title: Ca2+/calmodulin-dependent translocation of sphingosine kinase: role in plasma membrane relocation but not activation.
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.
authors.affiliation: Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, UK. kwyl@le.ac.uk
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Young
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
authors.forename: M Janine
authors.initials: MJ
authors.lastname: Parkinson
authors.forename: Paula
authors.initials: P
authors.lastname: Bartlett
authors.forename: Sarah
authors.initials: S
authors.lastname: Spiegel
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Nahorski
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D001683:Biological Clocks / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D043587:Receptor, Muscarinic M3; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013110:Sphingosine / Q000096:biosynthesis; D014407:Tumor Cells, Cultured; D000099157:Sphingosine Kinase
chemical_list: D002118:Calcium; D002147:Calmodulin; D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D043587:Receptor, Muscarinic M3; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D013110:Sphingosine; D000099157:Sphingosine Kinase