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Molecular Endocrinology, doi:10.1210/me.2007-0281
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Molecular Endocrinology 22 (2): 317-330
Copyright © 2008 by The Endocrine Society

Phosphorylation of Activation Function-1 Regulates Proteasome-Dependent Nuclear Mobility and E6-Associated Protein Ubiquitin Ligase Recruitment to the Estrogen Receptor β

Nathalie Picard1, Catherine Charbonneau1, Mélanie Sanchez, Anne Licznar, Muriel Busson, Gwendal Lazennec and André Tremblay

Research Center (N.P., C.C., M.S., A.T.), Centre Hospitalier Universitaire Ste-Justine, and Departments of Biochemistry (N.P., C.C., M.S., A.T.), and Obstetrics & Gynecology (A.T.), University of Montreal, Montréal, Québec, Canada H3T 1C5; Institut National de la Santé et de la Recherche Médicale (A.L., M.B., G.L.), Unité 844, Site Saint Eloi, Montpellier F-34091, France; and University of Montpellier I, Montpellier F-34090, France

Address all correspondence and requests for reprints to: André Tremblay, Research Center, Ste-Justine Hospital, 3175 Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5. E-mail: andre.tremblay{at}recherche-ste-justine.qc.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ubiquitin-proteasome pathway has been recognized as an important regulator in the hormonal response by estrogen receptor (ER) {alpha}, but its impact on ERβ function is poorly characterized. In the current study, we investigated the role of the ubiquitin-proteasome pathway in regulating ERβ activity and identified regulatory sites within the activation function (AF)-1 domain that modulate ERβ ubiquitination and nuclear dynamics in a hormone-independent manner. Although both ER{alpha} and ERβ were dependent on proteasome function for their maximal response to estrogen, they were regulated differently by proteasome inhibition in the absence of hormone, an effect shown to be dependent on their respective AF-1 domain. Given the role of AF-1 phosphorylation to regulate ER activity, we found that sequential substitutions of specific serine residues contained in MAPK consensus sites conferred transcriptional activation of ERβ in a proteasome-dependent manner through reduced ubiquitination and enhanced accumulation of mutant receptors. Specifically, serines 94 and 106 within ERβ AF-1 domain were found to modulate subnuclear mobility of the receptor to transit between inactive clusters and a more mobile state in a proteasome-dependent manner. In addition, cellular levels of ERβ were regulated through these sites by facilitating the recruitment of the ubiquitin ligase E6-associated protein in a phosphorylation-dependent manner. These findings suggest a role for ERβ AF-1 in contributing to the activation-degradation cycling of the receptor through a functional clustering of phosphorylated serine residues that cooperate in generating signals to the ubiquitin-proteasome pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN PLAYS a central role in reproductive physiology but also in pathological events such as breast and endometrium cancers. Regulation of target gene expression by estrogen is mediated upon its interaction with estrogen receptor (ER) {alpha} and ERβ, which belong to the nuclear hormone receptor family of ligand-activated transcription factors. Although ER{alpha} shares similarities in terms of structure and response to hormone with ERβ, it is considered a strong predictive factor for endocrine therapy of reproductive cancers (1, 2), whereas ERβ was shown to display anti-tumorigenic properties (3, 4, 5).

The current model for transcriptional activation of estrogen-responsive genes by ERs involves a conformational change upon ligand binding that favors interaction with their cognate estrogen response element (ERE) and combinatorial recruitment of transcriptional cofactor complexes that mediate chromatin reorganization and essential interactions with the basal transcription machinery (6, 7). Increasing evidence indicates that upon activation of ER{alpha} by estrogen, the recruitment of cofactors involved in chromatin remodeling, posttranslational modifications and transcriptional activity among others, occurs in an ordered fashion, thereby integrating the response elicited by estrogen upon inducible promoters (8, 9, 10, 11). Such concerted recruitment between cofactors is predicted to limit transactivation by ER{alpha} in response to changes in hormone levels.

Recent studies have integrated the ER{alpha}-mediated response to estrogen with proteasome-directed degradation of the receptor, thus supporting a means by which target cells can sustain or limit a hormonal response through a continuous receptor turnover. ER{alpha} has been shown to be degraded through the 26S proteasome pathway in a ligand-dependent manner (12, 13). Blocking proteasome activity with inhibitors such as MG132 impaired the ability of ER{alpha} to mediate a hormone-dependent transcriptional response and contributed to immobilize ER{alpha} in an inactive state in the nucleus (14, 15). Interestingly, components of the ubiquitin-proteasome system, such as the 19S proteasome regulatory subunit Trip1/SUG1 (16, 17) and the E3 ubiquitin ligases Mdm2 (18) and E6-associated protein (AP) (19), have been shown to enhance the transcriptional activity of several nuclear receptors including ER{alpha}. Moreover, these components were shown to cyclically reside with ER{alpha} on the pS2-responsive promoter in both a ligand-dependent and -independent manner, an effect abolished by MG132 resulting in the inhibition of pS2 gene transcription (10, 11). These findings thus suggest that the proteasome-mediated degradation process is closely related to ER{alpha} transcriptional competence.

