S‐Nitrosylation of Caveolin‐1 Cys156 Stimulates Src‐dependent Caveolin‐1 Tyr14 Phosphorylation Required for Caveolae‐mediated Endocytosis in Endothelial Cells

Abstract

Membrane‐associated Caveolin‐1 (Cav‐1) oligomers are required for caveolae formation. We previously showed that during an inflammatory response, Src‐dependent Cav‐1 phosphorylation increases transendothelial transport via caveolae and breakdown of the endothelial barrier, both of which were abolished in Cav‐1−/− lung microvessels. Thus, in this study we tested the hypothesis that TNFα‐induced NO production induces Cav‐1 S‐nitrosylation (SNO) resulting in Src displacement and activation which increases caveolae trafficking. Endothelial cells (ECs) stimulated with TNFα or NO donor showed reduced Cav‐1 oligomerization (MW > 250 kDa) as measured by immunoblot analysis of non‐reduced SDS PAGE gels and also increased Cav‐1 Tyr14 phosphorylation. Of the 3 Cys residues in the C‐terminus (Cys 133, 143, and 156), we observed S‐nitrosylation of Cys156by biotin switch assay. eNOS dependent increase in Cav‐1 Tyr14 phosphorylation and Cav‐1‐GFP labeled caveolae trafficking were blocked by L‐NAME and PP2. Cav1−/− ECs expressing C156S Cav‐1 mutants did not form stable oligomers and showed increased Src activity but were unable to form caveolae. When expressed in normal endothelial cells, C156S‐Cav‐1‐GFP and Y14D‐Cav‐1‐GFP had a dominant positive effect whereas Y14F‐Cav‐1‐GFP reduced caveolae‐mediated uptake and trafficking. These data suggest that Cav‐1 Cys156 SNO leads to Src activation and phosphorylation of Cav‐1 Tyr14 which increases caveolae trafficking dynamics in endothelial cells.