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Identification of Regulatory Sites of Phosphorylation of the Bovine Endothelial Nitric-oxide Synthase at Serine 617 and Serine 635
Open accessAbstract
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.
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“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 [646]
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“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 [646]
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Cites
Site-directed mutagenesis by overlap extension using the polymerase chain reaction
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., & Pease, L. R. (1989) · Gene · doi:10.1016/0378-1119(89)90358-2 · Cited by 7,414
Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation
Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., & Zeiher, A. M. (1999) · Nature · doi:10.1038/21224 · Cited by 3,591
Vlahos, C. J., Matter, W. F., Hui, K. Y., & Brown, R. F. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)37680-9 · Cited by 3,248
Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., Reese, C. B., & Cohen, P. (1997) · Current Biology · doi:10.1016/s0960-9822(06)00122-9 · Cited by 2,874
Regulation of endothelium-derived nitric oxide production by the protein kinase Akt
Fulton, D. J., Gratton, J., McCabe, T. J. D., Fontana, J. T., Fujio, Y., Walsh, K. X., Franke, T. F., Papapetropoulos, A., & Sessa, W. C. (1999) · Nature · doi:10.1038/21218 · Cited by 2,616
Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A., & Kaneko, M. (1987) · Biochemical and Biophysical Research Communications · doi:10.1016/0006-291x(87)90293-2 · Cited by 939
AMP‐activated protein kinase phosphorylation of endothelial NO synthase
Chen, Z.-P., Mitchelhill, K. I., Michell, B. J., Stapleton, D., Rodríguez‐Crespo, I., Witters, L. A., Power, D. A., de Montellano, P. R. O., & Kemp, B. E. (1999) · FEBS Letters · doi:10.1016/s0014-5793(98)01705-0 · Cited by 839
Self-citationFleming, I., Fisslthaler, B., Dimmeler, S., Kemp, B. E., & Busse, R. (2001) · Circulation Research · doi:10.1161/hh1101.092677 · Cited by 770
Self-citationMichell, B. J., Chen, Z., Tiganis, T., Stapleton, D., Katsis, F., Power, D. A., Sim, A. T. R., & Kemp, B. E. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.c100122200 · Cited by 560
Self-citationMontagnani, M., Chen, H., Barr, V. A., & Quon, M. J. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m103702200 · Cited by 550
Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase
Ui, M., Okada, T., Hazeki, K., & Hazeki, O. (1995) · Trends in Biochemical Sciences · doi:10.1016/s0968-0004(00)89056-8 · Cited by 520
Fulton, D. J., Gratton, J., & Sessa, W. C. (2001) · Journal of Pharmacology and Experimental Therapeutics · doi:10.1016/s0022-3565(24)29198-9 · Cited by 499
The Akt kinase signals directly to endothelial nitric oxide synthase
Michell, B. J., Griffiths, J., Mitchelhill, K. I., Rodríguez‐Crespo, I., Tiganis, T., Bozinovski, S., de Montellano, P. R. O., Kemp, B. E., & Pearson, R. B. (1999) · Current Biology · doi:10.1016/s0960-9822(99)80371-6 · Cited by 491
Self-citationFéron, O., Dessy, C., Moniotte, S., Desager, J., & Balligand, J. (1999) · Journal of Clinical Investigation · doi:10.1172/jci4829 · Cited by 413
McCabe, T. J., Fulton, D., Roman, L. J., & Sessa, W. C. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.275.9.6123 · Cited by 387
Harris, M. B., Ju, H., Venema, V. J., Liang, H., Zou, R., Michell, B. J., Chen, Z.-P., Kemp, B. E., & Venema, R. C. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m100229200 · Cited by 363
Self-citationGallis, B. M., Corthals, G. L., Goodlett, D. R., Ueba, H., Kim, F., Presnell, S. R., Figeys, D., Harrison, D. G., Berk, B. C., Aebersold, R. H., et al. (1999) · Journal of Biological Chemistry · doi:10.1074/jbc.274.42.30101 · Cited by 326
Butt, E., Bernhardt, M., Smolenski, A., Kotsonis, P., Fröhlich, L. G., Sickmann, A., Meyer, H. E., Lohmann, S. M., & Schmidt, H. H. H. W. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.275.7.5179 · Cited by 284
An Autoinhibitory Control Element Defines Calcium-regulated Isoforms of Nitric Oxide Synthase
Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Pavel Martasek, E., Roman, L. J., Masters, B. S. S., Jones, C. L., et al. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.47.29769 · Cited by 248
Bernier, S. G., Haldar, S. M., & Michel, T. M. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.m005116200 · Cited by 190
Venema, R. C., Sayegh, H. S., Kent, J. D., & Harrison, D. G. (1996) · Journal of Biological Chemistry · doi:10.1074/jbc.271.11.6435 · Cited by 184
