Regulation of neuronal nitric-oxide synthase by calmodulin kinases
Regulation of neuronal nitric-oxide synthase by calmodulin kinases
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DOI：
10.1074/jbc.274.29.20597
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发表时间：
1999-07-16
影响因子：
4.8
通讯作者：
Watanabe, Y
中科院分区：
文献类型：
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作者：
Hayashi, Y;Nishio, M;Watanabe, Y
研究了Ca2+/钙调蛋白（CaM）依赖性蛋白激酶（CaM激酶）对神经元一氧化氮合成酶（nNOS）的磷酸化作用，包括CaM激酶I α (CaM- ki α)、CaM激酶II α (CaM- kii α)和CaM激酶IV （CaM- kiv）。纯化后的重组nNOS在体外可被cam - ki α、cam - kii α和cam - kiv磷酸化Ser（847）。用Ala （S847A）代替Ser（847）可以阻止CaM激酶的磷酸化，磷酸化的重组野生型nNOS在Ser（847）处（大约0.5 mol的磷酸盐掺入nNOS中）的V-max比未磷酸化的酶降低30%，l -精氨酸的K-m和CaM的K-act都没有变化。突变体S847D的活性下降到野生型酶的50-60%。磷酸化的nNOS在Ser（847）和突变体S847D上的NOS酶活性下降部分是由于CaM结合受到抑制，而不是由于二聚体形成受损，二聚体被认为是酶激活所必需的。缺乏cam结合能力的失活nNOS是由Lys(732)-Lys-Leu突变为Asp(732)-Asp-Glu产生的（Watanabe， Y., Hu， Y., and Hidaka， H.（1997）《生物多样性杂志》，403,75-78）。与野生型酶一样，它被CaM激酶磷酸化，这表明磷酸化反应不需要CaM- nnos结合。我们开发了抗体NP847，该抗体特异性识别Ser（847）磷酸化状态的nNOS。利用抗体NP847，我们获得了nNOS在大鼠脑中磷酸化Ser（847）的证据。因此，我们的研究结果表明，CaM激酶诱导的nNOS丝氨酸（847）磷酸化改变了该酶的活性控制。
Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase I alpha (CaM-K I alpha), CaM kinase II alpha (CaM-K II alpha), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K I alpha, CaM-K II alpha, and CaM-K IV at Ser(847) in vitro. Replacement of Ser(847) with Ala (S847A) prevented phosphorylation by CaM kinases, Phosphorylated recombinant wild-type nNOS at Ser(847) (approximate to 0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease of V-max with little change of both the K-m for L-arginine and K-act for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50-60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser(847) and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys(732)-Lys-Leu to Asp(732)-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75-78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser(847). Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser(847) in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser(847) alters the activity control of this enzyme.