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The Hormonal Response of Estrogen Receptor β Is Decreased by the Phosphatidylinositol 3-Kinase/Akt Pathway via a Phosphorylation-dependent Release of CREB-binding Protein

2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês

10.1074/jbc.m607908200

1083-351X

Mélanie Sanchez, Karine Sauvé, Nathalie Picard, André Tremblay,

Reproductive System and Pregnancy

The hormonal response of estrogen receptors (ER) α and ERβ is controlled by a number of cofactors, including the general transcriptional coactivator CREB-binding protein (CBP). Growing evidence suggests that specific kinase signaling events also modulate the formation and activity of the ER coactivation complex. Here we show that ERβ activity and target gene expression are decreased upon activation of ErbB2/ErbB3 receptors despite the presence of CBP. This inhibition of ERβ involved activation of the phosphatidylinositol 3-kinase/Akt pathway, abrogating the potential of CBP to facilitate ERβ response to estrogen. Such reduced activity was associated with an impaired ability of ERβ to recruit CBP upon activation of Akt. Mutation of serine 255, an Akt consensus site contained in the hinge region of ERβ, prevented the release of CBP and rendered ERβ transcriptionally more responsive to CBP coactivation, suggesting that Ser-255 may serve as a regulatory site to restrain ERβ activity in Akt-activated cells. In contrast, we found that CBP intrinsic activity was increased by Akt through threonine 1872, a consensus site for Akt in the cysteine- and histidine-rich 3 domain of CBP, indicating that such enhanced transcriptional potential of CBP did not serve to activate ERβ. Interestingly, nuclear receptors sharing a conserved Akt consensus site with ERβ also exhibit a reduced ability to be coactivated by CBP, whereas others missing that site were able to benefit from the activation of CBP by Akt. These results therefore outline a regulatory mechanism by which the phosphatidylinositol 3-kinase/Akt pathway may discriminate nuclear receptor response through coactivator transcriptional competence. The hormonal response of estrogen receptors (ER) α and ERβ is controlled by a number of cofactors, including the general transcriptional coactivator CREB-binding protein (CBP). Growing evidence suggests that specific kinase signaling events also modulate the formation and activity of the ER coactivation complex. Here we show that ERβ activity and target gene expression are decreased upon activation of ErbB2/ErbB3 receptors despite the presence of CBP. This inhibition of ERβ involved activation of the phosphatidylinositol 3-kinase/Akt pathway, abrogating the potential of CBP to facilitate ERβ response to estrogen. Such reduced activity was associated with an impaired ability of ERβ to recruit CBP upon activation of Akt. Mutation of serine 255, an Akt consensus site contained in the hinge region of ERβ, prevented the release of CBP and rendered ERβ transcriptionally more responsive to CBP coactivation, suggesting that Ser-255 may serve as a regulatory site to restrain ERβ activity in Akt-activated cells. In contrast, we found that CBP intrinsic activity was increased by Akt through threonine 1872, a consensus site for Akt in the cysteine- and histidine-rich 3 domain of CBP, indicating that such enhanced transcriptional potential of CBP did not serve to activate ERβ. Interestingly, nuclear receptors sharing a conserved Akt consensus site with ERβ also exhibit a reduced ability to be coactivated by CBP, whereas others missing that site were able to benefit from the activation of CBP by Akt. These results therefore outline a regulatory mechanism by which the phosphatidylinositol 3-kinase/Akt pathway may discriminate nuclear receptor response through coactivator transcriptional competence. Estrogen mediates many aspects in growth, development, and reproduction, through its interaction with estrogen receptors ER 2The abbreviations used are: ER, estrogen receptor; PI3K, phosphatidylinositol 3-kinase; CBP, CREB binding protein; C/H3, cysteine- and