Fig. 3: TRIM21 is responsible for PI3K/AKT-regulated G6PD stability.

a The potential G6PD E3 ligases identified by affinity MS. b TRIM21 interacts with endogenous G6PD. PC3 cell lysates were reciprocally immunoprecipitated and immunoblotted with indicated antibodies. c TRIM21 controls G6PD ubiquitination. HEK293T cells were cotransfected with indicated plasmids; 24 h later, incubated with or without MG132 (5 µM) for 16 h, Total cell lysates were immunoprecipitated with a Flag antibody and immunoblotted with the indicated antibodies. d The enzymatic activity and protein levels of G6PD (left panel) and PPP metabolites and nucleotides (right panel) in the TRIM21 WT and knockout A549 cells, and knockout cells re-expressing the WT or E3 ligase-dead mutant TRIM21 plasmid. n = 6 independent xenografts. e The growth kinetics of the isogenic TRIM21 WT and knockout A549 cell-derived xenografts in vivo. Equal numbers of TRIM21 WT and knockout cells were implanted into the bilateral flanks of nude mice. Relative tumor volumes are presented with growth kinetics. f TRIM21 controls G6PD ubiquitination-mediated degradation. HEK293T cells were transfected with si-scramble (WT) or siTRIM21 (KD) RNAs and 2 days later, cells were treated with or without CHX for 24 h (upper) or transfected with HA-ubiquitin and Flag-G6PD expression plasmids (lower). After 24 h, the cells were treated with or without PKI-587 for 24 h or MG132 for 16 h and total cell lysates were immunoprecipitated with a Flag antibody and immunoblotted with the indicated antibodies. g The Trim21 mRNA levels are lower in the null, CS and GE mES cells than in WT cells. h Left, PI3K/AKT inhibitors control TRIM21 and G6PD levels. PC3 cells were treated with PKI-587 (1 µM), BAY1082439 (5 µM), GDC-0980 (40 µM), GDC-0068 (10 µM) or rapamycin (100 nM) for 24 h. Right panel, HEK293T cells were transfected with AKT-WT, AKT-T308D/S473D or AKT-T308A/S473A plasmids. Metabolites were measured using LC-MS. Data are presented as fold changes and the mean ± SD and were compared with WT or untreated cells. Each experiment was performed n = 3 (d, g) and n = 4 (h) independent times. *p < 0.05, **p < 0.001, and ***p < 0.001, based on Student’s t test (two-sided ANOVA). See also Supplementary Fig. 3. Source data are provided as a Source Data file.