FTY720-dependent PP2A activation in myeloid cells depends on the SET K209–FTY720 interaction but not on SET dimerization or increased ceramide levels. (A) PP2A activity in untreated and FTY720-treated Ba/F3 cells expressing WT or K209D SET proteins. (B) Pull-down assays demonstrate FTY720-SET interaction in NBD immunoprecipitates of Ba/F3-Jak2V617F cell lysates treated with NBD-conjugated FTY720 (top left) and in avidin-mediated affinity chromatography with lysates of Ba/F3-Jak2V617F cells treated with biotin-tagged FTY720 (bottom left). PP2A activity in Ba/F3-Jak2V617F cells treated with FTY720 (2.5 µM), FTY720-NBD (2.5 µM), and FTY720-biotin (2.5 µM) (right). (C) Diacylglycerol kinase assay shows gross ceramide levels in untreated and FTY720-treated Ba/F3-Jak2V617F cells (top). LC-MS/MS measurement of levels of specific ceramides in FTY720-treated Ba/F3-Jak2V617F cells expressed as percentage of those in untreated cells (bottom). The ceramide C18 able to induce PP2A activation is depicted as a solid bar. (D) PP2A activity in parental and WT or nondimerizing (TAF-1β PME mutant) SET-expressing Ba/F3 cells (left). Western blot show levels of GFP-SET in FLAG immunoprecipitates from lysates of Ba/F3-FLAG-SET coincubated with equal amount of Ba/F3-GFP-SET cell lysates in the presence of DMSO or FTY720 (right). DMSO, dimethylsulfoxide.
