Fig. 6.
GTPBP4 regulates translocation of PKM2 into the nucleus by sumoylation. (A) Co-IP was performed to verify that PKM2 SIM mutants (PKM2I267&268A) could abolish SUMO1-induced PKM2 sumoylation. n = 3. (B) Monomeric and dimeric PKM2 expression was analyzed using Western blot in PLC/PRF/5 (left) and SMMC-7721 cells (right) grouped and treated as showed. n = 3. (C) Western blot was used to detect PKM2 expression in the nucleus in GTPBP4 OE PLC/PRF/5 cells and GTPBP4 KD HCCLM3 cells and corresponding control cells. n = 3. (D) PLC/PRF/5 cells were transfected with Flag-tagged GTPBP4, GST-tagged PKM2, Myc-tagged SUMO1, and HA-tagged SENP1 and subjected to Western blot assay. (E) PLC/PRF/5 cells were transfected with Flag-tagged GTPBP4, GST-tagged PKM2, His-tagged PKM2I267&268A, and Myc-tagged SUMO1 and subjected to Western blot assay. n = 3. (F) ECAR was detected in GTPBP4 OE PLC/PRF/5 and control cells transfected with or without PKM2I267&268A. Error bars represent the mean ± SD from six independent experiments. n = 6. (G–H) Lactate production (G) and glucose consumption activity (H) were measured were detected in GTPBP4 OE PLC/PRF/5 cells and GTPBP4 OE SMMC-7721 cells and their respective control cells transfected both with or without PKM2I267&268A. n = 6. The data presented in (F)–(H) are compared among groups using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.