title: Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid, C8-beta-D-lactosylceramide.
abstract: Plasma membrane (PM) microdomains, including caveolae and other cholesterol-enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D-erythro-octanoyl-lactosylceramide (C8-D-e-LacCer), stimulates endocytosis via caveolae and induces the appearance of micron-size microdomains on the PM. To further understand the effects of C8-D-e-LacCer treatment on PM microdomains, we used a detergent-free method to isolate microdomain-enriched membranes from fibroblasts treated +/-C8-D-e-LacCer, and performed 2-DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain-enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho-associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D-e-LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8-D-e-LacCer-treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8-L-threo-lactosylceramide, a glycosphingolipid with non-natural stereochemistry. This study identifies new microdomain-associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic College of Medicine, Rochester, MN 55905-0001, USA.
authors.forename: Seong-Youl
authors.initials: SY
authors.lastname: Kim
authors.forename: Teng-ke
authors.initials: TK
authors.lastname: Wang
authors.forename: Raman Deep
authors.initials: RD
authors.lastname: Singh
authors.forename: Christine L
authors.initials: CL
authors.lastname: Wheatley
authors.forename: David L
authors.initials: DL
authors.lastname: Marks
authors.forename: Richard E
authors.initials: RE
authors.lastname: Pagano
mesh_terms: D015153:Blotting, Western; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007790:Lactosylceramides / Q000494:pharmacology*; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012867:Skin / Q000166:cytology; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D007790:Lactosylceramides; D008565:Membrane Proteins; D054460:rho-Associated Kinases