Ubiquination of ER{alpha} has been demonstrated to vary upon the presence of hormone or antiestrogens, and the extent of ubiquitination was shown to correlate with receptor degradation, suggesting that ligand-dependent conformational changes can modulate ER{alpha} ubiquitination (13, 20). Interestingly, unliganded ER{alpha} was also demonstrated to be ubiquitinated (10, 13), therefore providing evidence that mechanisms other than ligand binding may dictate ubiquitination. As such, other signaling events have also been proposed to regulate ER{alpha} and ERβ activity. Studies have reported that the ability of epidermal growth factor (EGF) to activate ER{alpha} involved a MAPK-mediated phosphorylation of serine-118 within ER{alpha} activation function (AF)-1 domain (21, 22). Such activation of ER{alpha} by phosphorylation was proposed to involve coactivator recruitment at the AF-1 domain of ER{alpha} (23). Similarly, we demonstrated that ERβ activity is also modulated by growth factor signaling through MAPK-directed phosphorylation of AF-1 (24). Such activation was found to involve a favored recruitment of steroid receptor coactivator (SRC)-1 and cAMP response element binding protein-binding protein (CBP) upon phosphorylation of ERβ AF-1 serines 106 and 124 (25, 26). However, the role of AF-1 in mediating ERβ response to nonhormonal stimuli remains poorly defined, and although both ER{alpha} and ERβ are regulated by growth factors, differences in terms of respective cofactor recruitment and AF-1 activity have been observed (27, 28).

In the present study, we investigated the function of the ubiquitin-proteasome pathway in modulating the AF-1-dependent transcriptional response of ERβ. We identified specific MAPK sites within ERβ AF-1 domain that regulate ERβ activity in a proteasome-dependent manner by modulating receptor ubiquitination, subnuclear mobility, and recruitment of E6-AP ubiquitin ligase. Our results demonstrate a role for the AF-1 domain to regulate the activation-degradation process of ERβ, thereby integrating its response to changes in kinase-activated pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} and ERβ-Mediated Transcription Are Dependent upon Proteasome Function
To assess the role of the proteasome pathway of degradation on ER{alpha} and ERβ transcriptional function, we first tested the effects of proteasome inhibition on the hormonal response of both receptors in human embryonic kidney 293 cells transfected with either ER{alpha} or ERβ and the estrogen-responsive luciferase reporter EREbLuc construct. As also reported (14), ER{alpha}-mediated transcriptional activation in the presence of estrogen was nearly abolished by MG132, an inhibitor of the 26S proteasome (Fig. 1AGo). The hormonal response of ER{alpha} was also decreased using clasto-lactacystin, an irreversible proteasome inhibitor. Under the same conditions, we also observed that ERβ was subjected to a similar dependence on the proteasome pathway in its response to estrogen (Fig. 1AGo). These results indicate that maximal activation of ER{alpha} and ERβ by estrogen requires proteasome function.


Figure 1
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  		Fig. 1. Transactivation by ER{alpha} and ERβ Is Variably Dependent upon Proteasome Function

A, Human 293 cells were transfected with an EREbLuc reporter gene and ER{alpha} or ERβ expression plasmid, and treated with vehicle, 10 nM E2, and proteasome inhibitors MG132 (1 µM) or clasto-lactacystin (1 µM) for 16 h. Cells were then harvested for luciferase activity measurements, and results normalized to β-galactosidase activity are expressed as relative luciferase units (RLU). B, 293 cells were transfected with EREbLuc reporter with ER{alpha} or ERβ expression plasmid and treated with increasing amounts of MG132 for 16 h. Values are expressed as the percentage of change in activity compared with untreated cells set at 100%. C, 293 cells were transfected with ER{alpha} or ERβ constructs and treated with 10 nM E2 or 1 µM MG132 for 16 h. Whole cell extracts were analyzed by Western blot using ER specific antibodies. Sample loading was normalized with β-actin content for each sample.

