| Abstrakt: |
Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoDSer200in the regulation of MyoD activity and protein turnover. In the present study, we show that in presence of p57Kip2, MyoDAla200, a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2–5-fold higher than of MyoDAla200alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1. Furthermore, p57Kip2increases the levels of MyoDAla200in cotransfected cells. This result implies that p57Kip2may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors. We report that overexpression of p57Kip2increased the half-life of MyoDAla200. This increased half-life of MyoD involves a physical interaction between MyoD and p57Kip2but not with p16Ink4a, as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57Kip2from C2C12 myogenic cells. Mutational and functional analyses of the two proteins show that the NH2domain of p57Kip2associates with basic region in the basic helix-loop-helix domain of MyoD. Competition/association assays and site-directed mutagenesis of the NH2terminus of p57Kip2identified the intermediate α-helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction. These data show that the α-helix domain of p57Kip2, which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57Kip2family members positively act on myogenic differentiation. |
