Sphingosine kinases (SK) 1 and 2 are unique lipid kinases that phosphorylate sphingosine to form sphingosine-1-phosphate (S1P). S1P is a bioactive molecule eliciting multiple effects both extracellularly via cell surface S1P receptors and intracellularly through a number of recently identified protein targets. The two enzymes arise from different genes, and differ in their cellular localisation, developmental expression, catalytic properties, and in at least some functional roles. Here, we describe methods for selectively detecting SK1 and SK2 activities in vitro, highlighting conditions that can discriminate between the activities of these two enzymes. The assays measure the production of 32P-labelled S1P following the addition of exogenous sphingosine and [γ32P] adenosine-5′-triphosphate. The S1P product can be purified by Bligh–Dyer solvent extraction, separated by thin-layer chromatography (TLC), and the radiolabelled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 and SK2 activities in tissue, cell, and recombinant protein samples.
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by the Fay Fuller Foundation and the National Health and Medical Research Council of Australia through a Senior Research Fellowship to S.M.P. (508098), and Project Grants 626937 and 1004695.
Strub GM, Maceyka M, Hait NC et al (2010) Extracellular and intracellular actions of sphingosine-1-phosphate. Adv Exp Med Biol 688:141–155PubMedCrossRefGoogle Scholar
Pitson SM, D’Andrea RJ, Vandeleur L et al (2000) Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes. Biochem J 350:429–441PubMedCrossRefGoogle Scholar
Liu H, Sugiura M, Nava VE et al (2000) Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J Biol Chem 275:19513–19520PubMedCrossRefGoogle Scholar
Wattenberg BW, Pitson SM, Raben DM (2006) The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function. J Lipid Res 47:1128–1139PubMedCrossRefGoogle Scholar
Allende ML, Sasaki T, Kawai H et al (2004) Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720. J Biol Chem 279:52487–52492PubMedCrossRefGoogle Scholar
Kharel Y, Lee S, Snyder AH et al (2005) Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720. J Biol Chem 280:36865–36872PubMedCrossRefGoogle Scholar
Mizugishi K, Yamashita T, Olivera A et al (2005) Essential role for sphingosine kinases in neural and vascular development. Mol Cell Biol 25:11113–11121PubMedCrossRefGoogle Scholar
Lai WQ, Irwan AW, Goh HH et al (2009) Distinct roles of sphingosine kinase 1 and 2 in murine collagen-induced arthritis. J Immunol 183:2097–2103PubMedCrossRefGoogle Scholar
Jo SK, Bajwa A, Ye H et al (2009) Divergent roles of sphingosine kinases in kidney ischemia-reperfusion injury. Kidney Int 75:167–175PubMedCrossRefGoogle Scholar
Wadgaonkar R, Patel V, Grinkina N et al (2009) Differential regulation of sphingosine kinases 1 and 2 in lung injury. Am J Physiol 296:L603–L613Google Scholar
Oskeritzian CA, Alvarez SE, Hait NC et al (2008) Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions. Blood 111:4193–4200PubMedCrossRefGoogle Scholar
Maceyka M, Sankala H, Hait NC et al (2005) SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J Biol Chem 280:37118–37129PubMedCrossRefGoogle Scholar
Hofmann LP, Ren S, Schwalm S et al (2008) Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner. Biol Chem 389: 1399–1407PubMedCrossRefGoogle Scholar
Okada T, Ding G, Sonoda H et al (2005) Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis. J Biol Chem 280:36318–36325PubMedCrossRefGoogle Scholar