Cyclin E-Cdk2 phosphorylation promotes late G1-phase degradation of MyoD in muscle cells

Proliferating myoblasts already express MyoD before the induction of differ entiation. Overexpression of MyoD in normal and transformed cell lines was shown to block cells from entering S phase, suggesting that the MyoD growth suppressive effect must be tightly controlled in growing myoblasts, Here w e show that during G1 phase, but not in G2, MyoD abundance is down-regulate d by the ubiquitin-proteasome pathway through phosphorylation of serine 200 . Roscovitine, a specific inhibitor of cyclin-Cdk2 complexes, prevents both phosphorylation and degradation of MyoD in G1. Inhibition of the ubiquitin -dependent proteasome pathway by MG132 results in stabilization of MyoD-wt, with little effect on a MyoD mutant where serine 200 is replaced by an ala nine. Our results show that MyoD Ser200 is the substrate for phosphorylatio n by cyclin E-Cdk2 stimulating its degradation by the ubiquitin-proteasome system which controls MyoD levels in G1. Phosphorylation/degradation of Myo D at the end of G1 thus represents the regulatory checkpoint in growing myo blasts allowing progression into S phase in a manner similar to the recentl y examplified cdk2-phosphorylation/degradation Of p27(Kip1). (C) 2000 Acade mic Press.