P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK.

Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression.