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Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation

Abstract

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 angiogenesis1,2,3. 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 layer4,5,6,7,8. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity9,10, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB11,12,13 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.

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Figure 1: Shear stress stimulates the phosphorylation of Akt.
Figure 2: Inhibition of PI(3)K prevents shear-stress-induced cGMP increase and NO release.
Figure 3: Inhibition of PI(3)K by wortmannin inhibits the shear-stress-induced phosphorylation of eNOS.
Figure 4: Akt phosphorylates Ser 1177 and increases eNOS enzyme activity.
Figure 5: Akt phosphoryiates SA177 and increases eNOS enzyme activity in cells.
Figure 6: Calcium dependence of eNOS activity and Akt phosphorylation.

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Acknowledgements

We thank C. Goebel, I. Winter and S. Ficus for expert technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft.

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Correspondence to Andreas M. Zeiher.

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Dimmeler, S., Fleming, I., Fisslthaler, B. et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605 (1999). https://doi.org/10.1038/21224

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