Inhibition of neuronal nitric-oxide synthase by calcium/calmodulin-dependent protein kinase IIα through ser847 phosphorylation in NG108-15 neuronal cells

Abstract

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser847 by Ca2+/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIα (CaM-K IIα) can directly phosphorylate nNOS on Ser847, leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Iα (CaM-K Iα), CaM-K IIα, and CaM-kinase IV (CaM-K IV) on Ser847 were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser-Asp-Ser- Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIα, 100; CaM-K Iα, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIα1-274 but not inactive CaM-K IIα1-274, generated by mutation of Lys42 to Ala, with nNOS into NG108-15 cells, resulted in increased Ser847 phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca2+ triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIα phosphorylating nNOS on Ser847, which in turn decreases the gaseous second messenger NO in neuronal cells.