The mechanisms of NO inhibition of CaMK [Ca2+/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser847 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 Ser847, 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.
Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II
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Tao Song, Naoya Hatano, Toshie Kambe, Yoshiaki Miyamoto, Hideshi Ihara, Hideyuki Yamamoto, Katsuyoshi Sugimoto, Kodai Kume, Fuminori Yamaguchi, Masaaki Tokuda, Yasuo Watanabe; Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II. Biochem J 1 June 2008; 412 (2): 223–231. doi: https://doi.org/10.1042/BJ20071195
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