Stabilization of MyoD by direct binding to p57(Kip2).
Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoD(Ser200) in the regulation of MyoD activity and protein turnover. In the present study, we show that in presence of p57(Kip2), MyoD(Ala200), a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2-5-fold higher than of MyoD(Ala200) alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1. Furthermore, p57(Kip2) increases the levels of MyoD(Ala200) in cotransfected cells. This result implies that p57(Kip2) may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors. We report that overexpression of p57(Kip2) increased the half-life of MyoD(Ala200). This increased half-life of MyoD involves a physical interaction between MyoD and p57(Kip2) but not with p16(Ink4a), as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57(Kip2) from C2C12 myogenic cells. Mutational and functional analyses of the two proteins show that the NH(2) domain of p57(Kip2) associates with basic region in the basic helix-loop-helix domain of MyoD. Competition/association assays and site-directed mutagenesis of the NH(2) terminus of p57(Kip2) identified the intermediate alpha-helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction. These data show that the alpha-helix domain of p57(Kip2), 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 p57(Kip2) family members positively act on myogenic differentiation.
Reynaud
E G
EG
Laboratoire de Génétique Oncologique UMR 1599, Centre National de la Recherche Scientifique, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif, France.
Leibovitch
M P
MP
Tintignac
L A
LA
Pelpel
K
K
Guillier
M
M
Leibovitch
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008809:Mice, Inbred C3H; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation
D017931:DNA Primers; D005656:Fungal Proteins; C075341:KIP2 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D017570:MyoD Protein; D011518:Proto-Oncogene Proteins; D029701:Saccharomyces cerevisiae Proteins; D019938:Cyclin D1; C495900:CDK4 protein, human; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases