Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Strub GM, Maceyka M, Hait NC et al (2010) Extracellular and intracellular actions of sphingosine-1-phosphate. Adv Exp Med Biol 688:141–155
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 350:429–441
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 275:19513–19520
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 47:1128–1139
Allende ML, Sasaki T, Kawai H et al (2004) Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720. J Biol Chem 279:52487–52492
Kharel Y, Lee S, Snyder AH et al (2005) Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720. J Biol Chem 280:36865–36872
Mizugishi K, Yamashita T, Olivera A et al (2005) Essential role for sphingosine kinases in neural and vascular development. Mol Cell Biol 25:11113–11121
Lai WQ, Irwan AW, Goh HH et al (2009) Distinct roles of sphingosine kinase 1 and 2 in murine collagen-induced arthritis. J Immunol 183:2097–2103
Jo SK, Bajwa A, Ye H et al (2009) Divergent roles of sphingosine kinases in kidney ischemia-reperfusion injury. Kidney Int 75:167–175
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–L613
Oskeritzian CA, Alvarez SE, Hait NC et al (2008) Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions. Blood 111:4193–4200
Maceyka M, Sankala H, Hait NC et al (2005) SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J Biol Chem 280:37118–37129
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: 1399–1407
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 280:36318–36325
Olivera A, Barlow KD, Spiegel S (2000) Assaying sphingosine kinase activity. Methods Enzymol 311:215–223
Olivera A, Spiegel S (1998) Sphingosine kinase. Assay and product analysis. Methods Mol Biol 105:233–242
Carman GM, Deems RA, Dennis EA (1995) Lipid signaling enzymes and surface dilution kinetics. J Biol Chem 270:18711–18714
Hait NC, Bellamy A, Milstien S et al (2007) Sphingosine kinase type 2 activation by ERK-mediated phosphorylation. J Biol Chem 282:12058–12065
Pitson SM, Moretti PA, Zebol JR et al (2003) Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J 22: 5491–5500
Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201:477–482
Kralik SF, Du XN, Patel C et al (2001) A method for quantitative extraction of sphingosine 1-phosphate into organic solvent. Anal Biochem 294:190–193
Vessey DA, Kelley M, Karliner JS (2005) A rapid radioassay for sphingosine kinase. Anal Biochem 337:136–142
Acknowledgements
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pitman, M.R., Pham, D.H., Pitson, S.M. (2012). Isoform-Selective Assays for Sphingosine Kinase Activity. In: Pébay, A., Turksen, K. (eds) Sphingosine-1-Phosphate. Methods in Molecular Biology, vol 874. Humana Press. https://doi.org/10.1007/978-1-61779-800-9_2
Download citation
DOI: https://doi.org/10.1007/978-1-61779-800-9_2
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-799-6
Online ISBN: 978-1-61779-800-9
eBook Packages: Springer Protocols