INDUCTION OF P18(INK4C) AND ITS PREDOMINANT ASSOCIATION WITH CDK4 ANDCDK6 DURING MYOGENIC DIFFERENTIATION

Terminal cell differentiation involves permanent withdrawal from the c ell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and ce ll differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CD K2, CDK4, and CDK6) associate with four CDK inhibitors: p18(INK4c) p19 (INK4d), P21, and p27(Kip1). During induced myogenesis, p21 and its as sociated CDK proteins underwent an initial increase followed by a decr ease as cells became terminally differentiated. The level of p27 prote in gradually increased, but the amount of total associated CDK protein s remained unchanged. p19 protein decreased gradually during different iation, as did its associated CDK4 protein. In contrast, p18 protein i ncreased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This i nitial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest l evel in terminally differentiated cells. Induction of p18 correlated w ith increased and sequential complex formation--first increasing assoc iation with CDK6 and then with CDK4 over the course of myogenic differ entiation. All of the CDK6 and half of the CDK4 were complexed with p1 8 in terminally differentiated C2C12 cells as well as in adult mouse m uscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C 2C12 cells differentiate, whereas the CDK6 kinase activity is low in b oth proliferating myoblasts and differentiated myotubes. Our results i ndicate that p18 may play a critical role in causing and/or maintainin g permanent cell cycle arrest associated with mature muscle formation.