Abstract
Oestrogen receptor-α (ERα) shuttles continuously between the nucleus and the cytoplasm, and functions as an oestrogen-dependent transcription factor in the nucleus and as an active mediator of signalling pathways, such as phosphatidylinositol 3-kinase (PI3K)/AKT, in the cytoplasm. However, little is known regarding the mechanism of ERα nucleocytoplasmic shuttling. In this study, we found that ERα is transported into the nucleus by importin-α/β1. Furthermore, we found that Transportin-2 (TNPO2) is involved in 17β-oestradiol (E2)-dependent cytoplasmic localisation of ERα. Interestingly, it was found that TNPO2 does not mediate nuclear export, but rather is involved in the cytoplasmic retention of ERα via the proline/tyrosine (PY) motifs. Moreover, we found that TNPO2 competitively binds to the basic nuclear localisation signal (NLS) of ERα with importin-α to inhibit importin-α/β-dependent ERα nuclear import. Finally, we confirmed that TNPO2 knockdown enhances the nuclear localisation of wild-type ERα and reduces PI3K/AKT phosphorylation in the presence of E2. These results reveal that TNPO2 regulates nucleocytoplasmic shuttling and cytoplasmic retention of ERα, so that ERα has precise functions depending on the stimulation.
Highlights
Oestrogen receptor-α (ERα) shuttles continuously between the nucleus and the cytoplasm, and functions as an oestrogen-dependent transcription factor in the nucleus and as an active mediator of signalling pathways, such as phosphatidylinositol 3-kinase (PI3K)/AKT, in the cytoplasm
More GFP-tagged mutants of each cluster were found in the cytoplasm as compared with the wild type; they were mainly localised in the nucleus, which was further enhanced in the presence of E2
In the absence of E2, whole cell lysates prepared from HeLa cells expressing a triple haemagglutinin (3 × HA)-ERα or 3 × HA-ERα-mNLS were incubated with recombinant GSTproteins. 3 × HA-ERα interacted with GST-importin-α4, but 3 × HA-ERα-mNLS mostly did not
Summary
Oestrogen receptor-α (ERα) shuttles continuously between the nucleus and the cytoplasm, and functions as an oestrogen-dependent transcription factor in the nucleus and as an active mediator of signalling pathways, such as phosphatidylinositol 3-kinase (PI3K)/AKT, in the cytoplasm. We confirmed that TNPO2 knockdown enhances the nuclear localisation of wild-type ERα and reduces PI3K/AKT phosphorylation in the presence of E2. These results reveal that TNPO2 regulates nucleocytoplasmic shuttling and cytoplasmic retention of ERα, so that ERα has precise functions depending on the stimulation. Our results show that Transportin-2 (TNPO2, importin-3) enhances the cytoplasmic localisation of ERα, not as a nuclear export receptor, but rather as a cytoplasmic retention factor, while it competitively inhibits the binding of importin-α. We propose that TNPO2 plays a critical role in determining the distribution of ERα between the nucleus and the cytoplasm in response to stimulation
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