent experiments and are plotted relative to RhoA-GTP levels in suspended FAK^−/− Pyk2 shRNA MEFs. (E) Elevated p190RhoGEF mRNA levels in FAK^−/− MEFs. RT-PCR analyses using p190RhoGEF- and β-actin–specific primers. Shown is an inverted ethidium bromide–stained gel. (F) p190RhoGEF protein expression is regulated by Pyk2. FAK^−/− Pyk2 shRNA MEFs were infected with Ad-FAK or Ad-Pyk2 for 48 h. p190RhoGEF or actin protein expression were analyzed by blotting and compared with lysates from FAK^+/+ and FAK^−/− MEFs. (G) Kinase- and Y402-dependent Pyk2 signaling promotes Pyk2 expression. FAK^−/− Pyk2 shRNA MEFs were infected with Ad-TA control, Ad-Pyk2 WT, Ad-Pyk2 F402, or Ad-Pyk2 A457 for 48 h. p190RhoGEF, Pyk2, and actin expression were analyzed by blotting.