An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes.
Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.
Roberts
Jane L
JL
Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia.
Moretti
Paul A B
PA
Darrow
Andrew L
AL
Derian
Claudia K
CK
Vadas
Mathew A
MA
Pitson
Stuart M
SM
D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008567:Membranes, Artificial; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D013110:Sphingosine / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry*; D013379:Substrate Specificity; D000099157:Sphingosine Kinase
D001426:Bacterial Proteins; D001710:Biotin; D008567:Membranes, Artificial; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D019809:Streptavidin; D000099157:Sphingosine Kinase