Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid, C8‐β‐ D ‐lactosylceramide

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.