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

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.