Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain
Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain
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DOI：
10.1128/mcb.13.10.5943-5956.1993
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发表时间：
1993-10
影响因子：
5.3
通讯作者：
S. Hardy;Y. Kong;S. F. Konieczny
中科院分区：
文献类型：
--
作者：
S. Hardy;Y. Kong;S. F. Konieczny
MRF 4是肌肉特异性碱性螺旋-环-螺旋转录因子家族的成员，该家族还包括MyoD、肌细胞生成素和Myf-5。当这些蛋白质中的每一种在成纤维细胞中过表达时，将细胞转化为表达几种骨骼肌基因的分化的肌纤维，例如α-肌动蛋白、肌肉肌酸激酶和肌钙蛋白I的基因。尽管事实上MRF 4起正转录调节剂的作用，但MRF 4蛋白受到多种试剂的负调节，最值得注意的是通过将细胞暴露于纯化的生长因子，如碱性成纤维细胞生长因子（bFGF）。为了确定bFGF是否通过特定的翻译后修饰抑制MRF 4活性，我们研究了MRF 4是否在体内作为磷蛋白存在，以及蛋白质的磷酸化状态是否调节其活性。我们的结果表明，MRF 4主要在丝氨酸残基上磷酸化，在苏氨酸残基上发生弱磷酸化。环腺苷酸依赖性蛋白激酶（PKA）和蛋白激酶C（PKC）在体外以及体内磷酸化MRF 4，并且每种激酶的过表达抑制MRF 4活性，从而阻断终末分化。PKC介导的位于DNA结合结构域内的保守苏氨酸残基（T-99）的磷酸化抑制MRF 4在体外与特异性DNA靶点结合。然而，虽然T-99本身是必不可少的生肌活性，我们的研究表明，T-99的磷酸化状态并没有发挥主要作用，在体内调节MRF 4的活性，因为PKA，PKC，和bFGF抑制MRF 4蛋白的活性，其中确定的PKA和PKC位点已发生突变。我们认为，负调控MRF 4施加bFGF不涉及直接修改的蛋白质在确定的PKA和PKC网站，而是可能涉及修改特定的coregulators与这种肌肉调节因子相互作用。
MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic activity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.