Neuronal nitric oxide synthase specific autophosphorylation in baculovirus/Sf9 insect cell system

Neuronal nitric oxide synthase (nNOS) is a calmodulin (CaM)-dependent enzyme, which generates the ubiquitous biological mediator nitric oxide. In this study, wild-type or mutant form of rat nNOS was overexpressed in a baculovirus/Sf9 insect cell system and examined for autophosphorylation. Incubation of purified wild-type nNOS in the presence of Mg2+ and ATP result in phosphorylation of nNOS. Deletional mutant of the nNOS amino terminus which contains G-Q-G-A-G-S sequence, however, shows loss of phosphorylation. Furthermore, purified wild-type rat liver cytokine-induced NOS (iNOS), which does not contain G-Q-G-A-G-S sequence, shows no phosphorylation activity. We also found that the ratio of wild-type nNOS phosphorylation was independent of nNOS concentration, consistent with an intramolecular reaction. These data suggest that nNOS-specific autophosphorylation pathway might be involved in the regulation of enzyme activity.