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

Abstract

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 (Ser847) by the calcium-calmodulin protein kinase II (CaMKII) inhibits nNOS activity, possibly by blocking the binding of Ca2+-CaM. Here we show that the NMDA mediates a novel bidirectional regulation of Ser847 phosphorylation. nNOS phosphorylated at Ser847 colocalizes with the NMDA receptor at spines of cultured hippocampal neurons. Treatment of neurons with 5 μM glutamate stimulated CaMKII phosphorylation of nNOS at Ser 847, whereas excitotoxic concentrations of glutamate, 100 and 500 μM, induced Ser847-PO4 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-PO4 dephosphorylation. Of particular note, stimulation with low glutamate that increased phosphorylation of nNOS at Ser847 could be reversed by subsequent high glutamate treatment which induced dephosphorylation. The reversibility of NMDA receptor-induced phosphorylation at Ser847 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.