Inhibition of neuronal nitric-oxide synthase by phosphorylation at Threonine1296 in NG108-15 neuronal cells

We demonstrate that neuronal nitric-oxide synthase (nNOS) is directly inhibited through the phosphorylation of Thr¹²⁹⁶ in NG108-15 neuronal cells. Treatment of NG108-15 cells expressing nNOS with calyculin A, an inhibitor of protein phosphatase 1 and 2A, revealed a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr¹²⁹⁶ residue. Cells expressing a phosphorylation-deficient mutant in which Thr ¹²⁹⁶ was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation mutant of nNOS in which Thr¹²⁹⁶ is changed to Asp showed a significant decrease in nNOS enzyme activity, being competitive with NADPH, relative to the wild-type enzyme. These data suggest that phosphorylation of nNOS at Thr¹²⁹⁶ may involve the attenuation of nitric oxide production in neuronal cells through the decrease of NADPH-binding to the enzyme.