NPM-ALK regulates the phosphorylation and activity of PKM2. (A) Changes in p-ALK and p-PKM2 in response to ALK inhibition. The spectral counts for ALK (all phosphorylated peptides) and the 2 identified PKM2 phosphopeptides are shown. (B) Immunoblots of lysates from SU-DHL-1 cells following CEP treatment of 6 hours at 300 nM CEP. (C) Immunoblots of 293T lysates that were transiently transfected with either WT NPM-ALK or K210R NPM-ALK. (D) In vitro kinase assay with purified His-PKM2 (WT, Y105F, or Y390F) and ALK immunoprecipitation using GFP-NPM-ALK (WT or K210R). (E) PKM2 activity assay for SUPM2 cells treated with 100 nM CEP for 4 hours. (F) PKM2 activity assay on lysates from 293T cells transfected with mock, WT NPM-ALK, or K210R NPM-ALK. (G) Stable expression of Flag-PKM2 in DEL cells. (H) PKM2 activity assay of DEL cells stably expressing WT or Y105F PKM2. Data are mean ± SD; **P < .01.