Ca2+/calmodulin-dependent translocation of sphingosine kinase: role in plasma membrane relocation but not activation

Cell Calcium. 2003 Feb;33(2):119-28. doi: 10.1016/s0143-4160(02)00205-1.

Abstract

Activation of sphingosine kinase (SPHK), thereby increasing cellular levels of sphingosine 1-phosphate (S1P), may be involved in a variety of intracellular responses including Ca(2+) signaling. This study uses mammalian SPHK1a, tagged with enhanced green fluorescent protein (eGFP), to examine whether translocation of this enzyme is linked with Ca(2+)-mobilizing responses. Real-time confocal imaging of SPHK1a-eGFP in human SH-SY5Y neuroblastoma cells visualized a relocation of the enzyme from the cytosol to the plasma membrane in response to Ca(2+)-mobilizing stimuli (muscarinic M(3)- or lysophosphatidic acid receptor activation, and thapsigargin-mediated store release). This redistribution was preceded by a transient increase in cytosolic SPHK1a-eGFP levels due to liberation of SPHK from localized higher intensity regions. Translocation was dependent on Ca(2+) mobilization from intracellular stores, and was prevented by pretreatment with the Ca(2+)/calmodulin inhibitor W-7, but not W-5 or KN-62. In functional studies, pretreatment with W-7 lowered basal and M(3)-receptor-mediated cellular S1P production. However, this pretreatment did not alter agonist-mediated Ca(2+) responses, and SPHK1a-eGFP activity itself appeared insensitive to Ca(2+)/calmodulin and W-7. These data suggest a role for Ca(2+)/calmodulin in controlling the subcellular distribution but not the activity of SPHK1a.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Clocks / drug effects
  • Biological Clocks / physiology
  • Calcium / metabolism*
  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology*
  • Calmodulin / antagonists & inhibitors
  • Calmodulin / metabolism*
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology*
  • Cytosol / drug effects
  • Cytosol / metabolism
  • Enzyme Inhibitors / pharmacology
  • Eukaryotic Cells / drug effects
  • Eukaryotic Cells / enzymology*
  • Humans
  • Phosphorylation / drug effects
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Protein Transport / drug effects
  • Protein Transport / physiology*
  • Receptor, Muscarinic M3
  • Receptors, Cell Surface / drug effects
  • Receptors, Cell Surface / metabolism
  • Receptors, G-Protein-Coupled*
  • Receptors, Lysophosphatidic Acid
  • Receptors, Muscarinic / drug effects
  • Receptors, Muscarinic / metabolism
  • Recombinant Fusion Proteins
  • Sphingosine / biosynthesis
  • Tumor Cells, Cultured

Substances

  • Calmodulin
  • Enzyme Inhibitors
  • Receptor, Muscarinic M3
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • Receptors, Lysophosphatidic Acid
  • Receptors, Muscarinic
  • Recombinant Fusion Proteins
  • Phosphotransferases (Alcohol Group Acceptor)
  • sphingosine kinase
  • Sphingosine
  • Calcium