Enhanced Electron Flux and Reduced Calmodulin Dissociation May Explain “Calcium-independent” eNOS Activation by Phosphorylation
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Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.
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NOX1 expression is activated by several factors including angiotensin II, PDGF, and thrombin.
“NOX1 expression is activated by several factors including angiotensin II, PDGF, and thrombin (382, 492, 630, 695, 999).”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [630]
The role of NOX1 in cardiovascular disease seems secondary to NOX2, but still significant, as shown in several models.
“Altogether, the role of NOX1 in cardiovascular disease seems secondary to NOX2, but still significant, as shown in several models (345, 380, 630, 854).”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [630]
NOX1 deletion limits the blood pressure increase elicited by angiotensin II.
“NOX1 deletion limits the blood pressure increase elicited by angiotensin II, although it is unclear if basal blood pressure levels are reduced (345, 346, 630).”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [630]
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“Phosphorylation of Ser(1179) activates eNOS by inducing a conformational change in eNOS that enhances the electron transfer rate (1, 24) or by inhibiting calmodulin dissociation from activated eNOS when calcium concentrations are low (25), thereby increasing NO production (26).”
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“Indeed, purified eNOS S1179D is more active than non-phosphorylated eNOS and has enhanced electron flux from the reductase to the oxygenase domain and reduced CaM dissociation at lower concentrations of calcium (McCabe et al., 2000).”
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Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium
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“Dysfunction of this pathway has been reported to contribute to the pathogenesis of many disorders, including hypertension and atherosclerosis (Ruetten et al., 1999; Mizuno et al., 2010).”
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Self-citationPhosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme
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“eNOS Serll77 phosphorylation further contributes to elevated eNOS enzyme activity by reducing CaM dissociation from activated eNOS [34,65] and allowing activation of eNOS at resting levels of calcium [45,66].”
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“catalyzed by protein kinase B (Akt) and a number of other kinases [16,17].”
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“Mutation of S1179 to D renders eNOS constitutively active, whereas S1179 to A is less activated (Dimmeler et al., 1999; Fulton et al., 1999; McCabe et al., 2000).”
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“The exact mechanism responsible for an increase of eNOS enzymatic activity is unclear, although the late phosphorylation at Ser1177 is thought to potentiate eNOS sensitivity for Ca2+/CaM complex (Dimmeler et al., 1999; Fulton et al., 1999; Gratton et al., 2000; McCabe et al., 2000).”
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“dissociation from activated eNOS [19].”
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Endothelial nitric oxide synthase transgenic models of endothelial dysfunction
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“Substitution of aspartate in place of S1177 (S1177D) mimics the increased catalytic activity caused by phosphorylation, since the aspartate side chain contains a negatively charged carboxyl group that mimics the negatively charged phosphate group of eNOS phosphorylated at S1177 [83, 85].”
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“The additional mutation of Ser(1177) to aspartate to increase electron flow through the reductase domain [17] slightly, but not significantly, enhanced O2 production.”
Mechanism of nitric oxide synthase regulation: Electron transfer and interdomain interactions
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“eNOS regulation by S1P involves another set of signalling pathways that modulate enzyme’s phosphorylation at the serine 1177 residue; this phosphorylation of eNOS at its C terminus ‘sensitizes’ the enzyme to activation at lower levels of calcium in vitro.(55) Full activation of eNOS by S1P requires a pathway comprisin…”
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Self-citationSrc Kinase Activates Endothelial Nitric-oxide Synthase by Phosphorylating Tyr-83
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Glycated Low Density Lipoproteins Modify Platelet Properties: A Compositional and Functional Study
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“During the experimental conditions of our study, insulin may have augmented the ACh vasodilator response because Ca2 -dependent activation of eNOS by ACh may be potentiated by Ca2 -independent eNOS phosporylation, through increased electron flux through the reductase domain of the enzyme at any concentration of intrac…”
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“The increase in electron flow, which occurs with phosphorylation of eNOS, increases the release of NO or O2 from NOS (23).”
BackgroundHypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium
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- Authors
- McCabe, T. J., Fulton, D., Roman, L. J., & Sessa, W. C.
- Affiliations
- Yale University (US)
- The University of Texas at San Antonio Health Science Center (US)
- Corresponding author
- Sessa, William C.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2000-03
- Volume
- 275
- Issue
- 9
- Pages
- 6123-6128
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2090688283
- Cited by
- 387
- References
- 32
- Citations by year
- 2025: 6 · 2024: 3 · 2023: 5 · 2022: 11 · 2021: 13 · 2020: 18 · 2019: 4 · 2018: 11 · 2017: 8 · 2016: 9 · 2015: 16 · 2014: 18 · 2013: 14 · 2012: 21
- FWCI
- 14.94
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- American Heart Association
- National Institutes of Health (HL61371, HL64793)
- National Heart, Lung, and Blood Institute (HL-61371, HL-64793, R01HL064793, HL 57665, R01HL057665)
- National Institute of General Medical Sciences (R01GM052419)
- Topics
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
- Keywords
- endothelial nitric oxide synthase, eNOS phosphorylation, Akt, nitric oxide production, electron flux, reductase domain, calcium-independent activation, cytochrome c reduction, L-arginine
- MeSH terms
- Animals, Calcium, Calmodulin, Cattle, Egtazic Acid, Electrons, Enzyme Activation, Kinetics, Mutation, NADH Dehydrogenase, NADP, Nitric Oxide, Phosphorylation, Proto-Oncogene Proteins, Protein Serine-Threonine Kinases, Nitric Oxide Synthase, Dimerization, Proto-Oncogene Proteins c-akt, Nitric Oxide Synthase Type III, Static Electricity
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