FIGURE 3.

SK1 is an oligomer. HEK 293 cells were transiently transfected with myc-tagged G81D SK1, FLAG-tagged D178N SK1, myc-tagged WT SK1, and/or FLAG-tagged WT SK1 plasmid constructs. A and B, Western blots (WB) of anti-FLAG immunoprecipitates (IP) (A) or anti-myc immunoprecipitates (B) probed with respective anti-myc and anti-FLAG antibodies. Lysate SK1 represents lysates of cells overexpressing both myc- and FLAG-tagged WT SK1. C, Western blot and SK1 activity of HEK 293 cell lysates showing the effect of overexpressing FLAG-tagged D178N SK1 on myc-tagged WT SK1 activity (using micrograms of plasmid constructs shown). FLAG-tagged D178N SK1-myc-tagged G81D SK1 oligomers were catalytically deficient. Results are representative of three experiments. SK1 activity was also measured in immunoprecipitates: anti-myc immunoprecipitates: myc-tagged G81D SK1-transfected cells, 0.23 pmol/min; myc-tagged G81D SK1/FLAG-tagged D178N SK1-transfected cells, 1.65 pmol/min; myc-tagged WT SK1-transfected cells, 33 pmol/min. Anti-FLAG immunoprecipitates: FLAG-tagged D178N SK1-transfected cells, 2.8 pmol/min; FLAG-tagged D178N SK1/myc-tagged G81D SK-transfected cells, 2 pmol/min; FLAG-tagged WT SK1-transfected cells, 20.55 pmol/min. Results are representative of two or three experiments. D, effect of (S)-FTY720 vinylphosphonate or Bdp-So (both at 25 and 50 μm and assayed using 10 μm sphingosine) on WT and G113A SK1 (both transiently over-expressed in HEK293 cells) activity. Results are expressed as % of SK1 activity in the absence of (S)-FTY720 vinylphosphonate or Bdp-So (control = 100% SK1 activity, n = 3 experiments).