 
To test whether the activity of ER{alpha} and ERβ in absence of hormone was also dependent upon proteasome function, 293 cells were treated with increasing doses of MG132 in absence of estrogen and ER-driven luciferase activity was measured. We found that, whereas basal ER{alpha}-mediated transcription was enhanced by MG132, similar doses of inhibitor had no significant effect on ERβ-dependent activity (Fig. 1BGo). To ensure that this difference in response of ER{alpha} and ERβ to MG132 is not related to variations in their cellular content, both ER{alpha} and ERβ are shown to accumulate in cells treated with MG132 compared with untreated cells, consistent with the inhibition of their degradation (Fig. 1CGo).

The AF-1 Domain Affects Differently ER{alpha} and ERβ Activity during Proteasome Inhibition
Given the apparent difference in the requirement of the proteasome function involved in ligand-independent activity of ER{alpha} and ERβ, we determined the relative contribution of each receptor AF-1 domain on ER activity in response to proteasome inhibition. Truncated forms of ER{alpha} and ERβ were transfected and tested for their activity in absence of hormone. We observed that for ER{alpha}, removal of the N-terminal AB domain (CDEF{alpha} construct in Fig. 2AGo) did not greatly alter ER{alpha} response to proteasome inhibition, whereas removal of the AB region of ERβ (CDEFβ construct) resulted in a marked activation in the presence of MG132. These results suggest that each respective AF-1 domain may regulate differently the activity of ER{alpha} and ERβ in a proteasome-dependent manner. In support of such a different role, cells expressing ABC{alpha} construct showed a stronger activation in response to proteasome inhibition compared with cells expressing ABCβ (Fig. 2AGo). This apparent diverse contribution of each AF-1 was also transposable, as demonstrated with the use of chimeric forms of ER in which the AB region for each receptor was fused to the CDEF portion of the other, thus creating an ER{alpha}β and ERβ{alpha} fusion chimeras. As such, the addition of the AB{alpha} region to CDEFβ did not significantly modify the activation levels observed for CDEFβ with MG132 or clasto-lactacystin (compare ER{alpha}β with CDEFβ in Fig. 2AGo), whereas fusion of ABβ to CDEF{alpha} decreased the activation of CDEF{alpha} by both inhibitors, resulting in an activation profile more closely related to ERβ than to ER{alpha}. These results demonstrate a role for the AF-1 domain in regulating ERβ transcriptional activity that is dependent upon proteasome function and further suggest that regulatory signals are contained within this region to mediate receptor turnover and activity. Consistent with these observations, the N-terminal ABCβ construct was shown to strongly accumulate when expressed in cells treated with MG132 compared with untreated cells as determined by Western analysis, suggesting that this region itself is subjected to proteasome-dependent degradation (Fig. 2BGo). Under similar conditions, ABC{alpha} also accumulated but to a lesser extent than ABCβ, whereas CDEFβ and CDEF{alpha} levels remained respectively unaffected or slightly increased upon MG132 treatment.


Figure 2
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  		Fig. 2. Contribution of the AF-1 Region to the Proteasome-Dependent Activity of ER{alpha} and ERβ

A, The transcriptional activities of full-length versions or truncated forms lacking the N-terminal (CDEF constructs) or the C-terminal region (ABC constructs) of ER{alpha} or ERβ were determined on an EREbLuc in 293 cells, in response to 1 µM MG132 or 1 µM clasto-lactacystin. Also tested were chimeric forms of ER{alpha} and ERβ in which the AB regions were swapped between each ER isoform. Values are expressed as fold response in luciferase activity compared with untreated cells set at 1.0 for each ER construct used in transfection. B, HA-tagged forms of C-terminal (ABC) or N-terminal (CDEF) truncated forms of ER{alpha} and ERβ were expressed in 293 cells and analyzed by Western blot using an anti-HA antibody. Cells were treated with 10 nM E2 or 1 µM MG132 for 16 h before harvest. Protein amounts were normalized to β-actin content. IB, Immunoblot.

