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.
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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.
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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
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DOI: https://doi.org/10.1007/978-1-61779-800-9_2
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