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RhoA/Rho Kinase Blocks Muscle Differentiation via Serine Phosphorylation of Insulin Receptor Substrate-1 and -2

Department of Biochemistry and Molecular Biology (BK21 Project) (M.J.L., K.J.C., Y.D., J.H.K., W.C., I.K., J.H., K.-S.Y., S.S.K.), Medical Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute (M.J.L., K.J.C., Y.D., J.H.K., W.C., I.K., J.H., K.-S.Y., S.S.K.), Department of Thoracic Surgery (B.S.K.), School of Medicine, Kyung Hee University, Seoul 130-701, Korea; Department of Anesthesiology and Pain Medicine (Y.H.K.), Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul 110-746, Korea; and Department of Biotechnology (J.L.), Dongseo University, Busan 617-716, Republic of Korea

Address all correspondence and requests for reprints to: Sung Soo Kim, Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, #1, Hoegi-dong, Dongdaemoon-gu, Seoul 130-701, Korea. E-mail: sgskim{at}khu.ac.kr.

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.

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