Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser⁸⁴⁷ phosphorylation of neuronal nitric oxide synthase

Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N-methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca²⁺/calmodulin-dependent protein kinase IIα (CaM-K IIα) directly phosphorylates nNOS at residue Ser⁸⁴⁷, and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser⁸⁴⁷ induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Iα and CaM-K IV failed to phosphorylate nNOS at Ser⁸⁴⁷ in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser⁸⁴⁷ by CaM-K II in neuronal cells.