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Part of the series Methods in Molecular Biology pp 1-12

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An Improved Isoform-Selective Assay for Sphingosine Kinase 1 Activity

  • Melissa R. PitmanAffiliated withMolecular Signalling Laboratory, Centre for Cancer Biology, University of South Australia and SA Pathology
  • , Lorena T. DaviesAffiliated withMolecular Signalling Laboratory, Centre for Cancer Biology, University of South Australia and SA Pathology
  • , Stuart M. PitsonAffiliated withMolecular Signalling Laboratory, Centre for Cancer Biology, University of South Australia and SA Pathology Email author 

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Abstract

Sphingosine kinases (SK) are the sole enzymes responsible for the production of sphingosine 1-phosphate (S1P). S1P is a signaling molecule with a plethora of targets, acting as both a second messenger intracellularly and extracellularly via a family of cell surface G-protein-coupled S1P receptors. The two sphingosine kinases, SK1 and SK2, arise from different genes and have some distinct and overlapping cellular functions that are regulated in part by differential cellular localization, developmental expression, and catalytic properties. Here, we describe an improved method for selectively detecting SK1 activity in vitro and cell lysates via the use of the zwitterionic detergent CHAPS, which effectively inhibits SK2 activity and thus allows selective analysis of SK1 activity in a range of cell samples. The assay measures the production of 32P-labeled S1P following the addition of exogenous sphingosine and [γ32P]ATP. The S1P product can be purified by Bligh–Dyer solvent extraction, separated by thin layer chromatography (TLC), and the radiolabeled S1P quantified by exposing the TLC plate to a storage phosphor screen. This sensitive, reproducible assay can be used to selectively detect SK1 activity in tissue, cell, and recombinant protein samples.

Keywords:

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