Differences in eNOS activity due to subcellular localization are dictated by phosphorylation state rather than the local calcium environment

doi:10.1074/jbc.M505968200

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Differences in eNOS activity due to subcellular localization are dictated by phosphorylation state rather than the local calcium environment

Jarrod E. Church and David Fulton

Vascular Biology Center and, Medical College of Georgia, Augusta, GA 30912-2500

Corresponding Author: dfulton{at}mcg.edu

Nitric oxide (NO) produced in the endothelium via the enzyme endothelial nitric oxide synthase (eNOS) is an important vasoactive compound. Wild type (WT) eNOS is localized to the plasma membrane and perinuclear/Golgi region by virtue of N-terminal myristoylation and palmitoylation. Acylation deficient mutants (G2AeNOS) remain cytosolic and release less NO in response to Ca2+ elevating agonists, a disparity that we hypothesized was due to the greater distance between G2AeNOS and plasma membrane Ca2+ influx channels. The reduced activity of G2AeNOS versus WT, was reversed upon disruption of cellular integrity with detergents or sonication. NO production from both constructs relied almost exclusively on the influx of extracellular Ca2+, and elevating intracellular Ca2+ to saturating levels with 10 mM ionomycin in the presence of 10 mM extracellular Ca2+, equalized NO production. To identify the contribution of calcium to the differences in activity between these enzymes, we created Ca2+/CaM independent eNOS mutants by deleting the two putative autoinhibitory domains of eNOS. There was no difference in NO production between WT or G2A targeted Ca2+ -independent eNOS, suggesting that Ca2+ was the factor responsible. When eNOS constructs were fused in frame to the bioluminescent probe aequorin, membrane bound probes were exposed to higher [Ca2+] in unstimulated cells but upon ionomycin stimulation the probes experienced equal amounts of Ca2+. The WT and G2A enzymes displayed significant differences in the phosphorylation state of S617, S635 and S1179 and mutating all three sites to alanine or restoring phosphorylation with the phosphatase inhibitor calyculin abolished the differences in activity. We therefore conclude that the disparity in NO production between WTeNOS and G2AeNOS is not due to different localized [Ca2+] upon stimulation with ionomycin, but rather differences in phosphorylation state between the two constructs.

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