histidine-rich 3; SRC, steroid receptor coactivator; ERR, estrogen-related receptor; GR, glucocorticoid receptor; PR, progesterone receptor; PPAR, peroxisome proliferator-activated receptor; EGFR, epidermal growth factor receptor; ERE, estrogen response element; CREB, cAMP-response element-binding protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; WT, wild type; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase; RT, reverse transcription; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; E2, estradiol; PBS, phosphate-buffered saline; AF, activation function; CatD1, cathepsin D1.2The abbreviations used are: ER, estrogen receptor; PI3K, phosphatidylinositol 3-kinase; CBP, CREB binding protein; C/H3, cysteine- and histidine-rich 3; SRC, steroid receptor coactivator; ERR, estrogen-related receptor; GR, glucocorticoid receptor; PR, progesterone receptor; PPAR, peroxisome proliferator-activated receptor; EGFR, epidermal growth factor receptor; ERE, estrogen response element; CREB, cAMP-response element-binding protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; WT, wild type; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase; RT, reverse transcription; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; E2, estradiol; PBS, phosphate-buffered saline; AF, activation function; CatD1, cathepsin D1. α and ERβ. Although encoded by unique genes, the two ERs share the functional domains characteristic of the nuclear hormone receptor family (1.Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6043) Google Scholar). These consist of an N-terminal region (also termed AB region), which confers ligand-independent activation of ERs through its activation function (AF)-1, a highly conserved DNA-binding domain (C) that allows specific binding to genomic response elements, a flexible hinge region (D) that includes signals for nuclear localization and the binding of heat shock proteins, and finally a C-terminal region (EF) that contains the ligand binding domain, and the AF-2 function that mediates hormone-dependent activation. Increasing evidence suggests that, beside hormonal activation, ER function can be modulated by phosphorylation-dependent mechanisms, involving a wide variety of protein kinases that mostly target the AF-1 domain (2.Pearce S.T. Jordan V.C. Crit. Rev. Oncol. Hematol. 2004; 50: 3-22Crossref PubMed Scopus (250) Google Scholar, 3.Sanchez M. Tremblay A. Sinnett D. Molecular Genetics of Cancer. Research Signpost, Kerala, India2005: 149-185Google Scholar). In particular, direct phosphorylation of ERα AF-1 by MAPK/ERK in response to EGF was shown to induce ERα transactivation in the absence of ligand (4.Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1705) Google Scholar, 5.Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (845) Google Scholar). Similarly, phosphorylation of Ser-167 by pp90RSK1 was described to promote ERα AF-1 activity (6.Joel P.B. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (310) Google Scholar). Activation of phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B also contributed to phosphorylate ERα and mediate its ligand-independent activation, an effect shown to oppose the tamoxifen-induced apoptosis in breast cancer cells (7.Campbell R.A. Bhat-Nakshatri P. Patel N.M. Constantinidou D. Ali S. Nakshatri H. J. Biol. Chem. 2001; 276: 9817-9824Abstract Full Text Full Text PDF PubMed Scopus (809) Google Scholar). Although phosphorylation of ERβ has not been examined in detail, ERβ has been proposed as a potential target for intracellular kinases that modulate its transactivation properties. It was found that the ability of EGF and the oncogene Ras to activate ERβ resulted from the MAPK-directed phosphorylation of Ser-106 and Ser-124 within the AF-1 domain leading to favored recruitment of coactivators SRC-1 and CBP (8.Tremblay A. Tremblay G.B. Labrie F. Giguere V. Mol. Cell. 1999; 3: 513-519Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 9.Tremblay A. Giguere V. J. Steroid Biochem. Mol. Biol. 2001; 77: 19-27Crossref PubMed Scopus (62) Google