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Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition
Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclea...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315804/ https://www.ncbi.nlm.nih.gov/pubmed/22323292 http://dx.doi.org/10.1091/mbc.E11-09-0811 |
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author | Chen, Zhenlong Bakhshi, Farnaz R. Shajahan, Ayesha N. Sharma, Tiffany Mao, Mao Trane, Andy Bernatchez, Pascal van Nieuw Amerongen, Geerten P. Bonini, Marcelo G. Skidgel, Randal A. Malik, Asrar B. Minshall, Richard D. |
author_facet | Chen, Zhenlong Bakhshi, Farnaz R. Shajahan, Ayesha N. Sharma, Tiffany Mao, Mao Trane, Andy Bernatchez, Pascal van Nieuw Amerongen, Geerten P. Bonini, Marcelo G. Skidgel, Randal A. Malik, Asrar B. Minshall, Richard D. |
author_sort | Chen, Zhenlong |
collection | PubMed |
description | Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca(2+) ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-N(G)-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca(2+)-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1–Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition. |
format | Online Article Text |
id | pubmed-3315804 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-33158042012-06-16 Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition Chen, Zhenlong Bakhshi, Farnaz R. Shajahan, Ayesha N. Sharma, Tiffany Mao, Mao Trane, Andy Bernatchez, Pascal van Nieuw Amerongen, Geerten P. Bonini, Marcelo G. Skidgel, Randal A. Malik, Asrar B. Minshall, Richard D. Mol Biol Cell Articles Endothelial nitric oxide synthase (eNOS)–mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1–mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca(2+) ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-N(G)-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca(2+)-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1–Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition. The American Society for Cell Biology 2012-04-01 /pmc/articles/PMC3315804/ /pubmed/22323292 http://dx.doi.org/10.1091/mbc.E11-09-0811 Text en © 2012 Chen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. |
spellingShingle | Articles Chen, Zhenlong Bakhshi, Farnaz R. Shajahan, Ayesha N. Sharma, Tiffany Mao, Mao Trane, Andy Bernatchez, Pascal van Nieuw Amerongen, Geerten P. Bonini, Marcelo G. Skidgel, Randal A. Malik, Asrar B. Minshall, Richard D. Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition |
title | Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition |
title_full | Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition |
title_fullStr | Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition |
title_full_unstemmed | Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition |
title_short | Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition |
title_sort | nitric oxide–dependent src activation and resultant caveolin-1 phosphorylation promote enos/caveolin-1 binding and enos inhibition |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315804/ https://www.ncbi.nlm.nih.gov/pubmed/22323292 http://dx.doi.org/10.1091/mbc.E11-09-0811 |
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