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S‐nitrosylation of Caveolin‐1 Cys156 Regulates Oligomer Stability and Caveolae Trafficking
Author(s) -
Bakhshi Farnaz,
Chen Zhenlong,
Husain Yawer,
Shajahan Ayesha,
Liu Guoquan,
Malik Asrar B.,
Minshall Richard D.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.797.6
Subject(s) - caveolae , caveolin 1 , microbiology and biotechnology , caveolin , chemistry , s nitrosylation , enos , mutant , immunoprecipitation , nitric oxide , biology , signal transduction , nitric oxide synthase , biochemistry , organic chemistry , cysteine , gene , enzyme
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.