Scholar). Furthermore, the ligand-dependent activation of ERβ by the protooncogene Brx was shown to involve phosphorylation of ERβ in a p38-dependent manner, although the exact site(s) were not described (10.Driggers P.H. Segars J.H. Rubino D.M. J. Biol. Chem. 2001; 276: 46792-46797Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). More recently, we reported that activation of ErbB2 and ErbB3, which belong to the EGFR/ErbB receptor tyrosine kinase family, by growth factor heregulin resulted in a decrease in the estrogen-dependent cell growth and activity of ERα and ERβ in breast cancer cells (11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar). However, unlike ERα, this transcriptional repression of liganded ERβ by heregulin was dependent upon ERβ AF-1 function, thereby supporting a repressive role for kinase-mediated pathways in regulating ERβ AF-1 and AF-2 functions. Taken together, the regulation of estrogen receptor activity by phosphorylation is intricate and could dictate receptor function, whether it involves activation or repression. Recent evidence has emerged suggesting that nuclear receptor coactivators may also serve as points of convergence between the ER and growth factor signaling pathways. Phosphorylation of SRC coactivators has been described to modulate their intrinsic activities in mediating nuclear receptor transcription (12.Wu R.C. Smith C.L. O'Malley B.W. Endocr. Rev. 2005; 26: 393-399Crossref PubMed Scopus (133) Google Scholar). Coregulatory proteins are often present in limiting concentrations in the nucleus so that modifications of their level of expression as well as their activity can lead to alterations in nuclear receptor signaling. The transcriptional coactivators CREB-binding protein (CBP) and p300 are evolutionary highly conserved proteins, and genetic evidence supports their availability to be critical. In humans, loss of one functional copy of cbp leads to Rubenstein-Taybi syndrome, a haploinsufficiency disorder resulting in mental retardation (13.Petrij F. Giles R.H. Dauwerse H.G. Saris J.J. Hennekam R.C. Masuno M. Tommerup N. van Ommen G.J. Goodman R.H. Peters D.J. Nature. 1995; 376: 348-351Crossref PubMed Scopus (1019) Google Scholar). Through their extremely versatile ability in bridging numerous transcription factors, including most nuclear receptors, with the basal transcription machinery, recruitment of CBP/p300 is important to maintain appropriate transcriptional events (14.Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). One of the likely mechanisms responsible for CBP/p300 recruitment involves phosphorylation. It was reported that phosphorylation of CBP promotes its interaction with several transcription factors, including CREB, Smad3, NFκB p65 subunit, and p53 (15.Vo N. Goodman R.H. J. Biol. Chem. 2001; 276: 13505-13508Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 16.Kalkhoven E. Biochem. Pharmacol. 2004; 68: 1145-1155Crossref PubMed Scopus (390) Google Scholar). We have recently shown that MAPK-dependent phosphorylation of ERβ also facilitates the recruitment of CBP to potentiate the ligand-independent activation of ERβ in response to growth factors (9.Tremblay A. Giguere V. J. Steroid Biochem. Mol. Biol. 2001; 77: 19-27Crossref PubMed Scopus (62) Google Scholar). Given such diversity in the signaling pathways integrated by CBP, it is believed that phosphorylation-mediated events may compete at various levels for the limited availability of CBP. Here we describe a molecular mechanism by which ErbB2/ErbB3 and PI3K/Akt signaling impairs the activity of ERβ by reducing its ability to recruit and use CBP as a coactivator. The repression by Akt was also found for other nuclear receptors, for which a conserved Akt site may also participate in a manner similar to ERβ. In contrast, nuclear receptors that do not share such homology yielded increased responsiveness to CBP and benefit from the enhanced intrinsic activity of CBP by Akt. Plasmid Constructs−Expression of pCMX plasmids coding for ERα, ERβ, CBP, ErbB2, its constitutive variant V659E and ErbB3 receptors, and luciferase