Mutant MyoD Lacking Cdc2 Phosphorylation Sites Delays M-Phase Entry

Abstract

The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G₂/M transition by controlling the expression of p21^Waf1/Cip1 (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G₂. In growing myoblasts, MyoD reaccumulates during G₂ concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G₂ and delays M-phase entry. This G₂ arrest is not observed in p21^−/− cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G₂/M transition.

We are grateful to Anne Fernandez and Ned Lamb for critical reading of the manuscript.

L. A. Tintignac is a fellow of Ministère de la Recherche et de la Technologie (MRT). V. Sirri is supported by the Institut Gustave Roussy. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, and grants from Ligue Nationale contre le Cancer, Association pour la Recherche sur le Cancer (A.R.C. no. 5921), and the Institut Gustave Roussy.