The DOI matches its Crossref record; 38 of the 38 references it cites were found in a registry or an index.
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 the citing paper
“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]
In the citing paper
“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]
In the citing paper
“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]
Citations
Cites
Molecular Cloning: A Laboratory Manual
Sambrook, J., Fritsch, E., & Maniatis, T. (1989) · UCLA - Biblioteca de Ciencias de la Salud · Cited by 133,589
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
Self-citationCloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase
Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C. J., Reed, R. R., & Snyder, S. H. (1991) · Nature · doi:10.1038/351714a0 · Cited by 2,354
Dynamic activation of endothelial nitric oxide synthase by Hsp90
García‐Cardeña, G., Fan, R., Shah, V. H., Sorrentino, R., Cirino, G., Papapetropoulos, A., & Sessa, W. C. (1998) · Nature · doi:10.1038/33934 · Cited by 1,029
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-citationNishida, K., Harrison, D. G., Navas, J. P., Fisher, A. A., Dockery, S., Uematsu, M., Nerem, R. M., Alexander, R. W., & Murphy, T. J. (1992) · Journal of Clinical Investigation · doi:10.1172/jci116092 · Cited by 735
García‐Cardeña, G., Fan, R., Stern, D. F., Liu, J., & Sessa, W. C. (1996) · Journal of Biological Chemistry · doi:10.1074/jbc.271.44.27237 · Cited by 533
Phosphorylation of Endothelial Nitric Oxide Synthase in Response to Fluid Shear Stress
Corson, M. A., James, N. L., Latta, S. E., Nerem, R. M., Berk, B. C., & Harrison, D. G. (1996) · Circulation Research · doi:10.1161/01.res.79.5.984 · Cited by 521
Calcium‐dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin
Busse, R., & Mülsch, A. (1990) · FEBS Letters · doi:10.1016/0014-5793(90)80902-u · Cited by 511
Self-citationMolecular cloning and characterization of human endothelial nitric oxide synthase
Marsden, P. A., Schappert, K. T., Chen, H. S., Flowers, M. A., Sundell, C. L., Wilcox, J. N., Lamas, S., & Michel, T. M. (1992) · FEBS Letters · doi:10.1016/0014-5793(92)80697-f · Cited by 489
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 481
Self-citationSignal transduction of eNOS activation
Fleming, I. (1999) · Cardiovascular Research · doi:10.1016/s0008-6363(99)00094-2 · Cited by 458
Self-citationMcCabe, 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
Phosphorylation and subcellular translocation of endothelial nitric oxide synthase.
Michel, T. M., Li, G. K., & Busconi, L. (1993) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.90.13.6252 · Cited by 323
Lückhoff, A., Pohl, U., Mülsch, A., & Busse, R. (1988) · British Journal of Pharmacology · doi:10.1111/j.1476-5381.1988.tb16564.x · Cited by 312
Self-citationPhosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress
Fisslthaler, Dimmeler, S., Hermann, Busse, & Fleming (2000) · Acta Physiologica Scandinavica · doi:10.1046/j.1365-201x.2000.00627.x · Cited by 308
Self-citationAyajiki, K., Kindermann, M., Hecker, M., Fleming, I., & Busse, R. (1996) · Circulation Research · doi:10.1161/01.res.78.5.750 · Cited by 285
Self-citationButt, 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
Nakane, M., Mitchell, J. A., Förstermann, U., & Murad, F. (1991) · Biochemical and Biophysical Research Communications · doi:10.1016/s0006-291x(05)81351-8 · Cited by 274
Fleming, I., Bauersachs, J. K., Fißlthaler, B., & Busse, R. (1998) · Circulation Research · doi:10.1161/01.res.82.6.686 · Cited by 265
Self-citationAn 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
Agonist-modulated Palmitoylation of Endothelial Nitric Oxide Synthase
Robinson, L. J., Busconi, L., & Michel, T. M. (1995) · Journal of Biological Chemistry · doi:10.1074/jbc.270.3.995 · Cited by 220
Calcium- and endothelial-mediated vascular smooth muscle relaxation in rabbit aorta.
