A rho-associated protein kinase, ROKα, binds insulin receptor substrate-1 and modulates insulin signaling

Insulin receptor substrate-1 (IRS-1) is physphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins, In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions, In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleekstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rho-associated kinase alpha (xROK alpha) as a potential xIRS-1-binding protein, The original clone contained. the carboxyl terminus of xROK alpha (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral. Ras (v-Ras) mRNA, These results suggest that xROK alpha may play a role in insulin signaling via a direct interaction with xIRS-1.