Phosphorylation of Thr 495 Regulates Ca 2+ /Calmodulin-Dependent Endothelial Nitric Oxide Synthase Activity
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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In resting endothelial cells, Thr495 is generally phosphorylated.
“In resting endothelial cells, Thr495 is generally phosphorylated”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [297]
Thr495 phosphorylation is attributed to protein kinase C and the residue is dephosphorylated by protein phosphatase 1.
“The phosphorylation has been attributed to protein kinase C (PKC) (297, 646), and the residue is dephosphorylated by protein phosphatase 1 (PP1)”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [297]
Phosphorylation of Thr495 is a negative regulator of CaM binding to eNOS.
“Phosphorylation of Thr495 is a negative regulator of CaM binding to eNOS (297, 407, 646).”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [297]
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CLINICAL AND TOXICOLOGICAL RELEVANCE OF CYP2C9: Drug-Drug Interactions and Pharmacogenetics
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Self-citationRole of 20-hydroxyeicosatetraenoic acid (20-HETE) in vascular system
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“…smooth muscle membranes by activating Ca2+-activated K+-channels (Gebremedhin et al. and Zou et al. were first in 1992 and 1996; Campbell et al., 1996; Popp et al., 1996; Eckman et al., 1998; Hayabuchi et al., 1998; Miura and Gutterman, 1998; Fisslthaler et al., 1999; Fleming et al., 2001).”
Mechanisms and consequences of endothelial nitric oxide synthase dysfunction in hypertension
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“They include decreased availability of the eNOS substrate L-arginine or the cofactor tetrahydrobiopterin (BH4), but also phosphorylation state (Ser1177, Thr495, Tyr657) or S-glutathionlyation of the protein (for review see [19]).”
BackgroundInfluentialLin, M. I., Fulton, D. J., Babbitt, R. W., Fleming, I., Busse, R., Pritchard, K. A., & Sessa, W. C. (2003) · Journal of Biological Chemistry · doi:10.1074/jbc.m302836200 · Cited by 236 · see citing works
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“Increases in intracellular calcium levels activate CaM that in turn activates CaM kinase II, which also phosphor- ylates S1177 (Fleming et al. 2001).”
BackgroundInfluentialNitric oxide and penile erectile function
Toda, N., Ayajiki, K., & OKAMURA, T. (2005) · Pharmacology & Therapeutics · doi:10.1016/j.pharmthera.2004.11.011 · Cited by 231 · see citing works
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Erdogdu, Ö., Nathanson, D., Sjöholm, Å., Nyström, T., & Zhang, Q. (2010) · Molecular and Cellular Endocrinology · doi:10.1016/j.mce.2010.04.022 · Cited by 229 · see citing works
The endothelium: influencing vascular smooth muscle in many ways
Triggle, C. R., Samuel, S. M., Ravishankar, S., Marei, I., Arunachalam, G., & Ding, H. (2012) · Canadian Journal of Physiology and Pharmacology · doi:10.1139/y2012-073 · Cited by 218 · see citing works
“…617 by either PKA or AKT appears important for the recruitment of eNOS to calmodulin binding, whereas phosphorylation at Thr 495 by PKC attenuates this association (Dimmeler et al. 1999; Fulton et al. 1999; Fleming et al. 2001; Fulton et al. 2001; Michell et al. 2002; Dudzinski and Michel 2007).”
BackgroundFulton, D. J., Fontana, J. T., Sowa, G., Gratton, J., Lin, M. I., Li, K.-X., Michell, B. J., Kemp, B. E., Rodman, D. M., & Sessa, W. C. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m106302200 · Cited by 217 · see citing works
Self-citationPossible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function
Bird, I. M., Zhang, L., & Magness, R. R. (2003) · American Journal of Physiology-Regulatory, Integrative and Comparative Physiology · doi:10.1152/ajpregu.00108.2002 · Cited by 214 · see citing works
“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).”
Stahmann, N., Woods, A., Carling, D., & Heller, R. (2006) · Molecular and Cellular Biology · doi:10.1128/mcb.00383-06 · Cited by 214 · see citing works
“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).”
