Fig. 3: GP73 promotes PKM2 secretion by enhancing Ubc9-mediated SUMO1 modification of PKM2.

A, B GP73 increases the PM location of PKM2. A IF merged images reveal more PM location of PKM2 in HCC cells transfected with GP73 compared to vector. Na, K-ATPase was used as a plasma membrane marker. PKM2 (red), Na, K-ATPase (blue). Scale bar, 5 μm. The white rectangles represent the location of PKM2 in the PM. B IF merged images reveal the PM location of PKM2 in HCC cells depending on the doses of GFP-GP73. Na, K-ATPase was used as a plasma membrane marker. GFP (green), PKM2 (red), Na, K-ATPase (blue). Scale bar, 2 μm. The white rectangles represent the location of PKM2 in the PM. C IF merged images reveal the colocalization of GP73 and PKM2 in PM of HCC cells. GFP (green), PKM2 (red), Na, K-ATPase (blue). Scale bar, 2 μm. The white rectangles represent the colocation of GP73 and PKM2 in the PM. D IF merged images reveal the colocalization of GP73 and PKM2 in Huh7 cells transfected with GFP-GP73 and mRuby-PKM2 at 8, 12, and 24 h. GFP-GP73 (green), mRuby-PKM2 (red), Na, K-ATPase (blue). Scale bar, 2 μm. The white rectangles represent the co-location of GP73 and PKM2. E GP73 overexpression increases the amount of PKM2 in the PM and decreases the amount in the cytoplasm of HCC cells as determined by western blotting. Na, K-ATPase was used as a PM protein loading control and β-actin was used as a cytoplasm loading control. F GP73 overexpression promotes the SUMO1 modification of PKM2 and GP73 KO attenuates the modification of PKM2. Huh7 cells co-transfected with FLAG-SUMO1, FALG-Ubc9, HA-PKM2, and control vector or GP73 were subjected to an in vivo SUMO1 conjugation assay (left panel). Huh7 control or GP73 KO cells in the right panel were transfected with FLAG-SUMO1, FALG-Ubc9, and HA-PKM2. G Deletion of 146-205 aa in domain III of GP73 attenuates the combination of PKM2 and Ubc9. Huh7 cells co-transfected with indicated plasmids were lysed with IP lysis buffer, and Co-IP assay was carried out. The arrows indicate the location of the target proteins. H Deletion of 146-205 aa in domain III of GP73 attenuates the SUMO1 modification of PKM2. Huh7 cells co-transfected with indicated plasmids were subjected to an in vivo SUMO1 conjugation assay. The arrows indicate the location of the target proteins. I The SUMO1 modification of PKM2 promotes the combination of PKM2 and GP73. Huh7 cells co-transfected with indicated plasmids were lysed with IP lysis buffer, and Co-IP assay was carried out. J, K The PM location of PKM2-SUMO1 in Huh7 cells was detected by Confocal IF as in (J) and western blotting as in (K). FLAG (green), DAPI (blue), nucleus. Scale bar, 5 μm. L PKM2-SUMO1 promotes the combination of GP73 and PKM2. Huh7 cells co-transfected with indicated plasmids were lysed with IP lysis buffer, and an IP assay was carried out.