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 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.
Michell
Belinda J
BJ
St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.
Harris
M Brennan
MB
Chen
Zhi-Ping
ZP
Ju
Hong
H
Venema
Virginia J
VJ
Blackstone
Michele A
MA
Huang
Wei
W
Venema
Richard C
RC
Kemp
Bruce E
BE
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016228:Endothelial Growth Factors / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D002147:Calmodulin; D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012694:Serine; D000255:Adenosine Triphosphate; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D001920:Bradykinin; D002118:Calcium