Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II
Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II
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DOI：
10.1042/bj20071195
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发表时间：
2008-06-01
影响因子：
4.1
通讯作者：
Watanabe, Yasuo
中科院分区：
文献类型：
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作者：
Song, Tao;Hatano, Naoya;Watanabe, Yasuo
研究了NO抑制CaMK [Ca ~（2+）/CaM（钙调素）依赖性蛋白激酶] 11活性的机制。在大鼠垂体瘤GH 3细胞中，促甲状腺激素（TSH）释放激素（TRH）刺激的nNOS（神经元型NOS（NO合酶））Ser（847）磷酸化对CaMK抑制剂KN-93敏感，并且通过7-硝基吲唑（7-nitroindazole）抑制nNOS而增强。用7 NI原位处理后的CaMKII的酶活性也增加。通过与nNOS和L-精氨酸或与NO供体SNAP（S-亚硝基-N-乙酰基-DL-青霉胺）和DEA-NONOate [二乙胺-NONOate（diazeniumdiolate）]共孵育抑制CaMK II的体外活性。一旦被这些处理抑制，观察到CaMKII在添加还原剂DTT（二硫苏糖醇）时经历完全再活化。在表达CaMKII和nNOS的转染细胞中，用钙离子载体A23187处理进一步揭示了nNOS在Ser（847）处的磷酸化，这被7 NI和CaMKII S-亚硝基化增强。突变的CaMKII（C6 A），其中CyS 6被替换为丙氨酸残基，是难治的7 NI诱导的增强nNOS磷酸化或CaMKII S-亚硝基化。此外，我们可以使用MS鉴定CyS 6为CaMKII S-亚硝基化的直接靶点。此外，谷氨酸处理导致大鼠海马切片中CaMKII S-亚硝基化增加。这种谷氨酸诱导的S-亚硝基化被7 NI阻断。这些结果表明，在CyS 6的S-亚硝基化介导的CaMKII的失活可能有助于NO诱导的神经毒性在大脑中。
The mechanisms of NO inhibition of CaMK [Ca2+/CaM (calmodulin)-dependent protein kinase] 11 activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which CyS6 was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify CyS6 as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at CyS6 may contribute to NO-induced neurotoxicity in the brain.