Fig. 6: CIP2A induces PKM2 phosphorylation at S287 and nuclear translocation.

a Knocking down PP2A blocks CIP2A depletion-induced S37 phosphorylation. A549 cells stably expressing CIP2A shRNA were transfected with PP2A-A α/β siRNA or control, followed by immunoblotting analysis. b, c H1299 and A549 cells were treated with celastrol (b) or TD52 (c) for 48 h, lysed, and cell lysates were analyzed by immunoblotting. d, e H1299 cells were transfected with exogenous CIP2A, and 48 h later treated with celastrol/TD52 for additional 48 h. The cells were lysed for western blot using the indicated antibodies (d) or cell proliferation was measured by an IncuCyte Live-Cell Analysis System (e). f Structural analysis of the human PKM2 tetramer (PDB code: 3SRD). The four PKM2 monomers are shown in cartoon mode with different colors, and the interfaces between two dimers are represented by dashed lines. The amino acid residues Ile429/Leu431 (ERK1/2 binding sites), Arg399/400 (importin α5 binding site), Ser287 and Ser37 are indicated by ball and stick models and colored yellow, purple, cyan and green, respectively. Magnified images with labeled residues are also presented. g Interaction among PKM2, ERK1/2, importin α5 and p-PKM2(S37). Flag-CIP2A and shRNA-resistant HA-tagged PKM2 (WT or S287A mutant) were transfected into PKM2-depleted H1299 cells, followed by IP and western blotting analysis with the indicated antibodies. h Cytosolic and nuclear fractions were prepared from H1299-shPKM2 cells transfected with shRNA-resistant HA-PKM2 (WT or S37A mutant) and siCIP2A. Nuclear lamin B and cytoplasmic tubulin were used as controls.