Identification of Regulatory Sites of Phosphorylation of the Bovine Endothelial Nitric-oxide Synthase at Serine 617 and Serine 635

Michell, Belinda J.; Harris, M. Brennan; Chen, Zhi Ping; Ju, Hong; Venema, Virginia J.; Blackstone, Michele A.; Huang, Wei; Venema, Richard C.; Kemp, Bruce E.

doi:10.1074/jbc.m205144200

Cited by 193 publications

(196 citation statements)

References 29 publications

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“…Phosphorylation of T495 reduces eNOS activity through reduced calmodulin association and increased O 2 Ϫ production (12,18,29). S633 phosphorylation of eNOS increases eNOS activity in cultured cells and in enzyme activity assays (2,32). There was no significant difference in the level of eNOS PO 4 at T494 [T495(hum)] or S632 [S633(hum)] in OZR vs. lean controls.…”

Section: Discussionmentioning

confidence: 71%

Insulin resistance does not diminish eNOS expression, phosphorylation, or binding to HSP-90

Fulton

Harris

Kemp

et al. 2004

American Journal of Physiology-Heart and Circulatory Physiology

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.-Previously, using an animal model of syndrome X, the obese Zucker rat (OZR), we documented impaired endothelium-dependent vasodilation. The aim of this study was to determine whether reduced expression or altered posttranslational regulation of endothelial nitric oxide synthase (eNOS) underlies the vascular dysfunction in OZR rats. There was no significant difference in the relative abundance of eNOS in hearts, aortas, or skeletal muscle between lean Zucker rats (LZR) and OZR regardless of age. There was no difference in eNOS mRNA levels, as determined by real-time PCR, between LZR and OZR. The inability of insulin resistance to modulate eNOS expression was also documented in two additional in vivo models, the ob/ob mouse and the fructose-fed rat, and in vitro via adenoviral expression of protein tyrosine phosphatase 1B in endothelial cells. We next investigated whether changes in the acute posttranslational regulation of eNOS occurs with insulin resistance. Phosphorylation of eNOS at S632 (human S633) and T494 was not different between LZR and OZR; however, phosphorylation of S1176 was significantly enhanced in OZR. Phosphorylation of S1176 was not different in the ob/ob mouse or in fructose-fed rats. The association of heat shock protein 90 with eNOS, a key regulatory step controlling nitric oxide and aberrant O 2 Ϫ production, was not different between OZR and LZR. Taken together, these results suggest that changes in eNOS expression or posttranslation regulation do not underlie the vascular dysfunction seen with insulin resistance and that other mechanisms, such as altered localization, reduced availability of cofactors, substrates, and the elevated production of O 2 Ϫ , may be responsible. syndrome X; Zucker; obesity INSULIN RESISTANCE, a prediabetic condition associated with obesity, is increasing at an alarming rate in Western cultures (26,30). Diabetes increases the incidence of many vasculopathies, including atherosclerosis, microvascular disease, and poor wound healing (44, 47). Of even greater concern is the mounting evidence that insulin-resistant states, independent of frank diabetes, also promote vascular dysfunction (43, 52). Thus the effect of insulin resistance on vascular signaling mechanisms is an area of increasing scrutiny. Insulin-resistant states and diabetes are associated with reduced endothelium-dependent relaxation, and this dysfunction of the endothelium is highly correlated with future cardiovascular events (1,14,34,49). In the obese Zucker rat (OZR), we previously showed that endothelium-dependent relaxation is impaired (14); however, the mechanisms underlying reduced synthesis or action of nitric oxide (NO) remain to be established.Endothelium-dependent relaxation is mediated by the synthesis and subsequent diffusion of NO from the endothelium to the underlying smooth muscle (21, 35). Of the three NO synthases (NOS), the endothelial isoform (eNOS) is uniquely positioned within the vascular endothelium and alone is responsible for endothelium-dependent NO-mediated relaxation (20)....

