RESEARCH ARTICLE Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid, C8-b-D-lactosylceramide Seong-youl Kim à , Teng-ke Wang à , Raman Deep Singh, Christine L. Wheatley, David L. Marks and Richard E. Pagano Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic College of Medicine, Rochester, MN, USA Received: February 6, 2009 Revised: May 15, 2009 Accepted: June 22, 2009 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 7C8-D-e-LacCer, and performed 2-DE and mass spec- trophotometry 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. Keywords: Caveolin-1 / Cell biology / Glycosphingolipid-enriched microdomains / RAP1 GTPase activating protein 1 / Sphingolipids 1 Introduction Plasma membrane (PM) microdomains enriched in cholesterol and sphingolipids, sometimes referred to as ‘‘lipid rafts’’, are subcompartments of the PM, which play roles in cell signaling and endocytosis [1–3]. Present evidence suggests that there are multiple types of microdomains at the Abbreviations: C8-D-e-LacCer, D-erythro-octanoyl-lactosylcera- mide; C8-L-t-LacCer, L-threo-octanoyl-lactosylceramide; Cav1, caveolin-1; dfBSA, defatted BSA; HSFs, human skin fibroblasts; HMEM, HEPES-buffered minimal essential medium; PM, plasma membrane; RAP1GAP, RAP1 GTPase activating protein 1; RAPGEF4, RAP guanine nucleotide exchange factor 4; ROCK, Rho-associated kinase; TIRF, total internal reflection fluores- cence à Current addresses: Dr. Seong-youl Kim, Molecular Diagnostic Research Center, Bioneer Corporation, Daejeon, 306-220, South Korea; Dr. Teng-ke Wang, Respiratory Division, Beijing Chao-Yang Hospital West Campus, Shijingshan District, Beijing 100043, P. R. China Correspondence: Dr. Richard E. Pagano, Mayo Clinic College of Medicine, Stabile 8, 200 First Street, S.W., Rochester, MN 55905- 0001, USA E-mail: pagano.richard@mayo.edu Fax: 11-507-266-4413 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 4321–4328 4321 DOI 10.1002/pmic.200900077