Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH4 is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O2·-). Although phosphorylatio...

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Bibliographic Details
Published in:The Journal of biological chemistry 2008-10, Vol.283 (40), p.27038-27047
Main Authors: Chen, Chun-An, Druhan, Lawrence J., Varadharaj, Saradhadevi, Chen, Yeong-Renn, Zweier, Jay L.
Format: Article
Language:English
Subjects:
Animals
Calcium - metabolism
Calmodulin - metabolism
Cattle
Cells, Cultured
Endothelial Cells - cytology
Endothelial Cells - enzymology
Humans
Mechanisms of Signal Transduction
Nitric Oxide - biosynthesis
Nitric Oxide Synthase Type III - genetics
Nitric Oxide Synthase Type III - metabolism
Oxidation-Reduction
Phosphorylation
Protein Kinase C-alpha - genetics
Protein Kinase C-alpha - metabolism
Proto-Oncogene Proteins c-akt - genetics
Proto-Oncogene Proteins c-akt - metabolism
Superoxides - metabolism
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Summary:In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH4 is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O2·-). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O2·- generation are unknown. Therefore, we phosphorylated recombinant BH4-free eNOS in vitro using native kinases and determined O2·- generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O2·- generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca2+ sensitivity of eNOS, such that O2·- generation became largely Ca2+-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Cα (PKCα) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O2·- generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O2·- generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O2·- and NO generation, altering both the Ca2+ sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O2·- generation from the enzyme at low Ca2+ concentrations, and PKCα-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M802269200