Pim2 phosphorylates TSC2 on Ser-1798 to regulate mTOR-C1 signaling. (A) Schematic representation of two sets of TSC2 mutants. One set of mutants contains a single mutation from Ser or Thr to Ala; the second set of mutants maintains 1 WT Ser or Thr while mutating the other 7 to Ala. (B) Serine to alanine mutation of 1798 abolished TSC2 phosphorylation by Pim2. WT-TSC2 or TSC2 with single Ser/Thr to Ala mutations were transfected into TSC2 null 293A cells with either GFP or Pim2. TSC2 was immunoprecipitated by TSC2 Ab and then analyzed for p-TSC2 level by p-Akt substrate Ab with immunoblotting. (C) Pim2 primarily phosphorylates TSC2 on Ser-1798. WT-TSC2, all mutant-TSC2, or TSC2 mutants with single WT-Ser/Thr were transfected into TSC2 null 293A cells with either GFP or Pim2. TSC2 was immunoprecipitated by TSC2 Ab and then analyzed for p-TSC2 level by p-Akt substrate Ab with immunoblotting. (D) In vitro kinase assay confirms that Pim2 phosphorylates TSC2 on Ser-1798. WT-TSC2, TSC2-S1798A, or TSC2-S1798WT were transfected into TSC2 null 293A cells and, after 24 hours, were switched to serum-free medium for another 36 hours. TSC2 was immunoprecipitated by TSC2 Ab and then incubated with either recombinant Pim2 or recombinant Pim2 with preincubation with LGB321. Reaction was terminated and analyzed for p-TSC2 (Ser-1798) by immunoblotting. (E) TSC2 Ser-1798 phosphorylation plays an important role in mediating Pim2’s modulation on mTOR-C1. WT-TSC2 or TSC2-S1798A was transfected into TSC2 null 293A cells with either GFP or Pim2, and the phosphorylation status of p-S6RP (S235/S236) was examined 48 hours post transfection. (F) Pim inhibition leads to reduced p-TSC2 (Ser-1798) in MM cells. KMS-11 and KMS-26 cells were treated with LGB321 (0, 0.33, and 1.0 μM) for 2 hours; TSC2 was immunoprecipitated and analyzed for p-TSC2 (Ser-1798).