Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme

Chun An Chen, Lawrence J. Druhan, Saradhadevi Varadharaj, Yeong Renn Chen, Jay L. Zweier

Research output: Contribution to journalArticle

Abstract

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 O 2.- 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.

Original languageEnglish (US)
Pages (from-to)27038-27047
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number40
DOIs
StatePublished - Oct 3 2008
Externally publishedYes

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Phosphorylation
Nitric Oxide Synthase Type III
Superoxides
Nitric Oxide Synthase
Enzymes
Nitric Oxide
Calmodulin
Protein Kinase C
Caveolins
Calcium
Spin Trapping
Cohort Effect
Oxidative stress
Endothelial cells
NADP
Vascular Endothelial Growth Factor A
Paramagnetic resonance
Oxidative Stress
Phosphotransferases
Endothelial Cells

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme. / Chen, Chun An; Druhan, Lawrence J.; Varadharaj, Saradhadevi; Chen, Yeong Renn; Zweier, Jay L.

In: Journal of Biological Chemistry, Vol. 283, No. 40, 03.10.2008, p. 27038-27047.

Research output: Contribution to journalArticle

Chen, Chun An ; Druhan, Lawrence J. ; Varadharaj, Saradhadevi ; Chen, Yeong Renn ; Zweier, Jay L. / Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 40. pp. 27038-27047.
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AU - Zweier, Jay L.

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AB - 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 O 2.- 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.

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