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 (P KA) protein kinases. In vitro phosphorylation studies indicate that bo th PKG and PKA phosphorylate NOS on a single site. Phosphoamino-acid a nalysis and peptide mapping demonstrate that phosphorylation by either cyclic-nucleotide kinase occurs on a similar serine residue. Phosphor ylation of purified NOS by either PKG or PKA diminishes catalytic acti vity. Stimulation by 8-Br-cGMP of HEK-293 cells stably transfected wit h the cDNA for neuronal NOS (293.NOS cells) results in phosphorylation of immunoprecipitated NOS. Incubation of 293-NOS cells with 8-bromo-c GMP or dibutyryl-cAMP reduces nitrite release in response to stimulati on with calcium ionophore A23187. Phosphorylation-induced decreases in NOS activity may counterbalance and modulate NOS activating signals.