Gα12 and Gα13 stimulate rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 Crk-associated substrate

We examined whether constitutively active mutants of the G alpha proteins G alpha(12) and G alpha(13), which together comprise the G(12) subfamily of G alpha proteins, induce Rho-dependent tyrosine phosphoaylation of the focal adhesion proteins p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate. We report that transient expression of the constitutively active mutants of G alpha(12) Or of G alpha(13) in human embryonic kidney 293 cells stimulates tyrosine phosphorylation of a set of proteins of M-r of 110,000-130,000, 97,000, and 60,000-70,000. We identified p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate as prominent tyrosine-phosphorylated proteins in human embryonic kidney 293 cells expressing constitutively active G alpha(12) and G alpha(13). In common with the increased tyrosine phosphorylation of these proteins mediated by mitogens acting through heptahelical receptors, the G alpha(12)- and G alpha(13)-mediated increase in tyrosine phosphorylation is blocked by cytochalasin D, which specifically disrupts the actin cytoskeleton, and by the Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho. Our results support the hypothesis that G alpha(12) and G alpha(13) activate Rho and suggest that G alpha(12) and G alpha(13) may mediate the tyrosine phosphorylation of p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate.