PKCα phosphorylation of RhoGDI2 at Ser 31 disrupts interactions with Rac1 and decreases GDI activity

Rho family GTPases control a diverse range of cellular processes, and their deregulation has been implicated in human cancer. Guanine nucleotide dissociation inhibitors (GDIs) bind and sequester GTPases in the cytosol, restricting their actions. RhoGDI2 is a member of the GDI family that acts as a metastasis suppressor in a variety of cancer types; however, very little is known about the regulation and function of this protein. Here we present a mechanism for inactivation of RhoGDI2 via PKC phosphorylation of Ser 31 in a region that contacts GTPases. In cells, RhoGDI2 becomes rapidly phosphorylated at Ser 31 in response to phorbol 12-myristate 13-acetate stimulation. Based on the effects of pharmacological inhibitors and knockdown by siRNA, we determine that conventional type PKCα is responsible for this phosphorylation. Phospho-mimetic S31E-RhoGDI2 exhibits reduced binding to Rac1 relative to wild type, with a concomitant failure to reduce levels of activated endogenous Rac1 or remove Rac1 from membranes. These results reveal a mechanism of down-regulation of RhoGDI2 activity through PKC mediated phosphorylation of Ser 31. We hypothesize that this mechanism may serve to neutralize RhoGDI2 function in tumors that express RhoGDI2 and active PKCα.