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Section: Discussionmentioning

confidence: 71%

Insulin resistance does not diminish eNOS expression, phosphorylation, or binding to HSP-90

Fulton

Harris

Kemp

et al. 2004

American Journal of Physiology-Heart and Circulatory Physiology

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“…eNOS phosphorylation as an activation mechanism was recognized only recently but the evidence supporting its importance has grown rapidly (Dimmeler et al, 1999;Fulton et al, 1999). Currently, 5 Ser/Thr residues (Ser116, Thr497, Ser617, Ser635 and Ser1179) are known to be phosphorylated by numerous protein kinases including PKA, protein kinase B (Akt), CaM kinase II, AMP-activated kinase and protein kinase C in response to physiological stimuli (Michell et al, 2002;. In our previous studies, the stimulation of PKA by treating BAECs with a cell-permeable cAMP analog, 8-Br-cAMP, was found to enhance the phosphorylation of eNOS at Ser635 and Ser1179 (Boo et al, 2002b).…”

Section: Resultsmentioning

confidence: 99%

“…It is currently viewed that while PKA phosphorylates eNOS at Ser1179 and Ser635, protein kinase B (Akt) phosphorylates Ser1179 and Ser617 in vitro and in cells (Dimmeler et al, 1999;Fulton et al, 1999;Butt et al, 2000;Boo et al, 2002a;Michell et al, 2002). Because both PKA and Akt can phosphorylate eNOS at Ser1179 in common, and they share consensus sequences, I further examined if the phospho-PKA substrate antibody detects eNOS phosphorylated by Akt.…”

Section: Resultsmentioning

confidence: 99%

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Boo

2006

Exp Mol Med

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Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.

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“…However, several lines of evidence have shown that PKA also regulates the phosphorylation of eNOS at Ser 1179 , Ser 635 , and Ser 617 in bovine eNOS (Ser 1177 , Ser 633 , and Ser 615 in humans) [24,[26][27][28][29] . Ser 633/635 phosphorylation is critical in maintaining NO synthesis after the initial sensitization by Ca 2+ flux and Ser 1177/1179 phosphorylation [30] , and it is stimulated via the PKA pathway in response to shear stress and acute statin treatment in aortic endothelial cells [24,27] , suggesting that PKA-mediated Ser 633/635 phosphorylation may be another mechanism by which statins protect against myocardial no-reflow and necrosis after ischemia and reperfusion.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

Yang

Geng³

et al. 2012

Acta Pharmacol Sin

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Aim: The cholesterol-lowering drugs statins could enhance the activities of endothelial nitric oxide synthase (eNOS) and protect myocardium during ischemia and reperfusion. The aim of this study was to examine whether protein kinase A (PKA) was involved in statinmediated eNOS phosphorylation and cardioprotection. Methods: 6-Month-old Chinese minipigs (20-30 kg) underwent a 1.5-h occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD). In the sham group, the LAD was encircled by a suture but not occluded. Hemodynamic and cardiac function was monitored using a polygraph. Plasma activity of creatine kinase and the tissue activities of PKA and NOS were measured spectrophotometrically. p-CREB, eNOS and p-eNOS levels were detected using Western blotting. Sizes of the area at risk, the area of no-reflow and the area of necrosis were measured morphologically. Results: Pretreatment of the animals with simvastatin (SIM, 2 mg/kg, po) before reperfusion significantly decreased the plasma activity of creatine kinase, an index of myocardial necrosis, and reduced the no-reflow size (from 50.4%±2.4% to 36.1%±2.1%, P<0.01) and the infarct size (from 79.0%±2.7% to 64.1%±4.5%, P<0.01). SIM significantly increased the activities of PKA and constitutive NOS, and increased Ser 133 p-CREB protein, Ser 1179 p-eNOS, and Ser 635 p-eNOS in ischemic myocardium. Intravenous infusion of the PKA inhibitor H-89 (1 μg·kg -1 ·min -1 ) partially abrogated the SIM-induced cardioprotection and eNOS phosphorylation. In contrast, intravenous infusion of the eNOS inhibitor L-NNA (10 mg·kg -1 ) completely abrogated the SIM-induced cardioprotection and eNOS phosphorylation during ischemia and reperfusion, but did not affect the activity of PKA. Conclusion: Pretreatment with a single dose of SIM 2.5 h before reperfusion attenuates myocardial no-reflow and infarction through increasing eNOS phosphorylation at Ser 1179 and Ser 635 that was partially mediated via the PKA signaling pathway.

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Identification of Regulatory Sites of Phosphorylation of the Bovine Endothelial Nitric-oxide Synthase at Serine 617 and Serine 635

Cited by 193 publications

References 29 publications

Insulin resistance does not diminish eNOS expression, phosphorylation, or binding to HSP-90

Insulin resistance does not diminish eNOS expression, phosphorylation, or binding to HSP-90

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

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