title: Ceramide kinase is a mediator of calcium-dependent degranulation in mast cells.
abstract: Ceramide kinase (CERK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P) and is known to be activated by calcium. Although several groups have examined the functions of CERK and its product C1P, the functions of C1P and CERK are not understood. We studied the RBL-2H3 cell line, a widely used model for mast cells, and found that CERK and C1P are required for activation of the degranulation process in mast cells. We found that C1P formation was enhanced during activation induced by IgE/antigen or by Ca(2+) ionophore A23187. The formation of C1P required the intracellular elevation of Ca(2+). We generated RBL-2H3 cells that stably express CERK, and when these cells were treated with A23187, a concomitant C1P formation was observed and degranulation increased 4-fold, compared with mock transfectants. The cell-permeable N-acetylsphingosine (C(2)-ceramide), a poor substrate of CERK, inhibited both the formation of C1P and degranulation, indicating that C1P formation was necessary for degranulation. Exogenous introduction of CERK into permeabilized RBL-2H3 cells caused degranulation. We identified a cytosolic localization of CERK that provides exposure to cytosolic Ca(2+). Taken together, these results indicate that C1P formation is a necessary step in the degranulation pathway in RBL-2H3 cells.
authors.affiliation: Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Mitsutake
authors.forename: Tack-Joong
authors.initials: TJ
authors.lastname: Kim
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Inagaki
authors.forename: Mariko
authors.initials: M
authors.lastname: Kato
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Yamashita
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Igarashi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006868:Hydrolysis; D007476:Ionophores / Q000494:pharmacology; D008407:Mast Cells / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010739:Phospholipase D / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
chemical_list: D007476:Ionophores; D012333:RNA, Messenger; D000001:Calcimycin; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D010739:Phospholipase D; D002118:Calcium