 
Contribution of MAPK Consensus Sites in Proteasome-Regulated Transcription by ERβ
Our earlier studies have determined that phosphorylation of AF-1 in response to MAPK activation resulted in ERβ transcriptional activation (24, 25). More specifically, phosphorylation at positions 106 and 124 in mouse ERβ contributed in a favored recruitment of coactivators SRC-1 and CBP and subsequent ligand-independent activation (25, 26). Besides serines 106 and 124, other serine residues are also contained within consensus MAPK phosphorylation sites in ERβ AF-1 domain and may thus also function as putative functional targets of MAPK-associated signaling pathways. We tested the contribution of several of these MAPK sites in the proteasome-dependent regulation of ERβ activity, using serine to alanine mutants. As shown in Fig. 3AGo, single replacement of Ser-94, -106, or -124 conferred transcriptional activation of ERβ in the presence of MG132, whereas the Ser-69 substitution had no effect. Higher levels of activation were observed with the Ser-106 variant, reaching a near 4-fold increase. Interestingly, when Ser-94 was also substituted, thus creating a S94,106A double mutant, the increase in activity by proteasome inhibition was even stronger with a 7-fold increase compared with each respective single mutant. Other multiple variants containing the S106A mutation, such as the S106,124A and S94,106,124A variants, were also induced, although to a lesser extent than the S94,106A mutant, in response to proteasome inhibition (Fig. 3AGo). To ensure whether the apparent deregulation to proteasome inhibition in ERβ activity upon serine modifications resulted from the AF-1 domain itself, as suggested from the results presented in Fig. 2AGo, we performed luciferase assay with corresponding serine mutants in which the DEF region of ERβ was removed. A comparable activation profile of the ABC constructs carrying successive substitutions of Ser-94, -106, and -124 was obtained (Fig. 3BGo). In addition, removal of the DEF region conferred an even greater transcriptional potential to the S94,106A mutant in response to proteasome inhibition, reaching a 14-fold increase, compared with its corresponding full-length version (Fig. 3Go, A and B). A role for the AF-2 function in regulating transcription in a manner dependent on proteasome activity has already been described for ER{alpha} (13, 14), and our results support a similar role for ERβ as well. However, coordinate removal of specific serine residues within ERβ AF-1 domain confers transcriptional activation when proteasome function is disrupted, raising the possibility that AF-1 regulatory sites participate in ERβ degradation by the proteasome.


Figure 3
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  		Fig. 3. MAPK Consensus Sites within the AF-1 Modulate ERβ Activity upon Proteasome Inhibition

A, 293 cells were transfected with an EREbLuc reporter in the presence of wild-type ERβ or various serine to alanine substituted mutants at the indicated positions within the AF-1 region of ERβ. After transfection, cells were treated with 1 µM MG132 for 16 h and harvested for luciferase assay. Values are expressed as fold response compared with untreated cells set at 1.0 for each ERβ construct used in transfection. B, Similar experiment as in panel A except that wild-type (wt) and serine mutants of ERβ C-terminal truncated ABC constructs were used in transfection. C, Western analysis of wild-type and serine mutated ERβ expressed in cells treated with vehicle or 1 µM MG132 for 16 h. Protein amounts were normalized to β-actin content. D, Similar to panel C except that ABC truncated forms of ERβ were expressed and analyzed by Western blot.

 
To test how the AF-1 serine substitutions may affect the steady-state levels of ERβ, we performed Western analysis on cells expressing wild-type and mutated ERβ in response to proteasome inhibition. Whereas wild-type ERβ levels were markedly increased in cells treated with MG132, the ERβ variants carrying the Ser-94 and/or Ser-106 substitutions, in the context of full-length or DEF-truncated receptor, were less prone to accumulate (Fig. 3Go, C and D). Therefore, the changes in cellular levels of the S94,106A and S94,106,124A mutants did not account for the increase in their transcriptional activity after inhibition of proteasome function, but rather these changes indicate an inverse relationship between receptor activity and cellular content. Such correlation has also been described for ER{alpha} in response to hormone and shown to be required to achieve maximal receptor activation (13, 14).

Contribution of AF-1 Serine Residues in ERβ Stability and Ubiquitination
Because we found that the S94,106A mutation rendered ERβ less prone to accumulate upon the presence of MG132, we performed a cycloheximide chase to evaluate the impact of the mutation on ERβ protein turnover. As shown in Fig. 4Go, A and B, the half-life of ERβ increased from approximately 8 h to approximately 12 h upon disruption of Ser-94 and -106, indicating that at least these residues are involved in ERβ degradation. Ubiquitination is the posttranslational process commonly used to tag and direct proteins to degradation by the 26S proteasome, and the pattern and the extent to which a protein is ubiquitinated determine its rate of degradation (29). We analyzed whether ERβ was ubiquitinated in absence of ligand, and how the S94,106A substitution may affect ERβ ubiquitination in vivo. By expressing ERβ in cells with tagged-ubiquitin, we observed several ubiquitinated forms of ERβ, which accumulated in the presence of MG132 (Fig. 4CGo). Interestingly, reduced levels of ubiquitination were observed for the S94,106A mutant in untreated cells, and treatment with MG132 had moderate effect on receptor ubiquitination, when compared with wild-type ERβ. The apparent lower levels of ubiquitination of the S94,106A mutant suggest that it has a reduced ability to be targeted by ubiquitination, which may then result in enhanced stability and transcriptional activity. This indicates that specific residues within the AF-1 of ERβ modulate receptor ubiquitination in absence of ligand and therefore contribute to ERβ degradation by the proteasome.


