Inhibition of Neuronal Nitric-oxide Synthase by Phosphorylation at Threonine1296 in NG108-15 Neuronal Cells

Abstract

Neuronal nitric-oxide synthase (nNOS) has been shown to function as signal generators controlled by electrons supplied by NADPH and Ca²⁺-dependent binding of calmodulin (CaM). In the present study, we established that nNOS is directly inhibited through the phosphorylation of Thr1296 in NG108-15 neuronal cells. The treatment of calyculin A or okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, resulted in a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr1296 residue in NG108-15 cells expressing nNOS. Cells expressing a phosphorylation-deficient mutant in which Thr1296 was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation at Thr1296 by introduction of a Thr to Asp mutation resulted in a 40-50% decrease in nNOS enzyme activity and 20-fold increase in the Km for NADPH, with little change in the Kact for CaM relative to the wild-type enzyme. The mutant and Thr1296 -phosphorylated nNOS showed decrease of ADP-agarose binding ability relative to wild-type and un-phosphorylated enzymes, respectively. These data suggests that phosphorylation of nNOS at Thr1296 may involve the attenuation of NO production in neuronal cells through the decrease of NADPH-binding to the enzyme. [Jpn J Physiol 55 Suppl:S206 (2005)]