Self-citationPosttranslational Modifications of the 5′-AMP-activated Protein Kinase β1 Subunit
Mitchelhill, K. I., Michell, B. J., House, C. M., Stapleton, D., Dyck, J. R. B., Gamble, J., Ullrich, C., Witters, L. A., & Kemp, B. E. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.39.24475 · Cited by 158
Self-citationFleming, I., Hecker, M., & Busse, R. (1994) · Circulation Research · doi:10.1161/01.res.74.6.1220 · Cited by 150
Subunit Interactions of Endothelial Nitric-oxide Synthase
Venema, R. C., Ju, H., Zou, R., Ryan, J. W., & Venema, V. J. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.2.1276 · Cited by 148
Self-citationConus, N. M., Hemmings, B. A., & Pearson, R. B. (1998) · Journal of Biological Chemistry · doi:10.1074/jbc.273.8.4776 · Cited by 129
Scotland, R. S., Morales‐Ruiz, M., Chen, Y., Yu, J., Rudic, R. D., Fulton, D. J., Gratton, J., & Sessa, W. C. (2002) · Circulation Research · doi:10.1161/01.res.0000016506.04193.96 · Cited by 110
Endothelial nitric oxide synthase interactions with G-protein-coupled receptors
Marrero, M. B., Venema, V. J., Ju, H., He, H., Liang, H., Caldwell, R. B., & Venema, R. C. (1999) · Biochemical Journal · doi:10.1042/bj3430335 · Cited by 105
Self-citationVenema, R. C., Sayegh, H. S., Arnal, J., & Harrison, D. G. (1995) · Journal of Biological Chemistry · doi:10.1074/jbc.270.24.14705 · Cited by 96
Self-citationCalmodulin-dependent Regulation of Inducible and Neuronal Nitric-oxide Synthase
Lee, S., & Stull, J. T. (1998) · Journal of Biological Chemistry · doi:10.1074/jbc.273.42.27430 · Cited by 69
Flagged first, then by citations
Research works printed but not linked: 2
- Fulton, 2001, J. Pharmacol. Exp. Ther., 299, 818
- Fleming, 2001, Circ. Res., 88, e68
Cited by
Sorted by citations
Sessa, W. C. (2004) · Journal of Cell Science · doi:10.1242/jcs.01165 · Cited by 561
“…AMP kinase (AMPK) to phosphorylate eNOS on S1179 (Chen et al., 1999; Dimmeler et al., 1998; Fleming et al., 2001; Fulton et al., 1999; Harris et al., 2001; Haynes et al., 2000; Igarashi and Michel, 2001; Lin et al., 2003; Michell et al., 2002; Morales-Ruiz et al., 2001; Simoncini et al., 2000).”
BackgroundSources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation
Tejero, J., Shiva, S., & Gladwin, M. T. (2019) · Physiological Reviews · doi:10.1152/physrev.00036.2017 · Cited by 541
Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation
Mount, P. F., Kemp, B. E., & Power, D. A. (2007) · Journal of Molecular and Cellular Cardiology · doi:10.1016/j.yjmcc.2006.05.023 · Cited by 498
“The effects of phosphorylation at eNOS-Ser and eNOS-Ser remain controversial [11,12].”
BackgroundSelf-citationBalligand, J.-L., Feron, O., & Dessy, C. (2009) · Physiological Reviews · doi:10.1152/physrev.00042.2007 · Cited by 477
Nitric Oxide Signaling in Brain Function, Dysfunction, and Dementia
Steinert, J. R., Chernova, T., & Forsythe, I. D. (2010) · The Neuroscientist · doi:10.1177/1073858410366481 · Cited by 471
Metadata
- Authors
- 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.
- Affiliations
- St Vincents Institute of Medical Research (AU)
- Center for Vascular Biology Research (US)
- Augusta University (US)
- Corresponding author
- Venema, Richard C.
- Kemp, Bruce E.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2002-11
- Volume
- 277
- Issue
- 44
- Pages
- 42344-42351
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2024185487
- Cited by
- 212
- References
- 29
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 2 · 2022: 1 · 2021: 7 · 2020: 4 · 2019: 5 · 2018: 4 · 2017: 8 · 2016: 9 · 2015: 5 · 2014: 13 · 2013: 7 · 2012: 15
- FWCI
- 9.39
- 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
- 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 › Surgery › Cardiovascular, Neuropeptides, and Oxidative Stress Research
- Keywords
- endothelial nitric oxide synthase, eNOS phosphorylation, protein kinase A, Akt, bradykinin signaling, phosphatidylinositol 3-kinase, enzyme activation, phosphopeptide mapping, bovine aortic endothelial cells
- MeSH terms
- Adenosine Triphosphate, Amino Acid Sequence, Animals, Bradykinin, Calcium, Calmodulin, Cattle, Humans, Lymphokines, Molecular Sequence Data, Phosphorylation, Proto-Oncogene Proteins, Serine, Endothelial Growth Factors, Protein Serine-Threonine Kinases, Cyclic AMP-Dependent Protein Kinases, Nitric Oxide Synthase, Intercellular Signaling Peptides and Proteins, Vascular Endothelial Growth Factors, Vascular Endothelial Growth Factor A, Proto-Oncogene Proteins c-akt, Nitric Oxide Synthase Type III
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