Fig. 1. Agonist-induced phosphorylation of hSK1. Time course of in vivo hSK1 phosphorylation and activity following TNFα (1 ng/ml) and PMA (10 ng/ml) stimulation of cells. HEK293T cells over expressing hSK1 were metabolically labelled with ³²P prior to treatment with TNFα and PMA for the indicated times (min). Immunoprecipitated hSK1 from these cells was then subjected to SDS–PAGE and the incorporation of ³²P into hSK1 determined. Loading controls for hSK1 in the immunoprecipitates were visualized by western blot via their FLAG epitope. Quantitation of ³²P incorporation into hSK1 following 5, 10 and 30 min of TNFα treatments showed fold increases of 1.7 ± 0.3, 2.3 ± 0.3 and 3.4 ± 0.4, respectively, over that seen in the untreated cell extracts. Similarly, PMA treatment for 30 min resulted in a 2.9 ± 0.3-fold increase in ³²P incorporation into hSK1 compared with the untreated cell extracts. Sphingosine kinase activities in the extracts were determined prior to immunoprecipitation. All data are represented as means (± SD) from more than three experiments.