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.