Isoform-Selective Assays for Sphingosine Kinase Activity

  • Melissa R. Pitman
  • Duyen H. Pham
  • Stuart M. Pitson
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 874)

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.

Key words

Sphingosine kinase d-erythro-sphingosine Sphingosine-1-phosphate Thin-layer chromatography Bligh–Dyer extraction 

References

  1. 1.
    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
  2. 2.
    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
  3. 3.
    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
  4. 4.
    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
  5. 5.
    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
  6. 6.
    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
  7. 7.
    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
  8. 8.
    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
  9. 9.
    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
  10. 10.
    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
  11. 11.
    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
  12. 12.
    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
  13. 13.
    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
  14. 14.
    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
  15. 15.
    Olivera A, Barlow KD, Spiegel S (2000) Assaying sphingosine kinase activity. Methods Enzymol 311:215–223PubMedCrossRefGoogle Scholar
  16. 16.
    Olivera A, Spiegel S (1998) Sphingosine kinase. Assay and product analysis. Methods Mol Biol 105:233–242PubMedGoogle Scholar
  17. 17.
    Carman GM, Deems RA, Dennis EA (1995) Lipid signaling enzymes and surface dilution kinetics. J Biol Chem 270:18711–18714PubMedCrossRefGoogle Scholar
  18. 18.
    Hait NC, Bellamy A, Milstien S et al (2007) Sphingosine kinase type 2 activation by ERK-mediated phosphorylation. J Biol Chem 282:12058–12065PubMedCrossRefGoogle Scholar
  19. 19.
    Pitson SM, Moretti PA, Zebol JR et al (2003) Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J 22: 5491–5500PubMedCrossRefGoogle Scholar
  20. 20.
    Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201:477–482PubMedCrossRefGoogle Scholar
  21. 21.
    Kralik SF, Du XN, Patel C et al (2001) A method for quantitative extraction of sphingosine 1-phosphate into organic solvent. Anal Biochem 294:190–193PubMedCrossRefGoogle Scholar
  22. 22.
    Vessey DA, Kelley M, Karliner JS (2005) A rapid radioassay for sphingosine kinase. Anal Biochem 337:136–142PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Melissa R. Pitman
    • 1
  • Duyen H. Pham
    • 1
  • Stuart M. Pitson
    • 1
    • 2
  1. 1.Molecular Signalling LaboratoryCentre for Cancer Biology, SA PathologyAdelaideAustralia
  2. 2.School of Molecular and Biomedical ScienceUniversity of AdelaideAdelaideAustralia

Personalised recommendations