Fig 2: Degradation of non-phosphorylated CDC73 at Ser465 by UBR5-mediated UPS.A The protein levels of UBR5 and CDC73 in 4T1/GFP and 4T1/Ubr5-/- cells reconstituted with EGFP, UBR5, or UBR5 C2768A mutant were evaluated by western blotting. B The turnover of CDC73 in 4T1/GFP and 4T1/Ubr5-/- cells was measured by CHX treatment (50 µg/ml; 0, 6, 12 and 24 h) and then detected by western blotting and quantified by ImageJ software. The protein level of CDC73 was detected by western blotting in both 4T1/GFP and 4T1/Ubr5-/- cells treated with vehicle, MG132 (10 µM, 8 h) (C), STS (0.1 µM, 6 h) (D), VX-11e (1 µM or 10 µM, 24 h) (E), and VX-11e (10 µM, 24 h) or/and MG132 (10 µM, 8 h) (F). G The protein level of CDC73 was detected by western blotting in both 4T1/GFP and 4T1/Ubr5-/- cells transfected with control siRNA or siErk2 and treated with vehicle or MG132 (10 µM, 8 h). H Co-IP with anti-Myc antibody analyzed the physical interaction of endogenous UBR5 and exogenous CDC73 in HEK293T cells transfected with empty vector or Myc-CDC73 plasmids and treated with vehicle or VX-11e (10 µM, 24 h) in the presence of MG132 (10 µM, 8 h). I Ubiquitination assays analyzed the ubiquitination level of CDC73 modified by exogenous UBR5 in the presence of vehicle, VX-11e (10 µM, 24 h) or/and MG132 (10 µM, 8 h) under denaturing conditions in HEK293T cells transfected with the indicated plasmids. The phosphorylation levels of UBR5 and CDC73 were analyzed via the Phos-tag SDS-PAGE in 4T1 cells treated with vehicle or VX-11e (10 µM, 24 h) (J) and the control and Erk2 silenced 4T1 cells (K). L Ubiquitination assays analyzed the ubiquitination level of three mutants (T334A, T359A and S465A) of CDC73 modified with exogenous UBR5. The phosphorylation (M) and ubiquitination (N) levels of WT and S465A mutant of CDC73 were detected in HEK293T cells treated with vehicle or VX-11e (10 µM, 24 h). O The turnover of WT and the S465A mutant of CDC73. HEK293T cells were transfected with Myc-CDC73 and Myc-CDC73 (S465A) plasmids for 48 h and treated with CHX as described in (B).

Fig 3: (A) Survival rates according to the presence or absence of CDC73 mutation. The 10-year survival rates did not differ significantly between the two groups of patients. (B) Survival rates according to the loss of parafibromin. Loss of parafibromin staining was associated with a statistically significant decrease in the 10-year survival. (C) Survival rates according to the presence or absence of CDC73 mutation and loss of parafibromin. The presence of both abnormalities was associated with a statistically significant decrease in the 10-year survival.

Fig 4: (A) Effect of CDC73 mutational status on local recurrence and/or metastases in 32 patients with parathyroid carcinoma. Mut+, mutation positive; Mut-, mutation negative; Rec/Met+, development of recurrence and/or metastases; Rec/Met-, no development of recurrence and/or metastases. (B) Correlation of parafibromin staining results with local recurrence and/or metastases in 34 patients with parathyroid carcinoma. Staining-, loss of parafibromin; Staining+, retained parafibromin expression; Rec/Met+, development of recurrence and/or metastases; Rec/Met-, no development of recurrence and/or metastases.

Fig 5: UBR5 inhibits the protein level of CDC73 in breast cancer cells.A The mRNA expression of Ubr5 (i) and Cdc73 (ii) was measured by qPCR, and their protein level (iii) was analyzed by western blotting in 4T1/GFP and 4T1/Ubr5-/- cells. shNC and UBR5-knocked down MCF-7 cells (B), MDA-MB-231 cells (C), and BT549 cells (D) were analyzed for the mRNA expression of UBR5 (i) and CDC73 (ii), and their protein level (iii) by qPCR and western blotting, respectively. Data were shown as mean ± SEM for triplicates; ns not significant; **p < 0.01; ***p < 0.001.