Bidirectional regulation of neuronal nitric-oxide synthase phosphorylation at serine 847 by the N-methyl-D-aspartate receptor.
At glutamatergic synapses, the scaffolding protein PSD95 links the neuronal isoform of nitric-oxide synthase (nNOS) to the N-methyl-d-aspartate (NMDA) receptor. Phosphorylation of nNOS at serine 847 (Ser(847)) by the calcium-calmodulin protein kinase II (CaMKII) inhibits nNOS activity, possibly by blocking the binding of Ca(2+)-CaM. Here we show that the NMDA mediates a novel bidirectional regulation of Ser(847) phosphorylation. nNOS phosphorylated at Ser(847) colocalizes with the NMDA receptor at spines of cultured hippocampal neurons. Treatment of neurons with 5 microm glutamate stimulated CaMKII phosphorylation of nNOS at Ser(847), whereas excitotoxic concentrations of glutamate, 100 and 500 microm, induced Ser(847)-PO(4) dephosphorylation by protein phosphatase 1. Strong NMDA receptor stimulation was likely to activate nNOS under these conditions because protein nitration to form nitrotyrosine, a marker of nNOS activity, correlated in individual neurons with Ser(847)-PO(4) dephosphorylation. Of particular note, stimulation with low glutamate that increased phosphorylation of nNOS at Ser(847) could be reversed by subsequent high glutamate treatment which induced dephosphorylation. The reversibility of NMDA receptor-induced phosphorylation at Ser(847) by different doses of glutamate suggests two mechanisms with opposite effects: 1). a time-dependent negative feedback induced by physiological concentrations of glutamate that limits nNOS activation and precludes the overproduction of NO; and 2). a pathological stimulation by high concentrations of glutamate that leads to unregulated nNOS activation and production of toxic levels of NO. These mechanisms may share pathways, respectively, with NMDA receptor-induced forms of synaptic plasticity and excitotoxicity.
Rameau
Gerald A
GA
Howard Hughes Medical Institute, Department of Biochemistry, New York University School of Medicine, New York, New York 10016, USA.
Chiu
Ling-Yu
LY
Ziff
Edward B
EB
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018698:Glutamic Acid / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D009473:Neuronal Plasticity / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013569:Synapses / Q000201:enzymology
D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D012694:Serine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1