Fig 2: RNF31 stabilizes MDM2 and inhibits proteasome-mediated MDM2 degradation. (a) RNF31 increases MDM2 protein levels. HEK293 cells were transfected with 0.2 μg enhanced green fluorescent protein (EGFP) plasmid, 0.5 μg myc-MDM2 plasmid and the indicated amounts of Flag-RNF31 plasmid. After 24 h, whole-cell extracts were prepared for western blot analysis. (b) RNF31 increases MDM2 half-life in HEK293 cells. HEK293 cells were transfected with 0.5 μg myc-MDM2 plasmid and 0.5 μg Flag-tag or Flag-RNF31 plasmids. After 24 h, cells were treated with 100 μM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. (c) RNF31 inhibits proteasome-mediated MDM2 degradation. HEK293 cells were transfected with 0.5 μg MYC-MDM2 plasmid and 0.5 μg Flag-tag/Flag-RNF31 plasmid. After 24 h, cells were treated with 10 μM MG132/vehicle. Cell lysis was prepared for western blot analysis. (d) RNF31 inhibits MDM2 polyubiquitination in HEK293 cells. HEK293 cells were transfected with 0.5 μg myc-MDM2 plasmid and 0.5 μg Flag-tag or Flag-RNF31 plasmids. After 24 h, cells were treated with 10 μM MG132/vehicle for 4 h. Cell lysates were prepared for western blot analysis. MDM2 was measured by MYC antibody. The expected ubiquitin bands are indicated. (e) Co-IP assay shows that RNF31 inhibits MDM2 polyubiquitination in HEK293 cells. HEK293 cells were transfected with 0.5 μg MYC-MDM2 plasmid and 0.5 μg Flag-tag or Flag-RNF31 plasmids. After 24 h, cells were treated with 10 μM MG132 or vehicle for 4 h. Ubiquitin-binding beads were used to enrich the ubiquitin-ligated MDM2. MYC antibody was used to detect the ubiquitinated and unmodified MDM2. (f) RNF31 depletion facilitates MDM2 polyubiquitination. MCF-7 cells were transfected with siRNF31 or siControl. After 48 h, cells were treated with MG132 for 4 h. MDM2 antibody was used to detect MDM2. The possible ubiquitin-ligated MDM2 bands are indicated.

Fig 3: The ubiquitin ligase activity of RNF31 is required for ERα stabilization, signaling and mono-ubiquitination. (a) The mono-ubiquitination function of RNF31 is required for the RNF31-mediated increase of endogenous ERα protein levels. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31, Myc-tagged RNF31 R1/2M, in which cysteine residues responsible for the transfer of ubiquitin to substrates have been mutated or the Myc-tag alone, as indicated. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31 variants, ERα and the loading control GAPDH are indicated. (b) The mono-ubiquitination function of RNF31 is required to increase ERα signaling. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31, Myc-tagged RNF31 R1/2M or the Myc-tag alone, as indicated, along with an ERE-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection and calculated from experiments performed in triplicates. Data are shown as mean±s.d. (n=3). ***P<0.001 for wt-RNF31 versus RNF31 R1/2M or Myc-tag. RNF31 deletion domains/empty vector; NA, P>0.05 for wt-RNF31 versus RNF31 deletion domains. (c, d) Mutation of the RNF31 ubiquitin ligase domain abolishes mono-ubiquitination of ERα. (c) HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged RNF31, RNF31 R1/2M or the Myc-tag alone. Forty-eight hours post transfection, cell extracts were prepared and ERα was detected by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and the internal control GAPDH are indicated. (d) HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged full-length RNF31, RNF31 R1/2M or the Myc-tag alone. After 48 h, whole-cell extracts were subjected to immunoprecipitation of ERα and subsequently analyzed for ubiquitinated ERα forms by western blot analysis using anti-ubiquitin antibodies. The predicted molecular weights of RNF31, IgG, ERα and mono-ubiquitinated ERα are indicated.

Fig 4: RNF31 triggers ERα mono-ubiquitination. (a) Detection of a potentially mono-ubiquitinated form of ERα upon RNF31 overexpression. HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged RNF31 or the Myc-tag alone. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and of the internal control GAPDH are indicated. (b) Detection of endogenous mono-ubiquitinated ERα upon RNF31 depletion. MCF-7 cells were transfected with siRNF31 or siControl. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and the internal control GAPDH are indicated. (c) Deletion of the RNF31 RBR domain abolishes the potentially mono-ubiquitinated form of ERα. HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged full-length RNF31 derivatives or the Myc-tag alone. Forty-eight hours post transfection, cell extracts were prepared and ERα forms were detected by western blot analysis. The predicted molecular weights of RNF31, ERα, mono-ubiquitinated ERα and of the internal control GAPDH are indicated. (d) Direct evidence for ERα mono-ubiquitination. Immunoprecipitation of ubiquitinated proteins from MCF-7 cell extracts upon overexpression of RNF31. Ubiquitinated ERα species were detected by western blots using anti-ERα, identifying a prominent 75 kDa mono-ubiquitinated ER form. (e) ERα mono-ubiquitination requires the RNF31 RBR domain. Plasmids expressing Myc-tagged RNF31 derivatives were transfected into HEK-293 cells together with the ERα expression plasmid. Whole-cell extracts were subjected to immunoprecipitation of ERα and subsequently analyzed for ubiquitinated ERα forms by western blot analysis using anti-ubiquitin. The predicted molecular weight of mono-ubiquitinated ERα is indicated.

Fig 5: Intracellular localization analysis and model of crosstalk between RNF31 and ERα signaling in breast cancer cells. (a) MCF-7 cells were treated with 10 nM E2 or vehicle for 30 min before fixation. Intracellular localization of RNF31 (red) and ERα (green) was determined by immunofluorescence staining. Nuclei (blue) were stained with 4',6-diamidino-2-phenylindole (DAPI). Shown are representative images. For quantitative analysis see Supplementary Figure S5. (b) Co-IP assay reveals the interaction between RNF31 and ERα in the cytoplasm. The Subcellular Protein Fractionation Kit (Thermo Scientific, 78840) was used for extraction of cytoplasmic and nuclear proteins from MCF-7 cells. Vinculin and Histone 3 were used to identify the quality of cytoplasmic and nuclear fractions, respectively (left panel). Co-IP assays were performed with RNF31 antibody for precipitation and ERα antibody for detection (right panel). (c) Hypothetical model for the functional interplay of RNF31 with ERα signaling in breast cancer cells. RNF31 associates with ERα predominantly in the cytoplasm and promotes mono-ubiquitination of ERα, thereby counteracting proteasome-mediated degradation. The so elevated ERα protein levels cause increased genomic ERα signaling, for example, transcription of E2-target genes linked to the proliferation of breast cancer cells. RNF31, as part of the LUBAC ubiquitin ligase complex, has an established independent function in NFκB signaling. To which extent the two signaling pathways also crosstalk remains to be investigated.