Jak2V617F-driven PI-3Kγ/PKC–mediated signals induce inhibition of PP2A through SET phosphorylation. (A) Molecular pathway model of Jak2/Jak2V617F-mediated PP2A inhibition. Chemical inhibitors are indicated in black. (B) SET and Grb2 protein levels in MigR1-, Jak2V617F-, and BCR-ABL1-expressing Ba/F3 and 32Dcl3 cells (upper left); SET protein levels in CD34+ BM progenitors from healthy individuals (NBM) and PV and CML-BC patients (n = 3 each) (upper right). PP2A activity in Ba/F3 cells transduced with empty vector (pCDH), WT, nonphosphorylatable S9A, and phosphomimetic S9E mutant SET FLAG-tagged constructs (bottom panel). Inset shows ectopic WT and mutant SET proteins (nuclear fraction). (C) Levels of serine phosphorylated SET (pSET) in SET immunoprecipitates (SET) from lysates of untreated, kinase (Jak2, PI-3K, PI-3Kγ, PKC, and MEK1) inhibitors and FTY720-treated Ba/F3-Jak2V617F cells. (D) PP2A activity in untreated and drug-treated Ba/F3-Jak2V617F cells and in Ba/F3-Jak2V617F cells transduced with empty vector or expressing a S1PR1 shRNA and treated with 2.5 µM FTY720. Inset shows S1PR1 levels in parental and S1PR1 shRNA-expressing Ba/F3-Jak2V617F cells. *P ≤ .01.
