Figure 8
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.