reporter constructs vitA2-ERE-tkLuc and UAStkLuc have been described previously (8.Tremblay A. Tremblay G.B. Labrie F. Giguere V. Mol. Cell. 1999; 3: 513-519Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 9.Tremblay A. Giguere V. J. Steroid Biochem. Mol. Biol. 2001; 77: 19-27Crossref PubMed Scopus (62) Google Scholar, 11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar). ERβ fragments corresponding to the AB (amino acids 1-167) and DEF (amino acids 234-549) regions were obtained by PCR amplification and fused in-frame with the Gal4 DNA binding domain. The ERβ Ser-255 to alanine and the CBP Thr-1872 to alanine mutants were generated by PCR mutagenesis using Pfu polymerase (Stratagene). All constructs were verified by automated sequencing. The expression plasmid coding for the constitutively active PI3K p110α catalytic subunit was a kind gift from J. Downward, and plasmids expressing Akt and K179M kinase dead Akt were generously provided by T. Chan and P. Tsichlis. Cell Culture, DNA Transfection, and Luciferase Assay−Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS). The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. For transient transfection, cells were seeded in phenol red-free DMEM supplemented with 5% charcoal dextran-treated FBS, and plasmid constructs were introduced into cells using the calcium phosphate precipitation method as described (11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar). Typically, 50-60% confluent cells were transfected with 2 μg of DNA per well, which include 500 ng of reporter plasmid, 100 ng of receptor expression vector, 250 ng of CMX-βgal, 100 ng each of PI3K and Akt expression vector, and 30 ng of CBP plasmid when indicated. After 5-8 h, the medium was changed, and cells were stimulated with 10 nm estradiol (E2; Sigma) and/or 50 ng/ml heregulin-β (R&D Systems) for 16-20 h or left untreated. For luciferase assay, cells were lysed in potassium phosphate buffer containing 1% Triton X-100, and light emission was measured using a luminometer (Wallac) after the addition of luciferin. Luciferase assays were performed in duplicate from at least three independent experiments, and values were expressed as relative light units normalized to the β-galactosidase activity of each sample. Western Analysis and Immunoprecipitation Assay−Western analysis for the determination of phosphorylated and total Akt was performed as described with minor modifications (11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar). Briefly, transfected 293T cells were treated with 50 ng/ml heregulin-β for 20 min, washed in ice-cold PBS, and lysed in PBS containing 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 1mm phenylmethanesulfonyl fluoride, and protease inhibitors (Roche Applied Science). Cell lysates were then subjected to SDS-PAGE and proteins transferred to nitrocellulose for immunoblotting. Membranes were incubated at 4 °C with blocking reagent (Roche Applied Science) in TBS, probed with either a rabbit polyclonal antibody against phosphorylated Akt (Santa Cruz Biotechnology) or a mouse anti-Akt monoclonal antibody (Cell Signaling Technology), and signals revealed by ECL using appropriate horseradish peroxidase-conjugated secondary antibodies. The same procedure was used to determine the levels of ERβ, except that cells were transfected with HA-tagged ERβ (WT or S255A) and analyzed by Western using an anti-HA antibody (12CA5). For immunoprecipitation assay, transfected cells were washed in ice-cold PBS and lysed as described above. Cell lysates were precleared before incubation with an anti-CBP antibody (Santa Cruz Biotechnology) and protein A-Sepharose beads at 4 °C. Immunoprecipitates were then washed in lysis buffer, resolved by SDS-PAGE, and analyzed by Western blotting using an anti-HA antibody. Membranes were also probed with an anti-CBP antibody for standardization of CBP levels in each well. Generation of Hs-ER-stable Clones and RT-PCR−ER-negative Hs578t breast cancer cells were maintained in DMEM containing 10% FBS and transfected with expression vectors for ERα and