Src-induced phosphorylation of Cav-1 is analyzed using live TIRF and FRET microscopy, as well as by biochemical analysis. Cav1 phosphorylation destabilizes plasma membrane–associated Cav-1 oligomers and thereby is crucial for regulating the fission of caveolae from the plasma membrane in vascular endothelial cells.

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Caveolin‐1 (Cav‐1) is required for caveolae formation but it also binds to signaling proteins such as endothelial nitric oxide synthase (eNOS), which generally negatively regulates their activity. Interestingly, activation of caveolae trafficking is associated with an increase in NO production. Here, we test the hypothesis that a NO feedback mechanism directly modifies the assembly and mobility of caveolae. We determined whether eNOS‐derived NO affects Cav‐1 oligomer stability by S‐nitrosylation (SNO) of Cys156 and thereby regulates vesicular trafficking. Endothelial cells stimulated with TNFα for up to 4 hr were lysed and used to measure Cav‐1 SNO by biotin‐switch assay as well as Cav‐1 oligomer stability in non‐reduced SDS PAGE gels. TNFα increased NO production and Cav‐1 SNO and also decreased Cav‐1 oligomerization, both of which were blocked by L‐NAME. We next generated Cav‐1−/− cells expressing GFP‐tagged wild type Cav‐1 or Cys156Ser‐Cav‐1 mutant to determine the role of Cys156 in caveolin oligomerization and vesicle trafficking as characterized by immunoblot analysis and live‐cell spinning disc confocal imaging. These studies revealed that Cys156Ser‐Cav‐1 mutant‐labeled caveolae were less mobile and that oligomers were less stable compared to WT Cav‐1. Thus, S‐nitrosylation of Cav‐1 may be an important regulatory mechanism of caveolae trafficking in endothelial cells. Supported by R01 HL071626, P01 HL060678, and T32 HL007829.

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