Fig. 5: S287 is a critical phosphorylation site of PKM2.

a S287A decreases PKM2 phosphorylation. The indicated HA-PKM2 mutant plasmids were transfected into 293T cells, and protein was immunoprecipitated for phosphorylation analysis. b MS shows PKM2 S287 phosphorylation. c S287 of PKM2 is evolutionarily conserved in the indicated species. The sequences around PKM2 S287 from different species were aligned. d CIP2A shifts WT PKM2 but not S287A mutant PKM2 to higher-molecular-weight fractions. The indicated plasmids were transfected into 293T cells, and cell lysates were separated by size exclusion chromatography, followed by western blot analysis. Fraction numbers and relative molecular weights (Mr) are indicated. Ectopic expression of Flag-CIP2A and HA-tagged WT PKM2 or S287A PKM2 mutant in 293T cells was detected by western blot. e Size exclusion chromatography of purified recombinant WT, S287A and S287D PKM2 that were expressed in bacteria. The molecular mass standards and theoretical molecular mass of PKM2 tetramer (240 kDa)/dimer (120 kDa) are marked on the lower axis. f Western blot analyses of PKM2 phosphorylation at Y105, S37 and S287 in A549 and H1299 cells transfected with shCIP2A. g Knockdown of CIP2A inhibits tumor growth and suppresses PKM2 S287 phosphorylation in tumor samples of mice inoculated with shCIP2A-expressing A549 cells. A549 cells transfected with shNC are used as a control. h Western blot analysis of PKM2 phosphorylation at S287 and S37 in H1299 cells that were transfected with Flag-CIP2A and Flag-B56α. i Effects of B56α on phosphorylation of PKM2 S287 by an in vitro dephosphorylation assay using HA-tagged PKM2 and FLAG-B56α proteins purified from 293T cells. j IHC staining experiments were performed in 15 human lung adenocarcinoma specimens using anti-CIP2A and anti-PKM2 pSer287 antibodies, and the correlation between immunoreactivity scores (IRSs) of CIP2A and PKM2 pS287 was analyzed. Note that some scores overlap, and the dots on the graphs indicate no less than one sample. Scale bars, 50 μm. k Cytosolic and nuclear fractions were prepared from H1299 cells transfected with shPKM2, shRNA-resistant HA-PKM2 (WT or S287D), and siCIP2A. Nuclear lamin B and cytoplasmic tubulin were used as controls. l, m Flag-CIP2A and reconstituted shRNA-resistant HA-PKM2 (WT or S287A mutant) were transfected into shPKM2-treated H1299 cells. Pyruvate kinase activity (l) and OCR mitochondrial respiration parameters (m) were measured. PKM2 silencing and re-expression efficiency were verified by western blot. Data represent means ± SD of three independent experiments. EV empty vector. Two-sided unpaired Student’s t-test; **P < 0.01; n.s. not significant.