Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation.
Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.
Harris
M B
MB
Vascular Biology Center, Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Ju
H
H
Venema
V J
VJ
Liang
H
H
Zou
R
R
Michell
B J
BJ
Chen
Z P
ZP
Kemp
B E
BE
Venema
R C
RC
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001920:Bradykinin / Q000494:pharmacology*; D065095:Calcineurin Inhibitors; D002417:Cattle; D002867:Chromones / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009025:Morpholines / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin
D000730:Androstadienes; D065095:Calcineurin Inhibitors; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D011518:Proto-Oncogene Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D016572:Cyclosporine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D001920:Bradykinin; D000077191:Wortmannin