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Identification of Regulatory Sites of Phosphorylation of the Bovine Endothelial Nitric-oxide Synthase at Serine 617 and Serine 635

2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês

10.1074/jbc.m205144200

1083-351X

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,

Protein Kinase Regulation and GTPase Signaling

Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser 1179 and dephosphorylation at Thr 497 activates the enzyme, whereas inhibition results when Thr 497 is phosphorylated and Ser 1179 is dephosphorylated. We have identified two further phosphorylation sites, at Ser 617 and Ser 635 , 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 Ser 617 . In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser 617 is Ca 2+ -dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser 635 is Ca 2+ -independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser 635 , but not Ser 617 , phosphorylation. Mimicking phosphorylation at Ser 635 by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser 617 does not alter maximal activity but significantly increases Ca 2+ -calmodulin sensitivity. These data show that phosphorylation of both Ser 617 and Ser 635 regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.