Fig. 2: PHGDH transmits low 3-PGA signal to Ser46 phosphorylation of p53.

a PHGDH interacts with p53. Cell lysates of HEK293 cells, regularly cultured or glucose starved for 2 h, were subjected to immunoprecipitation of endogenous PHGDH (left panel) or p53 (right panel), followed by immunoblotting of co-precipitated p53 (left panel) or PHGDH (right panel). TCL, total cell lysate. b Addition of 3-PGA to lysates of glucose-starved cells disrupts the PHGDH–p53 interaction. The 2-h-glucose-starved HEK293 cells were lysed, followed by addition of 3-PGA at indicated final concentrations into the lysates. Endogenous PHGDH was then immunoprecipitated, followed by immunoblotting with antibodies indicated. c PHGDH is required for p-p53-Ser46 induction in low glucose. HEK293 cells with knockout of PHGDH were glucose starved for 2 h, followed by immunoblotting to determine p-p53-Ser46. d Lack of occupancy of 3-PGA by PHGDH underlies the levels of p-p53-Ser46. PHGDH^–/– HEK293 cells were re-introduced with PHGDH-T57A and PHGDH-T78A that are defective in binding to 3-PGA, or PHGDH-R135W and PHGDH-V261M that are constitutively occupied with 3-PGA, and were glucose starved for 2 h, followed by immunoblotting to determine levels of p-p53-Ser46. e, f Glucose starvation induces the formation of PHGDH–AXIN–TIP60–HIPK2–p53 complex. HEK293 cells (e) or human primary HCC cells (f) were glucose starved for 2 h, followed by immunoprecipitation of endogenous p53 and immunoblotting with antibodies indicated. g, h AXIN tethers the upstream kinase HIPK2 for phosphorylation of p53-Ser46 in low glucose. HEK293 cells with knockdown of HIPK2 (h), HEK293 cells with knockout of AXIN or with re-introduced AXIN truncation mutants defective in interacting with p53 (Δp53) or HIPK2 (ΔHIPK2) (g), were glucose starved for 2 h, followed by immunoblotting to determine p-p53-Ser46. Experiments were performed three times, except four times in c and d.