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 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.