title: The calmodulin-binding site of sphingosine kinase and its role in agonist-dependent translocation of sphingosine kinase 1 to the plasma membrane.
abstract: Sphingosine kinases catalyze the formation of sphingosine 1-phosphate, a bioactive lipid involved in many aspects of cellular regulation, including the fundamental biological processes of cell growth and survival. A diverse range of cell agonists induce activation of human sphingosine kinase 1 (hSK1) and, commonly, its translocation to the plasma membrane. Although the activation of hSK1 in response to at least some agonists occurs directly via its phosphorylation at Ser225 by ERK1/2, many aspects governing the regulation of this phosphorylation and subsequent translocation remain unknown. Here, in an attempt to understand some of these processes, we have examined the known interaction of hSK1 with calmodulin (CaM). By using a combination of limited proteolysis, peptide interaction analysis, and site-directed mutagenesis, we have identified that the CaM-binding site of hSK1 resides in the region spanned by residues 191-206. Specifically, Phe197 and Leu198 are critically involved in the interaction because a version of hSK1 incorporating mutations of both Phe197 --> Ala and Leu198 --> Gln failed to bind CaM. We have also shown for the first time that human sphingosine kinase 2 (hSK2) binds CaM, and does so via a CaM binding region that is conserved with hSK1 because comparable mutations in hSK2 also ablate CaM binding to this protein. By using the CaM-binding-deficient version of hSK1, we have begun to elucidate the role of CaM in hSK1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of hSK1 from the cytoplasm to the plasma membrane, while having no effect on hSK1 phosphorylation and catalytic activation.
authors.affiliation: Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, University of Adelaide, Adelaide, South Australia 5000, Australia.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Sutherland
authors.forename: Paul A B
authors.initials: PA
authors.lastname: Moretti
authors.forename: Niamh M
authors.initials: NM
authors.lastname: Hewitt
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Bagley
authors.forename: Mathew A
authors.initials: MA
authors.lastname: Vadas
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Pitson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008246:Lysophospholipids / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099157:Sphingosine Kinase
chemical_list: D002147:Calmodulin; D002273:Carcinogens; D008246:Lysophospholipids; D010446:Peptide Fragments; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate