The ubiquitin-proteasome system is responsible for the regulation and turnover of the nuclear transcription factor MyoD. The degradation of MyoD can occur via an NH 2 terminus-dependent pathway or a lysine-dependent pathway, suggesting that MyoD ubiquitination may be driven by different mechanisms. To understand this process, deletion analysis was used to identify the region of MyoD that is required for rapid proteolysis in the lysine-dependent pathway. Here we report that the basic helix-loop-helix domain is required for ubiquitination and lysine-dependent degradation of MyoD in the nucleus. Site-directed mutagenesis in MyoD revealed that lysine 133 is the major internal lysine of ubiquitination. The half-life of the MyoD K133R mutant protein was longer than that of wild type MyoD, substantiating the implication of lysine 133 in the turnover of MyoD in myoblasts. In addition, the MyoD K133R mutant displayed activity 2-3-fold higher than the wild type in transactivation muscle-specific gene and myogenic conversion of 10T1/2 cells. Taken together, our data demonstrate that lysine 133 is targeted for ubiquitination and rapid degradation of MyoD in the lysine-dependent pathway and plays an integral role in compromising MyoD activity in the nucleus.Ubiquitination and degradation of many proteins are essential for cell cycle progression, signal transduction, and development (1). However, it is now clear that the ubiquitin-proteasome pathway can also carry out various non-proteolytic functions, controlling activities as diverse as transcription, nucleotide excision repair, receptor internalization, and ribosome function (see review, Ref. 2). The ubiquitination of the target protein is mediated by a cascade of ubiquitin transfer reactions that require a ubiquitin-activating enzyme (E1), a ubiquitinconjugating enzyme (E2), and a ubiquitin ligase (E3) that acts in the last step of the cascade (3). Ubiquitin protein ligases regulate the timing and substrate specificity in protein degradation. MyoD is a basic helix-loop-helix (b-HLH) 1 transcription factor that controls proliferation and differentiation (4). The mechanism by which MyoD induces myogenesis involves both the withdrawal from the cell cycle and the activation of musclespecific genes expression (5, 6). The HLH domain is required for dimerization with the ubiquitously expressed E-proteins, and the basic domain is responsible for DNA binding. Heterodimers bind to the consensus E-box (CANNTG) DNA sequence motif found in the promoters of many muscle-specific genes (7). Structurally, MyoD contains several functionally distinct domains responsible for transcriptional activation, chromatin remodeling, and nuclear localization (8, 9). Recent data demonstrate a direct link between MyoD levels and cell cycle regulation (10, 11), development (12), and regeneration (13). In this last process, MyoD is required for myogenic stem cell function in adult skeletal muscle (14). The interplay between G 1 cyclins and Cdk inhibitors on one hand and MyoD and its cofactors on the other...
