Isoform-selective assays for sphingosine kinase activity

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 identifi ed 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 32 P-labelled S1P following the addition of exogenous sphingosine and [ƴ32P] adenosine-5′-triphosphate. The S1P product can be purifi ed by Bligh–Dyer solvent extraction, separated by thin-layer chromatography (TLC), and the radiolabelled S1P quantifi ed 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.