ERβ as described previously (11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar), and resistant clones were isolated in the presence of G418 (0.6 mg/ml; Invitrogen) to generate Hs-ERα and Hs-ERβ cell lines, respectively. Stable clones were functionally validated for their respective expression of ERα or ERβ by Western analysis and for their estrogenic response by luciferase assay, compared with mock-transfected Hs578t cells. Total RNA was isolated from cells using TRIzol reagent (Invitrogen), and RT-PCR analysis was performed as described (17.Avallone R. Demers A. Rodrigue-Way A. Bujold K. Harb D. Anghel S. Wahli W. Marleau S. Ong H. Tremblay A. Mol. Endocrinol. 2006; 20: 3165-3178Crossref PubMed Scopus (65) Google Scholar). The relative signal intensity was analyzed (Alpha Innotech, San Leandro, CA) from three separate experiments. In Vitro Phosphorylation Assay−Bacterially expressed and purified GST fusions of wild type and S255A mutated ERβ were prepared as described (18.Tremblay G.B. Tremblay A. Copeland N.G. Gilbert D.J. Jenkins N.A. Labrie F. Gigue`re V. Mol. Endocrinol. 1997; 11: 353-365Crossref PubMed Scopus (825) Google Scholar). For in vitro phosphorylation assay, GST-ERβ fusions immobilized on glutathione-Sepharose 4B beads were resuspended in kinase buffer containing [γ-32P]ATP (Amersham Biosciences) and active Akt1 (Cell Signaling) and incubated at 30 °C for 30 min according to the manufacturer's instructions. Beads were then washed twice in kinase buffer and twice in PBS, and 32P incorporation was determined following SDS-PAGE and autoradiography. Gels were stained with Coomassie Blue to monitor for equal loading. Fluorescence Microscopy−Cells were seeded on coverslips in a 6-well plate overnight prior to transfection in phenol red-free DMEM supplemented with 5% charcoal dextran-treated FBS. Transient transfections were carried as above using the expression plasmids YFP-CBP and CFP-ERβ. 20 h after transfection, cells were washed twice with cold PBS and fixed in 4% formaldehyde. The coverslips were mounted on microscope slides and examined in fluorescence with excitation/emission filters of 435/470 nm (for CFP) and 480/535 nm (for YFP) using a Nikon TE-2000 inverted microscope. ErbB2/ErbB3 Receptor Dimer Activation Impairs the Hormonal Response and Coactivation of ERβ by CBP−Activation of the epidermal growth factor receptor EGFR/ErbB1, a member of the ErbB receptor tyrosine kinase family, is well recognized to promote ERα and ERβ transcriptional activation (4.Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1705) Google Scholar, 8.Tremblay A. Tremblay G.B. Labrie F. Giguere V. Mol. Cell. 1999; 3: 513-519Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). However, we have recently reported that activation of the ErbB2/ErbB3 heterodimer combination led to a decreased transcriptional activity of ERβ (11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar). Given the ability of CBP to associate and promote the activation of ERβ by a growth factor such as EGF (9.Tremblay A. Giguere V. J. Steroid Biochem. Mol. Biol. 2001; 77: 19-27Crossref PubMed Scopus (62) Google Scholar), we addressed how CBP could modulate ERβ activity in response to ErbB2/ErbB3 activation. ER-negative human embryonic kidney 293T cells were transfected with an EREtkLuc reporter and an ERβ plasmid. Cotransfection with CBP conferred a 2-fold increase in ERβ basal activity and a 7-fold increase in the presence of hormone (Fig. 1A). As reported previously (11.St Laurent V. Sanchez M. Charbonneau C. Tremblay A. J. Steroid Biochem. Mol. Biol. 2005; 94: 23-37Crossref PubMed Scopus (27) Google Scholar), the activation of the ErbB2/ErbB3 heterodimer by growth factor heregulin-β, which binds ErbB3, resulted in a reduced activation of ERβ by estrogen. Such impaired response was also mimicked using a constitutive variant of human ErbB2 (V659E), which corresponds to the natural mutation found in the rat Neu oncogene (19.Weiner D.B. Liu J. Cohen J.A. Williams W.V. Greene M.I. Nature. 