Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells

J Biol Chem. 2000 Sep 8;275(36):28139-43. doi: 10.1074/jbc.M003198200.

Abstract

We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/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 IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) 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 IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) 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 Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Glioma
  • Hybrid Cells
  • Molecular Sequence Data
  • Neuroblastoma
  • Neurons / enzymology*
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Nitric Oxide Synthase / chemistry
  • Nitric Oxide Synthase / metabolism*
  • Nitric Oxide Synthase Type I
  • Okadaic Acid / pharmacology
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Serine*

Substances

  • Enzyme Inhibitors
  • Peptide Fragments
  • Okadaic Acid
  • Serine
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type I
  • Protein Kinases
  • (CaM)-dependent protein kinase Ia kinase
  • Protein-Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Kinase
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Phosphoprotein Phosphatases