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

Zhenlong Chen, Farnaz R. Bakhshi, Ayesha N. Shajahan, Tiffany Sharma, Mao, Andy Trane, Pascal Bernatchez, Geerten P. Van Nieuw Amerongen, Marcelo G. Bonini, Randal A. Skidgel, Asrar B. Malik, Richard D. Minshall*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

81 Scopus citations

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-NG-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 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.

Original languageEnglish (US)
Pages (from-to)1388-1398
Number of pages11
JournalMolecular biology of the cell
Volume23
Issue number7
DOIs
StatePublished - Apr 1 2012
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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