Cyclin E–Cdk2 Phosphorylation Promotes Late G1-Phase Degradation of MyoD in Muscle Cells Lionel A. Tintignac,* Marie Pierre Leibovitch,* Magali Kitzmann,† Anne Fernandez,† Bernard Ducommun,‡ Laurent Meijer,§ and Serge A. Leibovitch* ,1 *Laboratoire de Ge ´ne ´tique Oncologique, UMR 1599 CNRS, Institut Gustave Roussy, 94805 Villejuif, France; †Institut de Ge ´ne ´tique Humaine, UPR 1142 CNRS, 34396 Montpellier Cedex 5, France; ‡LBCMCP, EP 2079 CNRS, Universite ´ Paul Sabatier, 31062 Toulouse, France; and §CNRS, Station Biologique, BP 72, 29682 Roscoff Cedex, France Proliferating myoblasts already express MyoD before the induction of differentiation. 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 con- trolled in growing myoblasts. Here we show that during G1 phase, but not in G2, MyoD abundance is down-reg- ulated by the ubiquitin–proteasome pathway through phosphorylation of serine 200. Roscovitine, a specific inhibitor of cyclin–Cdk2 complexes, prevents both phos- phorylation 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 alanine. Our results show that MyoD Ser200 is the substrate for phosphorylation by cyclin E–Cdk2 stimu- lating its degradation by the ubiquitin–proteasome sys- tem which controls MyoD levels in G1. Phosphorylation/ degradation of MyoD at the end of G1 thus represents the regulatory checkpoint in growing myoblasts allow- ing progression into S phase in a manner similar to the recently examplified cdk2-phosphorylation/degradation of p27 Kip1 . © 2000 Academic Press Key Words: cyclin E–Cdk2; degradation; G1 phase; MyoD; phosphorylation; ubiquitination. INTRODUCTION MyoD is a basic helix–loop– helix (bHLH) transcrip- tion factor that controls proliferation versus differen- tiation [1]. The mechanism by which MyoD induces myogenesis involves both the withdrawal from the cell cycle and the activation of muscle-specific gene expres- sion [2, 3]. Consistent with the negative role of MyoD in cell cycle progression [4, 5], MyoD activity must be tightly controlled in proliferating myoblasts. The an- tagonism between proliferation and differentiation im- plies that signaling pathways driving proliferation must be suppressed to allow the induction of differen- tiation. Cell cycle progression in eukaryotes is con- trolled by a series of cyclin-dependent kinases (Cdks) which are in turn modulated by binding to specific cyclins. D-type cyclins (D1, D2, D3)–Cdk4/Cdk6 and cyclin A/E–Cdk2 are involved in regulating G1/S tran- sition, whereas cyclin A/B–Cdk1 (also called cdc2) is implicated in the G2/M transition of the cell cycle [6, 7]. Recent data support the involvement of these com- plexes in the regulation of muscle differentiation [8, 9]. The cyclin-mediated inhibition of myogenesis by cyclin D1 implicates nuclear translocation of Cdk4 by cyclin D1 and the subsequent formation of a MyoD–Cdk4 complex that specifically inhibits the transactivation functions of MyoD in the absence of Cdk4 kinase activ- ity [10, 11]. On the other hand, phosphorylation of MyoD is one of the crucial mechanisms that controls its level and transcriptional activity in the cell cycle. MyoD is degraded by the ubiquitin–proteasome path- way [12], involving the attachment of ubiquitin to the N-terminal residue [13] and phosphorylation of MyoD at serine 200 plays a crucial role in modulating its half-life and transcriptional activity during myoblast proliferation [14, 15]. Cyclin-dependent inhibition of muscle gene transactivation requires Cdk activation and can be reversed by overexpression of Ckis [9, 16, 17]. Since cell cycle exit and induction of differentiation only take place in G1 [1], MyoD activity must be sup- pressed at this period of the cell cycle. Here we show that MyoD is subject to ubiquitin- dependent degradation, a process that is triggered by direct cyclin E–Cdk2-dependent phosphorylation of MyoD serine 200 at the end of G1 in a manner similar to the recently examplified Cdk2-phosphorylation/deg- radation of p27. MATERIALS AND METHODS Reagents. MG132 ( N-carbobenzoxyl-Leu-Leu-leucinal), chloro- quine, E-64 ( trans-epoxysuccinyl-L-leucylamido (4-guanidino)bu- tane, phosphocreatine kinase, creatine phosphate, and ubiquitin 1 To whom reprint requests should be addressed. Fax: 01 42 11 52 61. E-mail: leibovit@igr.fr. 300 0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 259, 300 –307 (2000) doi:10.1006/excr.2000.4973, available online at http://www.idealibrary.com on