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

Abstract

The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

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 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.