Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

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Abstract

<p>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 CaCl<sub>2</sub> and MgCl<sub>2</sub> by measuring the formation of fluorescent labeled C1P <i>in vitro</i>. CerK activities were 44.1±11.4 (pmol/µg/min) with vehicle, 137±29 with 2 mM CaCl<sub>2</sub>, and 144±32 with 2 mM MgCl<sub>2</sub> in the glycerol/albumin buffer. The addition of glycerophospholipids such as phosphatidylcholine, phosphatidylinositol (PI), PI 4,5-bisphosphate (PI(4,5)P<sub>2</sub>), and phosphatidic acid had no effect on CerK activity with CaCl<sub>2</sub>, although PI(4,5)P<sub>2</sub> and 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 CaCl<sub>2</sub>/MgCl<sub>2</sub>. In the lipid–protein overlay assay, cardiolipin bound to CerK and CerK lacking pleckstrin homology (PH) domain, but not PH domain of CerK, in CaCl<sub>2</sub>-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 <i>in vitro</i> and in cells.</p>

Journal

  • Biological and Pharmaceutical Bulletin

    Biological and Pharmaceutical Bulletin 39(10), 1708-1717, 2016

    The Pharmaceutical Society of Japan

Codes

  • NII Article ID (NAID)
    130005598502
  • NII NACSIS-CAT ID (NCID)
    AA10885497
  • Text Lang
    ENG
  • ISSN
    0918-6158
  • NDL Article ID
    027624032
  • NDL Call No.
    Z53-V41
  • Data Source
    NDL  J-STAGE 
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