Cyclic nucleotide dependent phosphorylation of neuronal nitric oxide synthase inhibits catalytic activity.
We have examined the regulation of neuronal nitric oxide synthase (NOS) by phosphorylation with cyclic-GMP (PKG) and cyclic-AMP-dependent (PKA) protein kinases. In vitro phosphorylation studies indicate that both PKG and PKA phosphorylate NOS on a single site. Phosphoamino-acid analysis and peptide mapping demonstrate that phosphorylation by either cyclic-nucleotide kinase occurs on a similar serine residue. Phosphorylation of purified NOS by either PKG or PKA diminishes catalytic activity. Stimulation by 8-Br-cGMP of HEK-293 cells stably transfected with the cDNA for neuronal NOS (293.NOS cells) results in phosphorylation of immunoprecipitated NOS. Incubation of 293-NOS cells with 8-bromo-cGMP or dibutyryl-cAMP reduces nitrite release in response to stimulation with calcium ionophore A23187. Phosphorylation-induced decreases in NOS activity may counterbalance and modulate NOS activating signals.
Dinerman
J L
JL
Department of Medicine (Division of Cardiology), Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Steiner
J P
JP
Dawson
T M
TM
Dawson
V
V
Snyder
S H
SH
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D006152:Cyclic GMP / Q000502:physiology*; D004586:Electrophoresis; D006801:Humans; D009474:Neurons / Q000201:enzymology*; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D009566:Nitrates; D010748:Phosphopeptides; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D011494:Protein Kinases; D006152:Cyclic GMP