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Molecular and Cellular Biology, April 1999, p. 3167-3176, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity

Magali Kitzmann, Marie Vandromme,* Valerie Schaeffer, Gilles Carnac, Jean-Claude Labbé, Ned Lamb, and Anne Fernandez

Institut de Génétique Humaine, Centre National de Recherche Scientifique, UPR 1142, 34396 Montpellier cedex 5, France

Received 2 October 1998/Returned for modification 6 November 1998/Accepted 30 December 1998

We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

* Corresponding author. Mailing address: Institut de Génétique Humaine, Centre National de Recherche Scientifique UPR 1142, 141 Rue de la Cardonille, 34396 Montpellier cedex 5, France. Phone: 33 (0)499 61 99 13. Fax: 33 (0)499 61 99 01. E-mail: Marie.Vandromme{at}igh.cnrs.fr.

Molecular and Cellular Biology, April 1999, p. 3167-3176, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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