Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences
Phosphorylation of calmodulin fragments by protein kinase CK2. Mechanistic aspects and structural consequences
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DOI：
10.1021/bi049365c
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发表时间：
2004-10-12
期刊：
影响因子：
2.9
通讯作者：
Pinna, LA
中科院分区：
文献类型：
--
作者：
Arrigoni, G;Marin, O;Pinna, LA
钙调蛋白在体内和体外被蛋白激酶 CK2 磷酸化，其磷酸化方式在 CK2 底物中是独特的，可被激酶的调节性 β 亚基抑制，并被多元肽显着增强。使用不同地包含 CK2 磷酸化位点的钙调蛋白合成片段，我们表明 Thr79、Ser81、Ser101 和 Thr117 的单独磷酸化受到肽的大小和组成的严重影响，并且钙调蛋白的 C 末端结构域与 β 亚基对钙调蛋白磷酸化的下调及其对聚赖氨酸的异常反应有关。 Far-Western 印迹分析揭示了钙调蛋白与 β 亚基 N 端结构域之间依赖于多聚赖氨酸的相互作用。我们还表明，Ser81 的磷酸化阻碍了 Thr79 随后的磷酸化，并且其本身促进了中央螺旋的展开，其灵活性有助于与钙调蛋白依赖性酶的相互作用。总的来说，我们的数据与通过不同 CaM 结构域、CK2 的催化和调节亚基以及模拟体内聚赖氨酸作用的聚阳离子效应子的协同作用对钙调蛋白磷酸化的多方面调节是一致的。
Calmodulin is phosphorylated in vivo and in vitro by protein kinase CK2 in a manner that is unique among CK2 substrates for being inhibited by the regulatory beta-subunit of the kinase and dramatically enhanced by polybasic peptides. Using synthetic fragments of calmodulin variably encompassing the CK2 phosphorylation sites here we show that individual phosphorylation of Thr79, Ser81, Ser101, and Thr117 is critically influenced by the size and composition of the peptides and that the C-terminal domain of calmodulin is implicated both in down-regulation of calmodulin phosphorylation by the beta-subunit and in its abnormal responsiveness to polylysine. A far-Western blot analysis discloses polylysine-dependent interaction between calmodulin and the N-terminal domain of the beta-subunit. We also show that phosphorylation of Ser81 hampers subsequent phosphorylation of Thr79 and by itself promotes the unfolding of the central helix, whose flexibility is instrumental to the interaction with calmodulin-dependent enzymes. Collectively taken, our data are consistent with a multifaceted regulation of calmodulin phosphorylation through the concerted action of distinct CaM domains, the catalytic and regulatory subunits of CK2, and polycationic effectors mimicking in vivo the effect of polylysine.