Fig. 6: RNF187 promotes P53 K48-linked polyubiquitination and degradation.

A In the presence of the proteasome inhibitor MG132, RNF187 does not further increase the P53 protein level. MCF-7 cells were transfected with 50 μM siControl or siRNF187. After 24 h, the cells were treated with 10 μM MG132/vehicle for 6 h. Cell lysates were prepared for western blot analysis. The results are representative of three independent experiments. B, C RNF187 depletion increases the P53 half-life in MCF-7 cells. MCF-7 cells were transfected with 50 nM siControl or siRNF187. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for the indicated times. Cell lysates were prepared for western blot analysis. The results are representative of three independent experiments. The relative P53 density was measured by ImageJ software. D RNF187 increases the K48-linked polyubiquitination of P53. HEK293 cells were transfected with 2 µg of P53 plasmid, 0.5 µg of HA-K48 Ubi plasmid, and 0.5 µg of Myc-tag or Myc-RNF187 plasmids. The cell extracts were immunoprecipitated with an anti-HA antibody. K48-specific polyubiquitinated P53 was detected via western blot analysis. E RNF187 decreases K63-linked polyubiquitination of P53. HEK293 cells were transfected with 2 µg of P53 plasmid, 0.5 µg of HA-K63 Ubi plasmid, and 0.5 µg of Myc-tag or Myc-RNF187 plasmids. The cell extracts were immunoprecipitated with an anti-HA antibody. K63-specific polyubiquitinated P53 was detected via western blot analysis. F RNF187 increases the total polyubiquitination of P53. HEK293 cells were transfected with 2 µg of P53 plasmid, 0.5 µg of HA-Ub plasmid, and 0.5 µg of Myc-tag or Myc-RNF187 plasmids. The cell extracts were immunoprecipitated with an anti-HA antibody. Total polyubiquitinated P53 was detected via western blot analysis. G The wild-type and mutant forms of RNF187 were used in the study. The mutation sites (C12A; C15A) in the RING domain diminished the ubiquitin ligase function of RNF187. H The ubiquitin ligase functional RING domain is required for RNF187 to promote P53 polyubiquitination. RNF187 increases the total polyubiquitination of P53. HEK293 cells were transfected with 2 µg of P53 plasmid, 0.5 µg of HA-Ub plasmid, and 0.5 µg of Myc-tag or Myc-RNF187 WT or RING mutant form plasmids. The cell extracts were immunoprecipitated with an anti-HA antibody. Total polyubiquitinated P53 was detected via western blot analysis. I The in vitro ubiquitination assay showed that RNF187 can directly promote P53 polyubiquitination. Ubiquitination was analyzed with a ubiquitination kit (Boston Biochem). The recombinant proteins were mixed with E1, ATP, ubiquitin solution, and E2 enzyme in a final volume of 20 µl reaction buffer. MDM2 was used as the positive control. Ubiquitination of P53 was analyzed with an anti-P53 antibody.