title: Mitogen-activated protein kinase kinase 1 (MEK1) stabilizes MyoD through direct phosphorylation at tyrosine 156 during myogenic differentiation.
abstract: Previously, we reported that mitogen-activated protein kinase kinase 1 (MEK1) activated in the mid-stage of skeletal muscle differentiation promotes myogenic differentiation. To elucidate the molecular mechanism, we investigated an activity of MEK1 for MyoD. Activated MEK1 associates with MyoD in the nucleus of differentiating myoblasts. In vitro kinase assay using active MEK1, a (32)P-labeled protein band corresponding to GST-MyoD was observed but not to mutant GST-MyoD-Y156F. Tyrosine phosphorylation of endogenous MyoD was detected with a specific anti-pMyoD-Y156 antibody; however, this response was blocked by PD184352, a MEK-specific inhibitor. These results indicate that activated MEK1 phosphorylates the MyoD-Y156 residue directly. Interestingly, the protein level of mutant MyoD-Y156F decreased compared with that of wild type but was recovered in the presence of lactacystin, a proteasome inhibitor. The protein level of MyoD-Y156E, which mimics phosphorylation at Tyr-156, was above that of wild type, indicating that the phosphorylation protects MyoD from the ubiquitin proteasome-mediated degradation. In addition, the low protein level of MyoD-Y156F was recovered over that of wild type by an additional mutation at Leu-164, a critical binding residue of MAFbx/AT-1, a Skp, Cullin, F-box (SCF) E3-ubiquitin ligase. The amount of MyoD co-precipitated with MAFbx/AT-1 also was reduced in the presence of active MEK1. Thus, these results suggested that the phosphorylation probably interrupts the binding of MAFbx/AT-1 to MyoD and thereby increases its stability. Collectively, our results suggest that MEK1 activated in differentiating myoblasts stimulates muscle differentiation by phosphorylating MyoD-Y156, which results in MyoD stabilization.
authors: Division of Brain Disease, Center for Biomedical Science, National Institutes of Health, Korea Center for Disease Control and Prevention, 187 Osongsaengmyeong2-ro, Gangoe-myeon, Cheongwon-gun, Chungcheongbuk-do 363-951, South Korea.
authors: Chulman
authors: C
authors: Jo
authors: Sun-Jung
authors: SJ
authors: Cho
authors: Sangmee Ahn
authors: SA
authors: Jo
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D055550:Protein Stability / Q000187:drug effects; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D001549:Benzamides; D044842:Cullin Proteins; D015853:Cysteine Proteinase Inhibitors; D009124:Muscle Proteins; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; C067713:lactacystin; C486691:Fbxo32 protein, mouse; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D000111:Acetylcysteine