A RHO-ASSOCIATED PROTEIN-KINASE, ROK-ALPHA, BINDS INSULIN-RECEPTOR SUBSTRATE-1 AND MODULATES INSULIN SIGNALING

Insulin receptor substrate-1 (IRS-1) is physphorylated on multiple tyr osine residues by ligand-activated insulin receptors. These tyrosine p hosphorylation sites serve to dock several Src homology 2-containing s ignaling proteins, In addition, IRS-1 contains a pleckstrin homology d omain and a phosphotyrosine binding domain (PTB) implicated in protein -protein and protein-lipid interactions, In a yeast two-hybrid screeni ng using Xenopus IRS-1 (xIRS-1) pleekstrin homology-PTB domains as bai t, we identified a Xenopus homolog of Rho-associated kinase alpha (xRO K alpha) as a potential xIRS-1-binding protein, The original clone con tained. the carboxyl terminus of xROK alpha (xROK-C) including the put ative 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 Xe nopus oocytes after microinjection of mRNA corresponding to xROK-C. Fu rthermore, microinjection of xROK-C mRNA inhibited insulin-induced mit ogen-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 matu ration induced by progesterone or by microinjection of viral. Ras (v-R as) mRNA, These results suggest that xROK alpha may play a role in ins ulin signaling via a direct interaction with xIRS-1.