Cited by 770Nitric Oxide and Endothelin EffectsProtein Kinase Regulation and GTPase SignalingCardiovascular Health and Disease Prevention

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Phosphorylation of Thr 495 Regulates Ca 2+ /Calmodulin-Dependent Endothelial Nitric Oxide Synthase Activity

Fleming, Ingrid, Fisslthaler, Beate, Dimmeler, Stefanie, Kemp, Bruce E., Busse, Rudi·2001·Circulation Research

DOI 10.1161/hh1101.092677

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Abstract

Abstract —The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca 2+ by the phosphorylation of Ser 1177 but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca 2+ -dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr 495 in the calmodulin (CaM)-binding domain. eNOS Thr 495 was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation. In the same cells, bradykinin enhanced the phosphorylation of Ser 1177 , which was maximal after 5 minutes, and abolished by the CaM-dependent kinase II (CaMKII) inhibitor KN-93. Bradykinin also enhanced the association of CaMKII with eNOS. Phosphorylation of Thr 495 was attenuated by the protein kinase C (PKC) inhibitor Ro 31-8220 and after PKC downregulation using phorbol 12-myristate 13-acetate. The agonist-induced dephosphorylation of Thr 495 was completely Ca 2+ -dependent and inhibited by the PP1 inhibitor calyculin A. Little CaM was bound to eNOS immunoprecipitated from unstimulated cells, but the agonist-induced dephosphorylation of Thr 495 enhanced the association of CaM. Mutation of Thr 495 to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp 495 mutant bound almost no CaM. Accordingly, NO production by the Ala 495 mutant was more sensitive to Ca 2+ /CaM than the aspartate mutant. These results suggest that the dual phosphorylation of Ser 1177 and Thr 495 determines the activity of eNOS in agonist-stimulated endothelial cells. Moreover, the dephosphorylation of Thr 495 by PP1 precedes the phosphorylation of Ser 1177 by CaMKII. The full text of this article is available at http://www.circresaha.org.

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“The phosphorylated residues are identified as Ser 116 and Thr 497 (495 in human) as well as Ser 1179 for bovine (equivalent to Ser 1177 for human) (28, 30).”
Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca2+/Calmodulin-Dependent Protein Kinase Kinase β
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“Thrombin has already been shown to activate this kinase (4), and bradykinin, another Gq-protein-coupled stimulus, has been shown to phosphorylate eNOS at serine 1177 via Ca(2) /calmodulindependent kinase II (13).”
Background
CaMK4 Gene Deletion Induces Hypertension
Santulli, G., Cipolletta, E., Sorriento, D., Giudice, C. D., Anastasio, A., Monaco, S., Maione, A. S., Condorelli, G., Puca, A. A., Trimarco, B., et al. (2012) · Journal of the American Heart Association · doi:10.1161/jaha.112.001081 · Cited by 212
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Dephosphorylation of Endothelial Nitric-oxide Synthase by Vascular Endothelial Growth Factor
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NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase
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The NO cascade, eNOS location, and microvascular permeability
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Paradoxes of nitric oxide in the diabetic kidney
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“For example, phosphorylation of Ser1177/1179 leads to activation of the enzyme (42), whereas phosphorylation of Thr495 has an opposite effect (33).”
Background
Recent Advances in Understanding Endothelial Dysfunction in Atherosclerosis
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Akt-Dependent Phosphorylation of Serine 1179 and Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase 1/2 Cooperatively Mediate Activation of the Endothelial Nitric-Oxide Synthase by Hydrogen Peroxide
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“This effect is associated with a concomitant increase in eNOS phosphorylation at serine 1179 (Fleming et al., 2001).”
Influential
Red wine polyphenols‐induced, endothelium‐dependent NO‐mediated relaxation is due to the redox‐sensitive PI3‐kinase/Akt‐dependent phosphorylation of endothelial NO‐synthase in the isolated porcine coronary artery
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eNOS phosphorylation in health and disease
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“6C), whereas eNOS phosphorylation at Thr495 remained unchanged (P NS; Fig.”
BackgroundInfluential
Proatherosclerotic Mechanisms Involving Protein Kinase C in Diabetes and Insulin Resistance
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“(Thr495) blunting eNOS activity [234, 235] and reduces”
Platelet-Derived Nitric Oxide Signaling and Regulation
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“…have also been shown to phosphorylate eNOS at S1179, including adenosine monophosphate-activated kinase (AMPK) (Chen et al., 1999), CaM protein kinase II (Fleming et al., 2001), protein kinase A (PKA) (Butt et al., 2000; Gangopahyay et al., 2011), and protein kinase G (PKG) (Butt et al., 2000).”
BackgroundInfluential
Selective blockade of endothelial Ca2+‐activated small‐ and intermediate‐conductance K+‐channels suppresses EDHF‐mediated vasodilation
Eichler, I., Wibawa, J., Grgic, I., Knorr, A., Brakemeier, S., Pries, A. R., Hoyer, J. D., & Köhler, R. (2003) · British Journal of Pharmacology · doi:10.1038/sj.bjp.0705075 · Cited by 174
“In di erent subsets of experiments, vessels were preincubated with 10 mM CLT or 10 mM sulphaphenazole (SPZ), a selective CYP 2C9 inhibitor (Fleming et al., 2001) for 30 min.”
Method
Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme
Chen, C., Druhan, L. J., Varadharaj, S., Chen, Y., & Zweier, J. L. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m802269200 · Cited by 173
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Self-citation
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Modulation of endothelial nitric oxide by plant-derived products
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“agonists – especially those increasing intracellular calcium – Thr can be rapidly dephosphorylated [18,19].”
Background
High-density lipoprotein and apolipoprotein AI increase endothelial NO synthase activity by protein association and multisite phosphorylation
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Self-citation
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“Thus, calcium-dependent kinases, such as CaMKII, can be activated that in turn activate eNOS [22].”
Organic Nitrate Therapy, Nitrate Tolerance, and Nitrate-Induced Endothelial Dysfunction: Emphasis on Redox Biology and Oxidative Stress
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Immediate and delayed VEGF‐mediated NO synthesis in endothelial cells: Role of PI3K, PKC and PLC pathways
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“However, two recent reports have suggested that the inhibition of PI3K with speci®c inhibitors had no or slight inhibitory e ect on NO release (Fleming et al., 2001; Thuringer et al., 2001).”
BackgroundMethodResult

