Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.
Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.
Dimmeler
S
S
Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Germany.
Fleming
I
I
Fisslthaler
B
B
Hermann
C
C
Busse
R
R
Zeiher
A M
AM
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism; D013552:Swine; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine; D000077191:Wortmannin; C496314:NOS3 protein, human; C494918:AKT1 protein, human