title: Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate.
abstract: Ceramide kinase (CerK) and ceramide-1-phosphate (C1P) are involved in various cellular functions, while regulation of the enzyme activity has not been well elucidated. We herein investigated the effects of several glycerophospholipids on human recombinant CerK activity with CaCl2and MgCl2by measuring the formation of fluorescent labeled C1P in vitro. CerK activities were 44.1±11.4 (pmol/µg/min) with vehicle, 137±29 with 2 mM CaCl2, and 144±32 with 2 mM MgCl2in the glycerol/albumin buffer. The addition of glycerophospholipids such as phosphatidylcholine, phosphatidylinositol (PI), PI 4,5-bisphosphate (PI(4,5)P2), and phosphatidic acid had no effect on CerK activity with CaCl2, although PI(4,5)P2and phosphatidic acid bound to CerK in the lipid-protein overlay assay. The addition of cardiolipin (diphosphatidylglycerol) at concentrations up to 0.1 µM increased, whereas those more than 1 µM decreased CerK activity with CaCl2/MgCl2. In the lipid-protein overlay assay, cardiolipin bound to CerK and CerK lacking pleckstrin homology (PH) domain, but not PH domain of CerK, in CaCl2-independent manner. Cardiolipin also bound to CerK in the multilamellar vesicle binding assay. A deviation from the normal range of cellular cardiolipin, both the decrease by phospholipase D6 expression and increase by an exogenous addition of the lipid, negatively regulated C1P formation in intact HepG2 cells. Our results revealed that cardiolipin bound to CerK and regulated the formation of C1P in vitro and in cells.
authors.affiliation: Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University.
authors.forename: Wataru
authors.initials: W
authors.lastname: Matsuzaki
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Takahashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Murayama
mesh_terms: D002122:Calcium Chloride / Q000494:pharmacology; D002308:Cardiolipins / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D020404:Glycerophospholipids / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D002308:Cardiolipins; D002518:Ceramides; D020404:Glycerophospholipids; D011994:Recombinant Proteins; C065576:ceramide 1-phosphate; D015636:Magnesium Chloride; D004533:Egtazic Acid; D004492:Edetic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D010739:Phospholipase D; D002122:Calcium Chloride