Authors: Fleming, I., Fisslthaler, B., Dimmeler, S., Kemp, B. E., & Busse, R.
Affiliations: Goethe University Frankfurt (DE)
ORCID: Fleming, Ingrid
Fisslthaler, Beate
Dimmeler, Stefanie
Kemp, Bruce E.
Venue: Circulation Research
Publisher: Ovid Technologies (Wolters Kluwer Health)
Published: 2001-06-08
Volume: 88
Issue: 11
Pages: E68-75
ISSN: 0009-7330 (ISSN-L)
1524-4571
DOI: 10.1161/hh1101.092677
OpenAlex: W2135297555
Cited by: 770
References: 38
Citations by year: 2026: 12 · 2025: 25 · 2024: 13 · 2023: 17 · 2022: 17 · 2021: 23 · 2020: 18 · 2019: 23 · 2018: 18 · 2017: 29 · 2016: 30 · 2015: 39 · 2014: 34 · 2013: 28 · 2012: 49
FWCI: 23.32
Type: journal article
Language: en
Open access: closed
Locations: Circulation Research (publishedVersion)
PubMed (publishedVersion)
Funders: National Heart Foundation of Australia
Deutsche Forschungsgemeinschaft (SFB 553)
Medical Research Council
National Health and Medical Research Council
Topics: Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiovascular Health and Disease Prevention
Keywords: endothelial nitric oxide synthase, calmodulin binding, CaMKII, PP1 phosphatase, calyculin A, bradykinin, protein kinase C, nitric oxide production, endothelial cells, dual phosphorylation, calmodulin-dependent kinase II, calmodulin
MeSH terms: Animals, Binding Sites, Bradykinin, Calcium, Calmodulin, Cells, Cultured, Coronary Vessels, Endothelium, Vascular, Enzyme Activation, Enzyme Inhibitors, Humans, Phosphorylation, Precipitin Tests, Protein Kinase C, Proto-Oncogene Proteins, Swine, Threonine, Blotting, Western, Mutagenesis, Site-Directed, Protein Serine-Threonine Kinases, Calcium-Calmodulin-Dependent Protein Kinases, Nitric Oxide Synthase, Mitogen-Activated Protein Kinases, Proto-Oncogene Proteins c-akt, Nitric Oxide Synthase Type III, Calcium-Calmodulin-Dependent Protein Kinase Type 2, In Vitro Techniques

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Phosphorylation of Thr 495 Regulates Ca 2+ /Calmodulin-Dependent Endothelial Nitric Oxide Synthase Activity

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