Singer, H. A., & Peach, M. J. (1982) · Hypertension · doi:10.1161/01.hyp.4.3_pt_2.19 · Cited by 202
Bernier, S. G., Haldar, S. M., & Michel, T. M. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.m005116200 · Cited by 190
Continuous Monitoring of Nitric Oxide Release from Human Umbilical Vein Endothelial Cells
Tsukahara, H., Gordienko, D. V., & Goligorsky, M. S. (1993) · Biochemical and Biophysical Research Communications · doi:10.1006/bbrc.1993.1685 · Cited by 188
Inhibition of Endothelial Nitric Oxide Synthase Activity by Protein Kinase C
Hirata, K., Kuroda, R., Sakoda, T., Katayama, M., Inoue, N., Suematsu, M., Kawashima, S., & Yokoyama, M. (1995) · Hypertension · doi:10.1161/01.hyp.25.2.180 · Cited by 188
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
Komeima, K., Hayashi, Y., Naito, Y., & Watanabe, Y. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.m003198200 · Cited by 181
Calcium-Dependent and Calcium-Independent Activation of the Endothelial NO Synthase
Fleming, I., Bauersachs, J. K., & Busse, R. (2008) · Journal of Vascular Research · doi:10.1159/000159220 · Cited by 151
Self-citationM�lsch, A., Bassenge, E., & Busse, R. (1989) · Naunyn-Schmiedeberg s Archives of Pharmacology · doi:10.1007/bf00169688 · Cited by 146
Self-citationAutoinhibition of Endothelial Nitric-oxide Synthase
Nishida, C. R., & de Montellano, P. R. O. (1999) · Journal of Biological Chemistry · doi:10.1074/jbc.274.21.14692 · Cited by 107
Someren, J. S., Faber, L. E., Klein, J. D., & Tumlin, J. A. (1999) · Biochemical and Biophysical Research Communications · doi:10.1006/bbrc.1999.0800 · Cited by 59
Protein kinase C modulates receptor-independent activation of endothelial nitric oxide synthase
Davda, R. K., Chandler, L. J., & Guzman, N. J. (1994) · European Journal of Pharmacology Molecular Pharmacology · doi:10.1016/0922-4106(94)90132-5 · Cited by 57
Fleming, I., Schermer, B., Popp, R., & Busse, R. (1999) · British Journal of Pharmacology · doi:10.1038/sj.bjp.0702381 · Cited by 56
Self-citation(100) Deformation twins in naturally deformed amphiboles
Biermann, C. (1981) · Nature · doi:10.1038/292821a0 · Cited by 46
Yamagata, Y., Czernik, A. J., & Greengard, P. (1991) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)98628-x · Cited by 43
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- 10.1161/hyp.25.2.180 · DOI
- 10.1161/res.79.5.984 · DOI
- 10.1161/res.82.6.686 · DOI
- Fulton D, Gratton J-P, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt [published correction appears in Nature. 1999;400:792]. Nature. 1999;399:597–601.
- 10.1161/res.78.5.750 · DOI
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“20 ) and increased association of eNOS with Hsp90 (ref”
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“For example, the agonist-induced activation of the CaM-dependent protein kinase II (CaMKII) is highly dependent on an increase in [Ca2 ]i, whereas the shear stress-induced activation of Akt is not affected by Ca2 removal (28, 36).”
InfluentialSelf-citationHambrecht, R., Adams, V., Erbs, S., Linke, A. H.-P., Kränkel, N., Shu, Y., Baither, Y., Gielen, S., Thiele, H., Gummert, J. F., et al. (2003) · Circulation · doi:10.1161/01.cir.0000074229.93804.5c · Cited by 941
Metadata
- 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 (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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