3b). have shown that the Brefeldin A-inhibited guanine nucleotide-exchange protein 3Cprohibitin 2 (BIG3-PHB2) complex has a crucial role in these cells. However, it remains unclear how BIG3 regulates the suppressive activity of PHB2. Here we demonstrate that BIG3 functions as an A-kinase anchoring protein that binds protein kinase A (PKA) and the isoform of the catalytic subunit of protein phosphatase 1 (PP1C), thereby dephosphorylating and inactivating PHB2. E2-induced PKA-mediated phosphorylation of BIG3-S305 and -S1208 serves to enhance PP1C activity, resulting in E2/ER signalling activation via PHB2 inactivation due to PHB2-S39 dephosphorylation. Furthermore, an analysis of independent cohorts of ER-positive breast cancers patients reveal that both BIG3 overexpression and PHB2-S39 dephosphorylation are strongly associated with poor prognosis. This is the first demonstration of the mechanism of E2/ER signalling activation via the BIG3CPKACPP1C tri-complex in breast cancer cells. Oestrogen (E2) CD247 has a crucial role in regulating the initiation, development and progression of breast cancer, with 70% of all breast cancer cells expressing oestrogen receptor alpha (ER)1,2. In these cells, the biological actions of E2 are mediated by both genomic effects on the transcriptional activation of nuclear ER and non-genomic effects on the activation of signalling pathways via plasma membrane-associated ER. In particular, genomic ER activation is influenced by coactivators and corepressors that positively or negatively modulate ER-mediated transcriptional activity. However, although the role of coactivators in E2-dependent ER-positive breast carcinogenesis has been well established, the pathophysiological role of corepressors remains highly debated. Prohibitin 2 (PHB2), also known as REA3, functions as both a modulator of the E2/ER signalling network and a corepressor of ER; however, its abundant expression in ER-positive breast cancers is not well understood. We previously reported that Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3); “type”:”entrez-protein”,”attrs”:”text”:”Q5TH69″,”term_id”:”147742985″Q5TH69 in UniProt kB annotation, which is exclusively overexpressed in the majority of breast cancers4,5, interacts with PHB2 in the cytoplasm, thereby inhibiting E2-dependent translocation to Tubastatin A the nucleus and plasma membrane, resulting in the constitutive activation of the E2/ER signalling pathways5,6,7,8,9. However, the pathophysiological role of BIG3 in the inactivation of PHB2 suppressive activity in breast cancer cells has not been elucidated. Accumulating evidence has revealed that other BIG family proteins (for example, BIG1 and BIG2) contain A-kinase anchoring protein (AKAP) sequences in their N-terminal regions that bind the regulatory subunits of protein kinase A (PKA) and the isoform of the catalytic subunit of protein phosphatase 1 (PP1C). These findings Tubastatin A suggest that BIG1 and BIG 2 contribute to the regulation of ADB ribosylation factor10,11. Anchoring proteins, such as AKAP, bind to Tubastatin A the catalytic subunit of protein phosphatase 1 (PP1C) to regulate its activity12. Indeed, several multivalent anchoring proteins form a complex and simultaneously co-localize with serine/threonine protein phosphatases and protein kinases12,13. A sequence comparison of BIG family proteins revealed that BIG3 showed only 21% identity with BIG1 and BIG2 (ref. 14). However, a detailed analysis predicted that, similar to BIG1 and BIG2, BIG3 contains several regions that bind to the RII subunit of cyclic AMP (cAMP)-dependent PKA. In addition, BIG3 has Tubastatin A been reported to potentially interact with the isoform of the catalytic subunit of protein phosphatase 1 (PP1C) analysis showed that BIG3 has no other PP1C-binding motifs such as G/SILK or MyPhoNe; however, both BIG1 and BIG2 contain G/SILK motifs. Therefore, BIG3 has been annotated as by the Hugo Gene Nomenclature (HGNC) and designated as a member of the phosphatase regulatory subunit family. However, the functional impact of BIG3 as a remains unknown. Therefore, understanding the properties of BIG3 as an AKAP, including PP1C interactor that contains a canonical PP1CCbinding motif RVxF sequence (1,228-KAVSF-1,232) (ref. 15), but not other PP1C-binding motifs such as G/SILK or MyPhoNe. We detected an endogenous interaction between BIG3 and PP1C in the ER-positive breast cancer cell lines MCF-7 and KPL-3C, which highly express both proteins (Supplementary Fig. Tubastatin A 1a), regardless of the presence of E2 (Fig. 1a). We further confirmed that the FLAG-tagged BIG3 mutant, which lacks an RVxF motif (PP1C), completely abolished the interaction with endogenous PP1C (Supplementary Fig. 1b), indicating an endogenous BIG3CPP1C interaction in breast cancer cells. Open in a separate window Figure 1 BIG3 phosphorylation functions as a regulatory subunit of PP1C.(a) Interaction of endogenous PP1C with BIG3 in MCF-7 and KPL-3C cells after E2 stimulation for 24?h. (b).