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Papers In Press, published online ahead of print March 1, 2008
J. Lipid Res., doi:10.1194/jlr.M700442-JLR200

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Journal of Lipid Research, Vol. 49, 531-542, March 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Ceramide kinase promotes Ca²⁺ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells

Vania Hinkovska-Galcheva^1,*,, Andrea Clark, Susan VanWay, Ji-Biao Huang, Miki Hiraoka^**, Akira Abe^**, Michael Borofsky^*, Robin G. Kunkel, Thomas Shanley, James A. Shayman^**, Frederick Lanni, Howard R. Petty^,*** and Laurence A. Boxer^*

^* Department of Pediatrics, Division of Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI 48109
Department of Pediatrics, Division of Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109
Department of Ophthalmology and Visual Science, University of Michigan Medical School, Ann Arbor, MI 48109
^** Department of Internal Medicine, Division of Nephrology, University of Michigan Medical School, Ann Arbor, MI 48109
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109
^*** Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109
Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, PA 15213

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one video.

Published, JLR Papers in Press, December 21, 2007.

¹ To whom correspondence should be addressed. e-mail: tzgalch{at}umich.edu

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcRIIA or both FcRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcRIIA/hCERK transfected cells displayed Ca²⁺ sparks around the phagosome. In contrast, cells expressing FcRIIA under identical conditions displayed little periphagosomal Ca²⁺ signaling. The enhanced Ca²⁺ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca²⁺ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca²⁺ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

Supplementary key words immunoglobulin G • ceramide-1-phosphate • store operated channels • transient potential channel • phagocytosis

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