Bidirectional Regulation of Neuronal Nitric-oxide Synthase Phosphorylation at Serine 847 by the N-Methyl-d-aspartate Receptor

• Published in: The Journal of biological chemistry Vol. 279; no. 14; pp. 14307 - 14314
• Main Authors: Rameau, Gerald A, Chiu, Ling-Yu, Ziff, Edward B
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 02.04.2004
• Subjects: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Cells, Cultured; Cerebral Cortex - cytology; Glutamic Acid - metabolism; Hippocampus - cytology; Neuronal Plasticity - physiology; Neurons - cytology; Neurons - enzymology; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type I; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Phosphatase 1; Rats; Receptors, N-Methyl-D-Aspartate - metabolism; Serine - metabolism; Synapses - enzymology
• DOI: 10.1074/jbc.M311103200
• ISSN: 0021-9258

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 μ m glutamate stimulated CaMKII phosphorylation of nNOS at Ser 847 , whereas excitotoxic concentrations of glutamate, 100 and 500 μ m , 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.