Fig. 3: MAPK13 phosphorylates PHGDH at S371 and promotes its protein degradation upon oxidative stress.

a SFB-PHGDH was pulled down from SK-Hep1 cells stably expressing SFB-PHGDH treated with or without 0.5 mM H₂O₂ and 20 μM CQ for 3 h. The mass spectra of peptide harboring modified S371 residue was presented. Predicted b-type and y-type ions (not including all) were listed above and below the peptide sequence, respectively. b c57BL/6 J mice were intraperitoneally injected with 250 mg/kg APAP. Mouse livers were collected at 0 h, 4 h or 8 h after APAP treatment. Immunohistochemistry (IHC) staining of liver section using anti-PHGDH pS371 antibody was performed. Representative images of IHC staining were presented on the left panel, and statistical analysis were shown on the right panel (n = 5 in each time point). c SK-Hep1 cells were transiently transfected with Flag-PHGDH or Flag-PHGDH S371A. Cells were then treated with 0.5 mM H₂O₂ and 50 μg/mL CHX as indicated time. IB, immunoblotting. d SK-Hep1 cells were transiently transfected with Flag-PHGDH WT or 5 times Flag-PHGDH S371D plasmid and then treated with 50 μg/mL CHX as indicated time. e SK-Hep1 cells were transiently transfected with Flag-PHGDH WT or Flag-PHGDH S371D and then treated with or without 20 μM CQ for 16 h. f Flag-PHGDH was immunoprecipitated from SK-Hep1 cells transiently transfected with Flag-PHGDH WT or twice dose of Flag-PHGDH S371D plasmid. 36 h after transfection, the endogenous interacting proteins (LAMP2A and HSC70) were detected. g SK-Hep1 cells were transiently transfected with Flag-PHGDH WT or Flag-PHGDH S371A and HA-HSC70. 24 h after transfection, the cells were treated with 20 μM CQ and Flag-PHGDH was immunoprecipitated from the cells after treated with or without 0.5 mM H₂O₂ for 12 h. The interacting proteins (LAMP2A and HA-HSC70) were detected. h Flag-PHGDH was immunoprecipitated from SK-Hep1 cells co-transfected with EV or Flag-PHGDH and HA-MAPK13. i In vitro kinase assays were performed by mixing purified His-MAPK13 F324S and His-PHGDH WT or His-PHGDH S371A protein. j PHGDH was immunoprecipitated from hepatocytes isolated from WT and Mapk13 KO mice which were then treated with or without 0.5 mM H₂O₂ and 10 μM CQ for 4 h. k Hepatocytes isolated from WT and Mapk13 KO mice were treated with or without 0.5 mM H₂O₂ for 16 h. l SK-Hep1 cells stably expressing shNT or shMAPK13 were transiently transfected with Flag-PHGDH, Flag-PHGDH S371A or Flag-PHGDH S371D, and then treated with 0.5 mM H₂O₂ for 24 h. m Hepatocytes isolated from WT and Mapk13 KO mice transiently transfected with Flag-Phgdh WT or Flag-Phgdh S371D. Cells were treated with 0.5 mM H₂O₂ for 16 h. Immunoblots are representative of three independent experiments. Data are means ± SD. Each point represents an individual mouse. P values were determined by two-tailed Student’s t-test.