Critical role for lysine 133 in the nuclear ubiquitin-mediated degradation of MyoD.
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 NH2 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.
Batonnet
Sabrina
S
Laboratoire de Génétique Oncologique, UMR 8125 CNRS, 94805 Villejuif, France.
Leibovitch
Marie Pierre
MP
Tintignac
Lionel
L
Leibovitch
Serge Alexandre
SA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D005822:Genetic Vectors; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017570:MyoD Protein / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D017570:MyoD Protein; D011500:Protein Synthesis Inhibitors; D025801:Ubiquitin; D004247:DNA; D003513:Cycloheximide; D008156:Luciferases; D008239:Lysine