Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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 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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Moncada, S. & Higgs, A. The L-arginine–nitric oxide pathway. N. Engl. J. Med. 329, 2002–2012 (1993).

    CAS  Article  Google Scholar 

  2. 2

    Rudic, R. D. et al. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J. Clin. Invest. 101, 731–736 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Murohara, T. et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J. Clin. Invest. 101, 2567–2578 (1998).

    CAS  Article  Google Scholar 

  4. 4

    Rubanyi, G. M., Romero, J. C. & Vanhoutte, P. M. Flow-induced release of endothelium-derived relaxing factor. Am. J. Physiol. 250, H1145–H1149 (1986).

    CAS  Article  Google Scholar 

  5. 5

    Kuchan, M. J. & Frangos, J. A. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am. J. Physiol. 266, C628–C636 (1994).

    CAS  Article  Google Scholar 

  6. 6

    Sessa, W. C., Pritchard, K., Seyedi, N., Wang, J. & Hintze, T. H. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ. Res. 74, 349–353 (1994).

    CAS  Article  Google Scholar 

  7. 7

    Davies, P. F. Flow-mediated endothelial mechanotransduction. Physiol. Rev. 75, 519–560 (1995).

    CAS  Article  Google Scholar 

  8. 8

    Busse, R. & Fleming, I. Pulsatile stretch and shear: physiological stimuli determining the production of endothelial-derived relaxing factor. J. Vasc. Res. 35, 73–84 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Corson, M. A. et al. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ. Res. 79, 984–991 (1996).

    CAS  Article  Google Scholar 

  10. 10

    Fleming, I., Bauersachs, J., Fissthaler, B. & Busse, R. Ca2+-independent activation of the endothelial nitric oxide synthase in response to tryosine phosphatase inhibitors and fluid shear stress. Circ. Res. 79, 984–991 (1996).

    Article  Google Scholar 

  11. 11

    Franke, T. F. et al. The protein kinase encoded by the Akt proto-oncogen is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81, 727–736 (1995).

    CAS  Article  Google Scholar 

  12. 12

    Burgering, B. M. T. & Coffer, P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599–602 (1995).

    ADS  CAS  Article  Google Scholar 

  13. 13

    Downward, J. Lipid-regulating kinases: some common themes at last. Science 279, 673–674 (1998).

    ADS  CAS  Article  Google Scholar 

  14. 14

    Zeiher, A. M. Endothelial vasodilator dysfunction: pathogenetic link to myocardial ischaemia or epiphenomenon? Lancet 348, S10–S12 (1996).

    CAS  Article  Google Scholar 

  15. 15

    Moroi, M. et al. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular responses to injury in mice. J. Clin. Invest. 101, 1225–1232 (1998).

    CAS  Article  Google Scholar 

  16. 16

    Bredt, D. S. et al. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351, 714–718 (1991).

    ADS  CAS  Article  Google Scholar 

  17. 17

    Busse, R. & Mülsch, A. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 265, 133–136 (1990).

    CAS  Article  Google Scholar 

  18. 18

    Ayajiki, K., Kindermann, M., Hecker, M., Fleming, I. & Busse, R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ. Res. 78, 750–758 (1996).

    CAS  Article  Google Scholar 

  19. 19

    Dimmeler, S., Assmus, B., Hermann, C., Haendeler, J. & Zeiher, A. M. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ. Res. 83, 334–342 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Alessi, D. R. et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15, 6541–6551 (1996).

    CAS  Article  Google Scholar 

  21. 21

    van der Zee, R. et al. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation 95, 1030–1037 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Ziche, M. et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J. Clin. Invest. 99, 2625–2634 (1997).

    CAS  Article  Google Scholar 

  23. 23

    Gerber, H. P., Dixit, V. & Ferrara, N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J. Biol. Chem. 273, 13313–13316 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Förstermann, U. et al. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochem. Pharmacol. 42, 1849–1857 (1991).

    Article  Google Scholar 

  25. 25

    Yano, S., Tokumitsu, H. & Soderling, T. R. Calcium promotes cell survival through CaM-K kinase activation of the protein kinase-B pathway. Nature 396, 584–587 (1998).

    ADS  CAS  Article  Google Scholar 

  26. 26

    Zeiher, A. M., Fisslthaler, B., Schray-Utz, B. & Busse, R. Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. Circ. Res. 76, 980–986 (1995).

    CAS  Article  Google Scholar 

  27. 27

    Khwaja, A., Rodriquez-Viciana, P., Wennström, S., Warne, P. H. & Downward, J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J. 16, 2783–2793 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Dimmeler, S., Haendeler, J., Nehls, M. & Zeiher, A. M. Suppression of apoptosis by nitric oxide via inhibition of ICE-like and CPP32-like proteases. J. Exp. Med. 185, 601–608 (1997).

    CAS  Article  Google Scholar 

  29. 29

    Kauffmann-Zeh, A. et al. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385, 544–548 (1997).

    ADS  CAS  Article  Google Scholar 

  30. 30

    Copper, J. A., Sefton, B. M. & Hunter, T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 99, 387–402 (1983).

    Article  Google Scholar 

Download references


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

Author information



Corresponding author

Correspondence to Andreas M. Zeiher.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing