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

Abstract

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 endo..