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Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

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Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition
Author(s): 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.
Abstract: 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 Ca2+ ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-NGnitroarginine 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 Ca2+-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.
Issue Date: 2012
Publisher: American Society for Cell Biology
Citation Info: Chen, Z., Bakhshi, F. R., Shajahan, A. N., Sharma, T., Mao, M., Trane, A., Bernatchez, P., van Nieuw Amerongen, G. P., Bonini, M. G., Skidgel, R. A., Malik, A. B., & Minshall, R. D. 2012. Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promotes eNOS/caveolin-1 binding and eNOS inhibition. Molecular Biology of the Cell. 23: 1388-1398. doi: 10.1091/mbc.E11-09-0811
Type: Article
Description: © 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 ( “ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.
Sponsor: This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute Grants R01 HL045638, R01 HL71626, and P01 HL60678.
Date Available in INDIGO: 2012-06-25

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