Abstract
Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.
Highlights
Endothelial nitric-oxide synthase1 is an important enzyme in the cardiovascular system producing nitric oxide (NO), a key regulator of blood pressure, platelet function, and vessel remodeling
Several laboratories have shown that Akt phosphorylation at Ser1179 mediates an increase in Endothelial nitric-oxide synthase (eNOS) activity in response to stimulation by vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (4, 9) and fluid shear stress (5, 6)
We have found that several protein kinases, including protein kinase C and AMP kinase, phosphorylate both Thr497 and Ser1179 in vitro, but only one site is accessible to each kinase in endothelial cells
Summary
Phosphorylation of eNOS and eNOS Peptides—Recombinant human eNOS (2.5 M) (7) was phosphorylated by GST-Akt in kinase assay buffer (50 mM Hepes, pH 7.5, 10 mM MgCl2, 5% glycerol, 1 mM dithiothreitol, 0.05% Triton X-100) containing 50 M [␥-32P]ATP (5000 cpm/pmol) for 4 h. Rat heart or recombinant eNOS was phosphorylated by PKA in kinase assay buffer containing 250 M ATP for Western blot analysis or 50 M [␥-32P]ATP (10,000 cpm/pmol) for phosphopeptide mapping, with either 1 mM EGTA or 100 M CaCl2 and 1 M CaM for 1– 4 h at 22 °C. Phosphopeptide Extraction and Purification of Tryptic Digests— Tryptic peptides of eNOS gel bands were prepared as described previously (7) and extracted with consecutive washes in 2% trifluoroacetic acid (TFA), 0.1% TFA with 30% acetonitrile, 0.1% TFA with 60% acetonitrile. Enzyme-liked immunosorbent assays and Western blot analyses confirmed that the purified antibodies did not recognize dephosphorylated eNOS. Wild-type and mutant virus stocks were used to express and purify the various forms of eNOS protein as described previously (18). Assay of eNOS Activity—Activities of purified wild-type and mutant forms of eNOS (1 g) were assayed by monitoring the rate of conversion of L-[14C]arginine to L-[14C]citrulline as previously described (18)
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