Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.
Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.
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DOI：
10.1042/bsr20120116
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发表时间：
2013-02-07
影响因子：
4
通讯作者：
Morgan KG
中科院分区：
文献类型：
--
作者：
Yilmaz M;Gangopadhyay SS;Leavis P;Grabarek Z;Morgan KG
CaMKII（Ca2+/钙调蛋白依赖性激酶 II）是一种丝氨酸/苏氨酸磷酸转移酶，由于其寡聚结构每个亚基内特定苏氨酸残基的自身磷酸化，能够长期保留活性。 CaMKII 的 γ 亚型是血管收缩力的重要调节剂。在这里，我们发现 CaMKII γ 在 Ser26（位于 ATP 结合位点内的残基）处的磷酸化终止了酶的持续活性。为了测试 Ser26 磷酸化的生理重要性，我们生成了磷酸化特异性 Ser26 抗体，并证明了在血管去极化和收缩时 Ser26 磷酸化增加。为了确定 Ser26 的磷酸化是否影响激酶活性，我们将 Ser26 突变为丙氨酸或天冬氨酸。模仿 CaMKII 磷酸化状态的 S26D 突变导致 Thr287 自磷酸化水平急剧下降，并大大降低对外源底物 (autocamtide-3) 的催化活性，而 S26A 突变则没有影响。这些数据与分子模型相结合表明，CaMKII γ Ser26 处的负电荷很可能通过阻断 ATP 结合来抑制酶对其 Thr287 处自磷酸化位点的催化活性。我们认为 Ser26 磷酸化构成了关闭 CaMKII 活性的重要机制。
CaMKII (Ca2+/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser26, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser26, we generated a phosphospecific Ser26 antibody and demonstrated an increase in Ser26 phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser26 affects the kinase activity, we mutated Ser26 to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr287 autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser26 of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr287 most probably by blocking ATP binding. We propose that Ser26 phosphorylation constitutes an important mechanism for switching off CaMKII activity.