Baudier J, Bergeret E, Bertacchi N, Weintraub H, Gagnon J, Garin J
Département de Biologie Moléculaire et Structurale, CFA, DBMS-BMCC, INSERM Unité 309, Grenoble, France.
Biochemistry. 1995 Jun 20;34(24):7834-46. doi: 10.1021/bi00024a007.
MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.
MyoD属于一个肌源性碱性螺旋-环-螺旋（bHLH）转录因子家族，可激活肌肉特异性基因。bHLH基序的碱性螺旋I序列包含蛋白激酶C（PKC）底物的共有序列。我们在此表明，MyoD在体外确实被PKC磷酸化，磷酸化位点位于bHLH基序碱性部分的苏氨酸115处。通过与钙调蛋白-靶肽模型类比，我们还在肌源性蛋白的共有碱性螺旋I基序中鉴定出一个保守的假定钙调蛋白/S100结合结构域。通过亲和层析和交联实验证明了MyoD与钙调蛋白以及丰富的肌肉S100a（αα）蛋白之间的钙依赖性相互作用。钙调蛋白和S100a的结合抑制了PKC对MyoD的磷酸化以及MyoD的DNA结合活性。发现S100a在拮抗DNA与MyoD的结合方面比钙调蛋白更有效。接下来，我们开发了一种使用钙调蛋白-琼脂糖柱通过亲和层析快速纯化细菌重组MyoD-bHLH结构域的方法，并研究了该肽被PKC的磷酸化及其与钙调蛋白和S100a的相互作用。我们证实PKC使MyoD-bHLH中的苏氨酸残基115磷酸化，Km为0.8微摩尔。钙调蛋白和S100a的结合抑制了PKC对MyoD-bHLH的磷酸化。通过天然凝胶电泳和使用5-（二甲基氨基）萘-1-磺酰基钙调蛋白的荧光光谱法鉴定了钙结合蛋白与MyoD-bHLH之间严格的钙依赖性相互作用。MyoD-bHLH与荧光标记的5-（二甲基氨基）萘-1-磺酰基钙调蛋白结合，解离常数约为20纳摩尔。S100a化学计量学地抑制了bHLH肽与标记钙调蛋白的结合，表明S100a对bHLH肽的亲和力比对钙调蛋白至少高1个数量级。为了支持S100a与MyoD在体内的相互作用，我们报告S100a和MyoD样免疫反应性在H9c2细胞中共定位，并且在存在钙的情况下，从粗制H9c2细胞提取物的S100a免疫沉淀物中回收了大量的MyoD样免疫反应性。我们提出肌源性蛋白代表了一个新的钙调蛋白/S100结合PKC底物家族，并且钙调蛋白/S100a可能参与bHLH肌源性蛋白活性的调节。
