p57(Kip2) stabilizes the MyoD protein by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts

We show that expression of p57(Kip2), a potent tight-binding inhibitor of s everal G(1) cyclin-cyclin-dependent kinase (Cdk) complexes, increases marke dly during C2C12 myoblast differentiation. We examined the effect of p57(Ki p2) on the activity of the transcription factor MyoD. In transient transfec tion assays, transcriptional transactivation of the mouse muscle creatine k inase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57 (Kip2), p21(Cip1), and p27(Kip1) but not p16(Ink4a) induced an increased le vel of MyoD protein, and we show that MyoD, an unstable nuclear protein, wa s stabilized by p57(Kip2). Forced expression of p57(Kip2) correlated with h ypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mut ant arrested cells at the G(1) phase transition and induced hypophosphoryla tion of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk 2 complexes was inhibited by p57(Kip2). In addition, the NH2 domain of p57( Kip2) necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulati on in proliferative myoblasts. Taken together, our data suggest that repres sion of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57(Kip2) could p lay an important role in the accumulation of MyoD at the onset of myoblast differentiation.