Presenter: Adel Elmoselhi,  St. Boniface Cardiovascular Research Centre, Winnipeg, MB, Canada.

Presenter’s Summary:
Endothelial-derived nitric oxide (NO) is critical in regulating vascular function in both physiological and pathological conditions. NO is produced within endothelial cells by eNOS (endothelial nitric oxide synthase) via L-arginine pathway. Several stimuli (vascular endothelial growth factor VEGF, shear stress, insulin, and receptor-operated agonists) phosphorylate e-NOS  and induce NO production. VEGF and insulin were shown to stimulate NO production via phosphatitylinositol-3-OH-kinase [PI(3)K] dependent pathway.  However, the exact kinase(s) and the role of their phosphorylation in NO production are not known.
Protein kinase B (Akt) is an important proto-oncogene that involved in glucose metabolism and cell survival. Akt is phosphorylated and activated via tyrosine kinase, G-coupled receptors, and shear stress that ultimately result in either protein activation or inactivation. This paper reports that Akt phosphorylates eNOS on Serine 1179 and this phosphorylation increases the production of NO.
Several lines of evidence were presented to show that Akt could phosphorylate eNOS on Ser 1179 and demonstrate its role in NO production:
1- Akt modulates eNOS and enhances NO production under basal conditions: Co-transfection of COS-7 cells with eNOS and the wild-type Akt (HA-Akt) enhance the accumulation of nitrate (measured by NO-specific chemiluminenscence), but not with the kinase inactive Akt (HA-Akt K179M). Similar results were obtained  using cyclic guanosine monophosphate (cGMP) bioassay, where cGMP accumulation were 5.5, 11.6, 5.8 pmol cGMP/mg protein in transfected cells with eNOS alone, or with eNOS and active-kinase Akt, or with eNOS and kinase-inactive Akt respectively.
2- eNOS and Akt association to the membrane is essential for NO production: in co-transfected COS-7 cells, Akt did not activate the non-acylated form of eNOS.
3- Specificity of Akt to eNOS: co-tansfection of Akt with neural NOS (nNOS) or inducible (iNOS) did not increase NO production.
4- eNOS is a substrate for Akt phosphorylation in vitro:  In transfected COS cells with active kinase (HA-Akt) or an inactive kinase (HA-Akt(K179M)), the active kinase phosphorylates histone 2B and eNOS while the inactive kinase did not.
5- Identify eNOS phosphorylation sites by Akt: (a) Indirectly, the two serine sites (ser 635 and 1179) were mutated to alanine residues. In intact COS cells, Akt resulted in twofold increase in the phosphorylation of the wild type vs mutant eNOS. Also, wortmannin, PI(3)K inhibitor, diminished Akt induced phosphorylation in eNOS/Akt transfected cells.  (b) Directly, by using high –performance liquid chromatography followed by matrix-assisted laser desorption,  ionization mass spectrometry for the wild-type eNOS incubated with immunopurified Akt, the tryptic phosphopeptide labeled with 32P co-elutes with a synthetic phosphopeptide ( a.a. 1177-1185, with phosphoserine at position 1179) and loss of H3PO4 indicating its phosphorylation.  Also, mutation of the Ser 1179 with alanine decreases Akt phosphorylation of eNOS compared to the wild type. Moreover, similar results were obtained using peptides ( a.a. 1174-1194) as a substrates for recombinant Akt and the wild type eNOS incorporated higher phosphate per mg compared to the alanine mutant peptide (24.6 vs. 0.22 nmol phosphate/mg respectively).
6- The functional importance of Ser 1179 in NO production: In COS cell transfection with double mutant S635 and 1179A  diminished Akt dependent NO release. Alanine mutant of Ser 635 did not affect NO production, while eNOS S1179 A diminished the its activity.
7- Akt mediates NO production from endothelial cells at resting level of Ca2+:  In bovine lung microvascular endothelial cells infected with adenoviruses expressing activated Akt(myr-Akt) stimulated NO production, while the cells infected with activated deficient Akt (AA-Akt) or beta-galactosidase (control) did not. Also, eNOS activity measured in lysates  of myr-Akt infected cells was enhanced by Ca2+(at fixed [calmodulin]) relative to cells infected with beta-galactosidase or AA-Akt viruses.
8- Akt participation in the signal transduction of NO production in endothelial cells via VEGF:   endothelial cells infected with myr-Akt showed increase in VEGF-induced NO production compared to cells infected with beta-galactosidase or AA-Akt viruses.

This evidence indicates that Akt can phosphorylate eNOS and consequently increase NO production. This phosphorylation occurs in serine 1179. Also their results shows the importance of compartmentalization of both eNOS and Akt to the membrane in order to achieve their functional interaction. Furthermore, the phosphorylation of the eNOS by Akt in the endothelial cells to induce NO occurs at resting level of [Ca2+]i . Akt is involved in the signal pathway of the VEGF-induced NO production.
 

Key Findings:
A) Protein kinase Akt can phosphorylate endothelium-derived nitric oxide synthase (eNOS) on serine 1179, which enhance nitric oxide (NO) production.
B) Akt phosphorylation of eNOS is involved in the signal pathway of different stimuli ( e.g. insulin, shear stress) which induce NO production via PI(3)dependent mechanism and independent of [Ca2+]i increase.

Why is this paper important ?
NO production via Akt-induced phosphorylation of eNOS can be use as a therapeutic target  in several cardiovascular diseases (e.g. hypertension, heart failure, atherosclerosis) where NO deficiency plays an essential role in its pathogenesis.

Questions unanswered:
Although this study shows nicely the specific site of phosphorylation of eNOS by Akt and relates it to NO production, many issues need to be consider further for example:
How much of the role Akt phosphorylation play in NO production in vivo model ?? How Akt interacts with different proteins induce NO such as Hsp90, CaMK, and AMP-activated kinase ?? Evidence is needed to explain how phosphorylation of eNOS by Akt increase NO production ??   In case of agonists induced NO production via activation of CaMK and increase [Ca2+]i , does Akt phosphorylate eNOS or may be other Ca2+-dependent protein(s) are involved ??
 

Authors’ Abstract:
Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.
 

Related references:
1- Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM , “Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation”. Nature 1999 Jun 10;399(6736):601-5.
2- Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE, “AMP-activated protein kinase phosphorylation of endothelial NO synthase”. FEBS Lett 1999 Jan 29;443(3):285-9