Caveolin‐1 Phosphorylation Negatively Regulates eNOS Activity in Endothelial Cells

Abstract

Endothelial nitric oxide synthase (eNOS) activity is regulated by phosphorylation, Ca2+ ‐calmodulin, and interaction with caveolin‐1 (Cav‐1). However, the mechanism by which Cav‐1 negatively regulates eNOS activity following stimulation is not clear. Here, we examined the kinetics of eNOS activation (phosphorylation and NO production) and binding to Cav‐1 in endothelial cells and mouse lung homogenates following addition of Ca2+ ionophore A23187. Ca2+increased Src, Akt, Cav‐1, and eNOS phosphorylation and NO production within minutes which returned to baseline levels within 30 min. We observed an increase in eNOS association (co‐IP) with Cav‐1 following stimulation which was blocked by NOS inhibitor L‐NAME and Src inhibitor PP2. Furthermore, siRNA‐mediated knockdown of Cav‐1 potentiated eNOS phosphorylation whereas depletion of eNOS blocked the increase in Src activity and Cav‐1 phosphorylation. FRET between Cav‐1‐YFP and eNOS‐CFP in co‐transfected CHO cells significantly increased after stimulation. Moreover, in co‐transfected HEK293 cells, WT and phospho‐mimicking S1179D‐ eNOS but not phospho‐defective S1179A‐ eNOS co‐IP'd with WT and phospho‐mimicking Y14D‐Cav‐1 but not phospho‐defective Y14F‐Cav‐1 mutant. These data suggest that Ca2+‐dependent activation of eNOS induces Cav‐1 phosphorylation via Src which in turn binds to eNOS and negatively regulates its activity. Supported by NIH HL071626 and HL060678.