1989; 339: 230-231Crossref PubMed Scopus (357) Google Scholar). However, although CBP strongly transactivates ERβ in control cells, it is unable to prevent the inhibition of the hormonal response of ERβ when the ErbB2/ErbB3 heterodimer is not only expressed but is also stimulated by heregulin-β (Fig. 1A). Despite the presence of CBP, the transcriptional activity of ERβ was decreased in both a hormone-independent and -dependent manner. This inhibition was even more pronounced in cells expressing the constitutive ErbB2 V659E mutant. Signaling of the EGFR/ErbB family members involves the activation of a variety of kinase pathways. More specifically, activation of the ErbB2/ErbB3 heterodimer has been shown to efficiently couple with the PI3K/Akt pathway, mainly through the intrinsic ability of numerous Src homology 2-binding motifs within ErbB3 that recognize the p85 regulatory subunit of PI3K (20.Fedi P. Pierce J.H. Di Fiore P.P. Kraus M.H. Mol. Cell. Biol. 1994; 14: 492-500Crossref PubMed Google Scholar, 21.Prigent S.A. Gullick W.J. EMBO J. 1994; 13: 2831-2841Crossref PubMed Scopus (319) Google Scholar). To evaluate the impact of ErbB2/ErbB3 activation on the Akt pathway, the activity of endogenous Akt was determined by Western analysis using a phospho-specific antibody against Ser-473. Although treatment of mock-transfected 293T cells with heregulin-β did not lead to activation of Akt, indicating that endogenous expression of ErbB3 is negligible, if not absent, an increase in phosphorylated Akt was observed in cells expressing ErbB2/ErbB3 and treated with heregulin-β (Fig. 1B). Similarly, cells expressing the ErbB2 V659E variant in the presence of ErbB3 also showed increased levels of phosphorylated Akt. Activation of the PI3K/Akt Pathway Mimics the Inhibition of ERβ Response to Hormone in the Presence of CBP through the C-terminal Region of ERβ−The possibility that ErbB2/ErbB3 activation by heregulin-β decreases ERβ activity and its coactivation by CBP by enhancing the activity of Akt was further tested by transient expression of a membrane-bound and constitutively active p110α subunit (CAAX) of PI3K. Expression of the p110α mutant was sufficient to activate endogenous Akt in 293T cells, which was further enhanced when cells were cotransfected with a plasmid for WT Akt, as determined by Western blot analysis (see Ref. 22.Marte B.M. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. Curr. Biol. 1997; 7: 63-70Abstract Full Text Full Text PDF PubMed Google Scholar; data not shown). Under these conditions, we found that the estrogen-dependent activation of ERβ in the presence of CBP, which reached almost 12-fold compared with untreated cells, was strongly impaired dropping to a 3-fold response in Akt-activated cells (Fig. 2A). As observed previously in ErbB2 V659E-expressing cells in response to Akt activation (Fig. 1A), the addition of CBP reduces further the response of ERβ to estrogen when compared with cells without exogenous CBP. These results suggest that ectopic expression of CBP could not relieve the inhibition of ERβ by the PI3K/Akt pathway, therefore mimicking the results in ErbB2/ErbB3-expressing cells. The repression of ERβ by activated Akt in the presence of CBP was partially relieved in cells expressing a dominant negative form of Akt (K179M), suggesting that the effects of activated PI3K on ERβ mainly transit through Akt (Fig. 2A). We next performed Western analysis to ascertain whether the modulation of ERβ activity was not a direct effect of its protein concentration under the conditions used. As shown in Fig. 2B, activation of the Akt pathway led to an accumulation of ERβ in untreated cells. A similar increase was also observed in the presence of estrogen, although the levels of ERβ were slightly lower compared with untreated cells, probably reflecting an increase in ER turnover in response to hormone as reported previously (23.Nawaz Z. Lonard D.M. Dennis A.P. Smith C.L. