Proteasome dependent degradation of ERa bound to E2 or SERDs ERa is a short lived protein. ERa degradation occurs in presence Pazopanib cost of natural ligands or pure antiestrogens such as ICI in a pro teasome dependent manner. The 26S proteasome is a large protein complex present in the cytoplasm and nucleus of eukaryotic cells. The catalytic core of this multi subunit complex, described as the 20S proteasome, contains a and b subunits. We visualized GFP ERa and the 20S proteasome subunit a2 in SK19 cells. SK19 cells grown on glass coverslips and treated as described were fixed, permeabilized and subjected to indirect immuno fluorescence using a monoclonal anti 20S proteasome subunit a2 primary antibody. Images acquired on an Olympus inverted wide field microscope in 3 D and subjected to deconvolution revealed punctuate nuclear staining of proteasome subunits throughout the nucleus.

We did not observe any cytoplasmic staining of this proteasome subunit under our culture conditions. In the presence of E2, GFP ERa accumulated at numerous nuclear sites that colocalized at least partially with proteasome foci. Next we used a double immuno nanogold labelling approach in MCF 7 cells to characterize the extent of ER a2 colocalization. Upon exposure to E2, at least four nuclear clusters per nuclear sections were detected. In the majority of clusters more than 3 gold particles for each protein were present. Endogenous ERa colocalized with the 20S proteasome sub unit a2 in nuclear microdomains of about 100 nm in diameter.

We then determined the effect of LMB, an inhibitor of the nuclear export receptor CRM1, and of ALLN, an inhi bitor of the proteasome, on SERD dependent degrada tion of ERa in SK19 cells. SK19 cells were pretreated with 10 nM LMB or 100 uM ALLN for 30 min. Figure 4C shows that LMB did not block E2, ICI or RU58 induced ERa degradation suggesting that SERD bound ERa is degraded in the nucleus. In the presence of E2, but not ICI or RU58, degradation was slightly less pro nounced in cells pretreated with LMB suggesting that a fraction of E2 bound ERa is also degraded by the cyto ICI and RU58 induced degradation of ERa confirming that SERD ERa complexes were degraded by the nuclear proteasome. Note that at the protein level, GFP ERa is degraded to a lesser extent than endogenous ERa which is likely to be a consequence of reduced transcription of ESR1 in the presence of E2 and SERDs. GFP ERa transcription is under the control of a CMV promoter which insensitive to antiestrogens. Finally, we investigated the distribution of GFP ERa and the 20S proteasome subunit a2 in SK19 cells trea ted with ICI or GSK-3 RU58. GFP ERa foci also significantly overlapped with accumulation sites of the 20S proteasome subunit a2 throughout the nucleus.