The Hormonal Response of Estrogen Receptor  Is Decreased by the Phosphatidylinositol 3-Kinase/Akt Pathway via a Phosphorylation-dependent Release of CREB-binding Protein * Received for publication, August 17, 2006, and in revised form, November 17, 2006 Published, JBC Papers in Press, December 12, 2006, DOI 10.1074/jbc.M607908200 Me ´ lanie Sanchez ‡ , Karine Sauve ´ ‡ , Nathalie Picard ‡ , and Andre ´ Tremblay ‡§1 From the Research Center, Sainte-Justine Hospital, and the ‡ Departments of Biochemistry and § Obstetrics and Gynecology, University of Montreal, Montre ´al H3T 1C5, Que ´bec, Canada The hormonal response of estrogen receptors (ER)  and ER 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 ER activity and target gene expression are decreased upon activation of ErbB2/ErbB3 receptors despite the presence of CBP. This inhibition of ER involved activation of the phosphatidylinositol 3-kinase/Akt pathway, abrogating the potential of CBP to facilitate ER response to estrogen. Such reduced activity was associated with an impaired ability of ER to recruit CBP upon activation of Akt. Mutation of serine 255, an Akt consensus site contained in the hinge region of ER, prevented the release of CBP and rendered ER transcriptionally more responsive to CBP coactivation, suggesting that Ser-255 may serve as a regulatory site to restrain ER 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 ER. Interestingly, nuclear receptors sharing a conserved Akt consensus site with ER 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 reg- ulatory 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 recep- tors ER 2  and ER. Although encoded by unique genes, the two ERs share the functional domains characteristic of the nuclear hormone receptor family (1). These consist of an N-ter- minal 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 activa- tion, ER function can be modulated by phosphorylation-de- pendent mechanisms, involving a wide variety of protein kinases that mostly target the AF-1 domain (2, 3). In particular, direct phosphorylation of ER AF-1 by MAPK/ERK in response to EGF was shown to induce ER transactivation in the absence of ligand (4, 5). Similarly, phosphorylation of Ser- 167 by pp90 RSK1 was described to promote ER AF-1 activity (6). Activation of phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B also contributed to phosphorylate ER and mediate its ligand-independent activation, an effect shown to oppose the tamoxifen-induced apoptosis in breast cancer cells (7). Although phosphorylation of ER has not been exam- ined in detail, ER has been proposed as a potential target for intracellular kinases that modulate its transactivation proper- ties. It was found that the ability of EGF and the oncogene Ras to activate ER resulted from the MAPK-directed phosphoryla- tion of Ser-106 and Ser-124 within the AF-1 domain leading to favored recruitment of coactivators SRC-1 and CBP (8, 9). Fur- thermore, the ligand-dependent activation of ER by the proto- oncogene Brx was shown to involve phosphorylation of ER in a p38-dependent manner, although the exact site(s) were not described (10). More recently, we reported that activation of ErbB2 and ErbB3, which belong to the EGFR/ErbB receptor * This work was supported by the Canadian Institutes of Health Research, the Cancer Research Society Inc., and the Canadian Foundation for Innovation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 New Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: Research Center, Ste-Justine Hospital, 3175 Co ˆ te Ste-Catherine, Montre ´al, Que ´bec H3T 1C5, Canada. Tel.: 514-345-4931, ex. 2830; Fax: 514-345-4988; E-mail: andre.tremblay@ recherche-ste-justine.qc.ca. 2 The abbreviations used are: ER, estrogen receptor; PI3K, phosphatidylinosi- tol 3-kinase; CBP, CREB binding protein; C/H 3 , cysteine- and histidine-rich 3; SRC, steroid receptor coactivator; ERR, estrogen-related receptor; GR, glu- cocorticoid receptor; PR, progesterone receptor; PPAR, peroxisome prolif- erator-activated receptor; EGFR, epidermal growth factor receptor; ERE, estrogen response element; CREB, cAMP-response element-binding pro- tein; 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; E 2 , estradiol; PBS, phos- phate-buffered saline; AF, activation function; CatD1, cathepsin D1. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 7, pp. 4830 –4840, February 16, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 4830 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 7 • FEBRUARY 16, 2007 by guest on January 28, 2018 http://www.jbc.org/ Downloaded from