Abstract
We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative "gain of function" mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions.
Highlights
We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase on basal and stimulated nitric oxide (NO) release, cooperative phosphorylation, and co-association with hsp90 and Akt
Specificity of Phospho-endothelial nitric-oxide synthase (eNOS)-specific Antibodies—We first set out to determine the specificity of the antibodies generated against phospho-eNOS serine 116 (Ser-116), serine 617 (Ser-617), and serine 635 (Ser-635)
vascular endothelial growth factor (VEGF)-stimulated Phosphorylation of eNOS—We examined the effects of VEGF stimulation on phosphorylation of Ser-1179, Ser-116, Ser-617, and Ser-635
Summary
Inhibitory loop and are phosphorylated in response to endothelial cell stimulation by VEGF, ATP, and bradykinin, in addition Ser-635 is phosphorylated in response to shear stress [23, 24]. A recent report showed that mutation of Ser-635 to aspartate causes a 2-fold increase in maximal activity of the purified enzyme, an effect comparable to S1179D [23]. Mutation of Ser-617 to aspartate was recently shown to increase calcium sensitivity without changing maximal enzyme activity of the purified enzyme [23]. Previous studies describing the role of these serine phosphorylation sites do so by examining eNOS enzyme activity of the purified enzyme or in detergent-solubilized cell lysates. Activity assays of the purified enzyme are done in the absence of eNOS-associated proteins (caveolin-1, heat shock protein 90, etc.) and without phosphorylation at other sites and, do not always accurately reflect eNOS activity or the production of NO in live cells. We determined the effect of the eNOS phosphorylation site mutations on the ability of the heat shock protein 90 (hsp90) and the kinase, Akt, to coassociate with eNOS
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