cdk1- and cdk2-mediated phosphorylation of MyoD Ser200 in growing C2 myoblasts: Role in modulating MyoD half-life and myogenic activity

We have examined the role of protein phosphorylation in the modulation of t he key muscle-specific transcription factor MyoD. We show that MyoD is high ly phosphorylated in growing myoblasts and undergoes substantial dephosphor ylation during differentiation. MyoD can be efficiently phosphorylated in v itro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated f rom proliferative myoblasts. Comparative two-dimensional tryptic phosphopep tide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addit ion, when the seven proline-directed sites in MyoD were individually mutate d, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-A la200) abolished the slower-migrating hyperphosphorylated form of MyoD, see n either in vitro after phosphorylation by cdk1-cyclin B or in vivo followi ng overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activit y threefold higher than that of wild-type MyoD in transactivation of an E-b ox-dependent reporter gene and promoted markedly enhanced myogenic conversi on and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the hal f-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, subst antiating a role of Ser200 phosphorylation in regulating MyoD turnover in p roliferative myoblasts. Taken together, our data show that direct phosphory lation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromisi ng MyoD activity during myoblast proliferation.