Liu Chuan-ju, Ding Bo, Wang Hong, Lengyel Peter
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8024, USA.
Mol Cell Biol. 2002 May;22(9):2893-905. doi: 10.1128/MCB.22.9.2893-2905.2002.
The murine p204 protein level is highest in heart and skeletal muscle. During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm. p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium. Here we report that p204 is required for the differentiation of C2C12 myoblasts. We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3. These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins. Our hypothesis is based on the following findings. (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium. (ii) p204 bound to the Id proteins in vitro and in vivo. (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved. (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro. (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes.
小鼠p204蛋白水平在心脏和骨骼肌中最高。在培养的成肌细胞融合形成肌管的过程中，由于依赖肌肉特异性MyoD蛋白的转录，p204水平升高，并且p204被磷酸化并从细胞核转运至细胞质。p204过表达加速了分化培养基中成肌细胞的融合，甚至在生长培养基中也能触发这一过程。在此我们报告，p204是C2C12成肌细胞分化所必需的。我们提出，它至少部分地通过克服Id蛋白（Id1、Id2和Id3）对MyoD和E47蛋白活性的抑制来实现分化。已知这些Id蛋白通过结合并阻断MyoD、E12/E47和其他生肌碱性螺旋-环-螺旋（bHLH）蛋白的活性来抑制骨骼肌分化。我们的假设基于以下发现。（i）通过反义RNA降低C2C12成肌细胞中的p204水平：（a）增加了Id2的水平；（b）抑制了依赖MyoD、E12/E47和其他bHLH蛋白的肌肉特异性肌球蛋白重链蛋白的积累；（c）抑制了分化培养基中成肌细胞与肌管的融合。（ii）p204在体外和体内均与Id蛋白结合。（iii）在p204与Id2的结合中，涉及p204的b段和Id2的HLH段。（iv）添加p204克服了Id蛋白在体外对MyoD和E47与DNA结合的抑制。（v）在成肌细胞中过表达p204：（a）即使在生长培养基培养中也降低了Id蛋白的水平；（b）克服了Id蛋白对MyoD和E47依赖性转录的抑制，并且也克服了Id2对成肌细胞与肌管融合的抑制。
