Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at Serine 617 and Serine 635

Belinda J. Michell, M. Brennan Harris, Zhi Ping Chen, Hong Ju, Virginia J. Venema, Michele A. Blackstone, Wei Huang, Richard C Venema, Bruce E. Kemp

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103 Scopus citations


Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser1179 and dephosphorylation at Thr497 activates the enzyme, whereas inhibition results when Thr497 is phosphorylated and Ser1179 is dephosphorylated. We have identified two further phosphorylation sites, at Ser617 and Ser635, by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser617. In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser617 is Ca2+-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser635 is Ca2+-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethyl-xanthine also causes Ser635, but not Ser617, phosphorylation. Mimicking phosphorylation at Ser635 by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser617 does not alter maximal activity but significantly increases Ca2+-calmodulin sensitivity. These data show that phosphorylation of both Ser617 and Ser635 regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.

Original languageEnglish (US)
Pages (from-to)42344-42351
Number of pages8
JournalJournal of Biological Chemistry
Issue number44
StatePublished - Nov 1 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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