BackgroundCaMK4 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 213 · see citing works
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Michell, B. J., Harris, M. B., Chen, Z.-P., Ju, H., Venema, V. J., Blackstone, M. A., Huang, W., Venema, R. C., & Kemp, B. E. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m205144200 · Cited by 212 · see citing works
Self-citationTranscriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression
Searles, C. D. (2006) · American Journal of Physiology-Cell Physiology · doi:10.1152/ajpcell.00457.2005 · Cited by 210 · see citing works
Dessy, C., Saliez, J., Ghisdal, P., Daneau, G., Lobysheva, I. I., Frérart, F., Belge, C., Jnaoui, K., Noirhomme, P., Féron, O., et al. (2005) · Circulation · doi:10.1161/circulationaha.104.532960 · Cited by 208 · see citing works
Deep insight into cytokine storm: from pathogenesis to treatment
Nie, J., Zhou, L., Tian, W., Liu, X., Yang, L., Yang, X., Zhang, Y., Wei, S., Wang, D. W., & Wei, J. (2025) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-025-02178-y · Cited by 224 · see citing works
NAD(P)H Oxidase-Derived Reactive Oxygen Species as Mediators of Angiotensin II Signaling
Hanna, I. R., Taniyama, Y., Szöcs, K., Ročić, P., & Griendling, K. K. (2002) · Antioxidants and Redox Signaling · doi:10.1089/152308602762197443 · Cited by 194 · see citing works
Cytochrome P450 pathways of arachidonic acid metabolism
Kroetz, D. L., & Zeldin, D. C. (2002) · Current Opinion in Lipidology · doi:10.1097/00041433-200206000-00007 · Cited by 193 · see citing works
Dephosphorylation of Endothelial Nitric-oxide Synthase by Vascular Endothelial Growth Factor
Kou, R., Greif, D. M., & Michel, T. M. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m204519200 · Cited by 192 · see citing works
Zimmermann, K., Opitz, N., Dedio, J., Renné, C., Müller‐Esterl, W., & Oess, S. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.252345399 · Cited by 192 · see citing works
The NO cascade, eNOS location, and microvascular permeability
Durán, W. N., Breslin, J. W., & Sánchez, F. A. (2010) · Cardiovascular Research · doi:10.1093/cvr/cvq139 · Cited by 191 · see citing works
Paradoxes of nitric oxide in the diabetic kidney
Komers, R., & Anderson, S. (2003) · American Journal of Physiology-Renal Physiology · doi:10.1152/ajprenal.00265.2002 · Cited by 190 · see citing works
“For example, phosphorylation of Ser1177/1179 leads to activation of the enzyme (42), whereas phosphorylation of Thr495 has an opposite effect (33).”
BackgroundRecent Advances in Understanding Endothelial Dysfunction in Atherosclerosis
Yang, Z., & Ming, X. (2006) · Clinical Medicine & Research · doi:10.3121/cmr.4.1.53 · Cited by 189 · see citing works
Cai, H. “., Li, Z., Davis, M. E., Kanner, W. A., Harrison, D. G., & Dudley, S. C. (2003) · Molecular Pharmacology · doi:10.1124/mol.63.2.325 · Cited by 187 · see citing works
“This effect is associated with a concomitant increase in eNOS phosphorylation at serine 1179 (Fleming et al., 2001).”
InfluentialNdiaye, M., Chataigneau, M., Lobysheva, I. I., Chataigneau, T., & Schini‐Kerth, V. B. (2004) · The FASEB Journal · doi:10.1096/fj.04-2146fje · Cited by 185 · see citing works
Nitric oxide, a protective molecule in the cardiovascular system
Lei, J., Vodovotz, Y., Tzeng, E., & Billiar, T. R. (2013) · Nitric Oxide · doi:10.1016/j.niox.2013.09.004 · Cited by 184 · see citing works
eNOS phosphorylation in health and disease
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Structural basis for endothelial nitric oxide synthase binding to calmodulin
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“6C), whereas eNOS phosphorylation at Thr495 remained unchanged (P NS; Fig.”
BackgroundInfluentialProatherosclerotic Mechanisms Involving Protein Kinase C in Diabetes and Insulin Resistance
Rask‐Madsen, C., & King, G. L. (2005) · Arteriosclerosis Thrombosis and Vascular Biology · doi:10.1161/01.atv.0000155325.41507.e0 · Cited by 176 · see citing works
Hyperglycemia and Endothelial Dysfunction in Atherosclerosis: Lessons from Type 1 Diabetes
Funk, S. D., Yurdagul, A., & Orr, A. W. (2012) · International Journal of Vascular Medicine · doi:10.1155/2012/569654 · Cited by 176 · see citing works
“(Thr495) blunting eNOS activity [234, 235] and reduces”
Platelet-Derived Nitric Oxide Signaling and Regulation
Gkaliagkousi, Ε., Ritter, J. M., & Ferro, A. (2007) · Circulation Research · doi:10.1161/circresaha.107.158410 · Cited by 175 · see citing works
Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium
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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).”
BackgroundInfluentialEichler, 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 · see citing works
“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.”
MethodPhosphorylation 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 · see citing works
The eNOS signalosome and its link to endothelial dysfunction
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Self-citationRegulation of eNOS Enzyme Activity by Posttranslational Modification
Heiß, E. H., & Dirsch, V. M. (2014) · Current Pharmaceutical Design · doi:10.2174/13816128113196660745 · Cited by 170 · see citing works
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].”
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Self-citationRamírez‐Sánchez, I., Maya-Ramos, L., Ceballos, G. M., & Villarreal, F. J. (2010) · Hypertension · doi:10.1161/hypertensionaha.109.147892 · Cited by 169 · see citing works
“Thus, calcium-dependent kinases, such as CaMKII, can be activated that in turn activate eNOS [22].”
Daiber, A., & Münzel, T. F. (2015) · Antioxidants and Redox Signaling · doi:10.1089/ars.2015.6376 · Cited by 167 · see citing works
Gélinas, D. S., Bernatchez, P. N., Rollin, S., Bazan, N. G., & Sirois, M. G. (2002) · British Journal of Pharmacology · doi:10.1038/sj.bjp.0704956 · Cited by 166 · see citing works
“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)
- Venue
- Circulation Research
- Published
- 2001-06-08
- Volume
- 88
- Issue
- 11
- Pages
- E68-75
- ISSN
- 0009-7330 (ISSN-L)
- 1524-4571
- 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 › 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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