Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants

Abstract

Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein. The functional diversity of the nNOS isoform is also linked to its splice variants. Of note is that determination of phosphorylation stoichiometry remains as a challenge. This study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSμ and nNOSα proteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively. A quantitative mass spectrometric approach was then developed to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, under l-Arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSμ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on NOS activity that may differ between variants and isoforms. Furthermore, the methodologies for incorporating pSer into large protein and quantifying the percentage of pSer should be applicable to other protein systems.