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1858-1862Crossref PubMed Scopus (493) Google Scholar). These results suggest that the inhibition in ERβ activity to Akt activation is not related to a decrease in ERβ protein levels. CBP is known to transactivate estrogen receptors through both its AF-1 and AF-2 activities (9.Tremblay A. Giguere V. J. Steroid Biochem. Mol. Biol. 2001; 77: 19-27Crossref PubMed Scopus (62) Google Scholar, 24.Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In an attempt to identify the functional domain within ERβ responsible for its impaired ability to be coactivated by CBP in response to Akt, we used Gal4 fusions of truncated forms of ERβ for which each respective AF-containing domain has been removed. Fig. 2C shows that in Akt-activated cells, the activation of a Gal4-ABβ (corresponding to ERβ amino acids 1-167) on a UAStkLuc reporter was further enhanced by CBP, reaching a near 5-fold increase compared with control cells. The N-terminal domain of ERβ is known to contain several serine residues that are conserved within the recognition motifs for Ser/Thr kinases of the MAPK family, and phosphorylation of specific residues was shown to allow for coactivators such as CBP to be recruited and to potentiate ERβ AF-1 activity (8.Tremblay A. Tremblay G.B. Labrie F. Giguere V. Mol. Cell. 1999; 3: 513-519Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 9.Tremblay A. Giguere V. J. Steroid Biochem. Mol. Biol. 2001; 77: 19-27Crossref PubMed Scopus (62) Google Scholar). However, none of the potential phosphorylation sites within ERβ AB region belongs to a consensus Akt site, suggesting that the enhanced activity of ERβ AF-1 by CBP in response to Akt might possibly result from other kinase pathways activated by Akt or direct effects on CBP itself. We next tested the role of the C-terminal region of ERβ in the same conditions. Cells transfected with a Gal4-DEFβ (amino acids 234-549) showed a reduced hormone-dependent activity to Akt activation in the presence of CBP, mimicking the response observed with full-length ERβ (Fig. 2C, right panel). These results indicate that the repressive effect of activated Akt on CBP-mediated transactivation of ERβ is mediated through a region contained in the C-terminal portion of ERβ, which in the context of the full-length receptor seems to counteract the positive effect on the AF-1 activity. Serine 255 in the Hinge Region Mediates ERβ Inhibition to ErbB2/ErbB3 Signaling−Our examination of the C-terminal sequence of mouse ERβ revealed a consensus sequence RQRSAS255 in the hinge region of ERβ that corresponds to the recognition motif RXRXX(S/T) for the kinase Akt (Fig. 3A). To determine whether Ser-255 is a direct target for Akt-mediated phosphorylation, we used site-directed mutagenesis to convert the serine at position 255 into an alanine and performed an in vitro kinase assay. Fig. 3B shows that disruption of Ser-255 strongly abolished the phosphorylation of ERβ by Akt compared with wild type, indicating that Ser-255 can be efficiently phosphorylated by Akt. We then tested whether Ser-255 was involved in the inhibition of ERβ activity to ErbB2/ErbB3 activation as observed in Fig. 1A. Using the S255A mutant in luciferase assay, we found that the inhibition observed for WT ERβ by either ErbB2/ErbB3 dimer expression or its activation with heregulin-β was completely abrogated by disruption of Ser-255 (compare Figs. 1A and 3C). Noticeably, the hormonal response of S255A was enhanced upon ErbB2/ErbB3 activation and further potentiated by CBP. This enhanced response to hormone by the S255A mutant was also observed in response to Akt activation using the constitutively active p110α PI3K construct in transfection (Fig. 3C). Therefore, the results indicate the hinge region of ERβ contains a specific site that not only can be targeted by Akt but also dictates responsiveness of ERβ to CBP coactivation in response to Akt signaling pathway. To determine whether Ser-255 is involved in the regulation of ERβ in terms of protein levels, we next performed Western analysis on cells expressing the ERβ S255A mutant. As compared with wild type ERβ (Fig. 2B), the disruption of Ser-255 completely abrogated the accumulation of ERβ in response to Akt activation (Fig. 3D), indicating