Live imaging of endogenous Ca2+/calmodulin-dependent protein kinase II in neurons reveals that ischemia-related aggregation does not require kinase activity
Live imaging of endogenous Ca2+/calmodulin-dependent protein kinase II in neurons reveals that ischemia-related aggregation does not require kinase activity
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DOI：
10.1111/jnc.13263
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发表时间：
2015-11-01
影响因子：
4.7
通讯作者：
Bayer, K. Ulrich
中科院分区：
文献类型：
--
作者：
Barcomb, Kelsey;Goodell, Dayton J.;Bayer, K. Ulrich
钙/钙调蛋白依赖的蛋白激酶II(CaMKII)形成12聚体全酶。这些全酶在缺血条件下在神经元内聚集成更大的聚集体，并在体外模拟缺血条件下。这种聚集被认为是通过不同全酶中一个激酶亚基的调节域与另一个激酶亚基的T位点之间的相互作用来调节的，这种相互作用需要钙/钙调素的刺激和核苷酸的诱导。这个模型做出了几个预测，并在这里得到了验证：体外聚集被CaMKII抑制剂talCN21和talCN19o(阻断T位点)以及KN93(CaM竞争性的)减少。值得注意的是，这些和之前测试的阻止CaMKII激活的操作都减少了聚集，这表明了一种替代机制，而不是需要激酶活性。然而，用核苷酸竞争性广谱激酶抑制剂星形孢菌素和H7进行的实验表明，情况并非如此。在体外，星孢菌素和H7即使在没有核苷酸的情况下也能使CaMKII聚集。在大鼠海马神经元内，体内能够实时监测内源性CaMKII聚集。这种聚集可被talCN21阻断，但不能被星状孢子素阻断，即使两者都有效地抑制CaMKII活性。这些结果支持CaMKII聚集的机制模型，并表明不需要激酶活性。
The Ca2+/calmodulin-dependent protein kinase II (CaMKII) forms 12meric holoenzymes. These holoenzymes cluster into larger aggregates within neurons under ischemic conditions and invitro when ischemic conditions are mimicked. This aggregation is thought to be mediated by interaction between the regulatory domain of one kinase subunit with the T-site of another kinase subunit in a different holoenzyme, an interaction that requires stimulation by Ca2+/CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation invitro was reduced by the CaMKII inhibitors tatCN21 and tatCN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block CaMKII activation all reduced aggregation, suggesting an alternative mechanism that instead requires kinase activity. However, experiments with the nucleotide-competitive broad-spectrum kinase inhibitors staurosporin and H7 showed thatthis is not the case. In vitro, staurosporine and H7 enabled CaMKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous CaMKII aggregation. This aggregation was blocked by tatCN21, but not by staurosporine, even though both effectively inhibit CaMKII activity. These results support the mechanistic model for CaMKII aggregation and show that kinase activity is not required.