eEF2K phosphorylates PKM2 at Thr129. a Possible eEF2K phosphorylation sites in PKM2 identified by LC-MS/MS analysis following eEF2K kinase assays (2 replicates: Rep 1 and Rep 2) and in-gel digestion of PKM2. MS analysis. Selected potential phosphorylation sites are circled. b HEK293 cells were transfected with empty vector, FLAG-Rheb or FLAG-PKM2 constructs, cells were lysed 48 h after transfection and samples were immunoblotted using anti-FLAG. c WT FLAG-PKM2 and the indicated FLAG-PKM2 mutant proteins were expressed in HEK293 cells. FLAG-PKM2 was immunoprecipitated and then incubated with recombinant eEF2K kinase (1 μg) for 30 min for an in vitro phosphorylation assay. d Sequence alignment of the region of PKM2 Thr129 (in Homo sapiens) with other species. e MS-spectra shows PKM2 T129 phosphorylation. f A549 cells expressing WT PKM2 or PKM2 T129A were treated with BS3 for 1 h before being lysed. Immunoblotting analyses were performed with the indicated antibodies. The relative levels of the PKM2 and P-STAT3 to actin were quantified. **P < 0.01, vs. PKM2 WT.