RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2.
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.
Lim
Min Jin
MJ
Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Dongdaemoon-gu, Seoul 130-701, Korea.
Choi
Kyu Jin
KJ
Ding
Yan
Y
Kim
Jin Hwan
JH
Kim
Bum Shik
BS
Kim
Yun Hong
YH
Lee
Jinhwa
J
Choe
Wonchae
W
Kang
Insug
I
Ha
Joohun
J
Yoon
Kyung-Sik
KS
Kim
Sung Soo
SS
D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D015536:Down-Regulation / Q000502:physiology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology*; D051379:Mice; D009132:Muscles / Q000166:cytology*; D032386:Myoblasts, Cardiac / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D012694:Serine / Q000378:metabolism*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000502:physiology*
D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C526298:Irs2 protein, mouse; C526299:Irs2 protein, rat; D010750:Phosphoproteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein