Gautel M, Castiglione Morelli M A, Pfuhl M, Motta A, Pastore A
EMBL, Heidelberg, Germany.
Eur J Biochem. 1995 Jun 1;230(2):752-9.
The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C-terminus which bears similarity to the substrate. A calmodulin-binding sequence has also been identified in the C-terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin-binding site in the titin kinase C-terminal sequence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C-terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interaction of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt alpha-helical conformations in solution. Helicity increases upon binding of calmodulin in a calcium-dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin-binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.
肌联蛋白家族的巨型肌肉蛋白是脊椎动物和无脊椎动物横纹肌所特有的，其共同特征是含有一个功能和调节机制迄今尚不清楚的催化蛋白激酶结构域。在与肌联蛋白进化相关的肌球蛋白轻链激酶家族中，激酶调节是通过钙调蛋白与激酶C末端一个与底物相似的区域结合来实现的。在海兔抽动激酶的C末端也鉴定出了一个钙调蛋白结合序列。类似地，我们在肌联蛋白激酶C末端序列中鉴定出一个假定的钙调蛋白结合位点。对表达的催化结构域本身以及该区域的一系列合成肽进行了结合钙调蛋白能力的测试。生化数据表明，在有钙存在的情况下，肌联蛋白激酶及其C末端的肽以等摩尔复合物的形式与钙调蛋白结合。然而，截短肽与钙调蛋白的相互作用比肌球蛋白轻链激酶的弱。核磁共振研究表明，这些肽在溶液中倾向于采用α-螺旋构象。根据圆二色光谱判断，钙调蛋白结合后，螺旋度以钙依赖的方式增加。因此，我们提出肌联蛋白的这个钙调蛋白结合区域可能对该酶起调节作用，其底物仍有待确定。
