Isoform-Selective Assays for Sphingosine Kinase Activity

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


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 



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.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

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

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