Figure 4
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  		Fig. 4. Serines 94 and 106 Regulate ERβ Stability and Ubiquitination

A, Cycloheximide chase experiment using 293 cells expressing ERβ or the S94,106A mutant. Transfected cells were treated with 50 µM cycloheximide and lysed at the indicated time points for Western analysis. β-Actin was used as a loading control. B, The S94,106A mutation confers an increased stability to ERβ. Quantitation of signal intensity of ERβ and S94,106A mutant derived from two separate experiments of cycloheximide chase described in panel A. Results are normalized to β-actin content and expressed as the percentage of change of time zero, which was set at 100%. C, 293 cells were transfected with either wild-type or the S94,106A mutant of ERβ in the presence of a HA-ubiquitin plasmid. Cells were treated with vehicle or 1 µM MG132 for 16 h, and harvested for immunoprecipitation (IP) using an anti ERβ antibody. Immunoprecipitates were analyzed by Western blot with an anti-HA antibody. IB, Immunoblot.

 
AF-1 Modifications Affect the Nuclear-Cytoplasmic Distribution of ERβ
Receptor redistribution between nuclear and cytoplasmic compartments has been described for many nuclear receptors in response to agonists and antagonists, and at least for ER{alpha}, such response was shown to affect receptor proteolysis (13, 20, 30). To test whether AF-1 substitutions may alter the nuclear-cytoplasmic distribution of ERβ in a proteasome-dependent manner, we determined the relative protein levels of ERβ compared with the S94,106A mutant in each cellular compartment. Both nuclear and cytoplasmic fractions were isolated and validated for the presence of selective markers poly-ADP-ribose polymerase (PARP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively (Fig. 5AGo). We found higher levels of the S94,106A variant in both nuclear and cytoplasmic fractions of untreated cells, when compared with wild-type ERβ (Fig. 5BGo), supporting a lesser propensity of the mutant to be ubiquitinated and a prolonged half-life in conditions where proteasome function is not impaired. In addition, the levels of nuclear S94,106A mutant were not significantly changed upon proteasome inhibition, whereas ERβ strongly accumulated in the same conditions (Fig. 5BGo), suggesting that serines 94 and 106 are involved in the degradation of nuclear ERβ by the proteasome. In addition, to determine why the S94,106A mutant failed to accumulate in the presence of MG132, the levels of wild-type ERβ and S94,106A mutant were analyzed by Western in nuclear insoluble (matrix) and soluble fractions from cells treated or not with MG132. The results presented in Fig. 5CGo demonstrate that ERβ accumulates in the matrix in response to MG132, whereas a large proportion of the mutant was not affected and therefore resides in the soluble nuclear fraction. These data suggest that Ser-94,106 regulate ERβ nuclear mobility to soluble/insoluble compartments and further support their role as recruiting signals in the clustering and immobilization of ERβ to the matrix in a manner independent of hormone.


Figure 5
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  		Fig. 5. Proteasome Function and Serine Modifications Affect the Nuclear Localization of ERβ

Panel A, Validation of the separation into cytoplasmic (C) and nuclear (N) protein extracts by Western analysis using antibodies against, respectively, PARP and GAPDH markers. Panel B, 293 cells were transfected with HA-tagged wild-type or the S94,106A ERβ construct, and treated with vehicle or 1 µM MG132 for 16 h. Both C and N fractions were isolated and analyzed by Western blot using an anti-HA antibody. Panel C, Cells were transfected as in panel B and nuclear extracts were separated into insoluble matrix fractions (M) and soluble nuclear fractions (S) for Western analysis.

 
Subnuclear Trafficking of ERβ Is Dependent on AF-1 Modifications and Proteasome Function
Earlier studies using fluorescence recovery after photobleaching (FRAP), have revealed the dynamic nature of steroid receptor movement within the nucleus and their association with active chromatin templates to engage transcriptional regulation (15, 31). Based on FRAP studies, the intranuclear mobility of ER{alpha} was found to be affected differently depending upon the addition of agonist or antagonist, indicating that ER{alpha} may associate to respectively transcriptionally active domains within the nucleus or immobilized to inactive clusters referred to as the nuclear matrix (10, 15, 32). Inhibition of proteasome activity was also shown to immobilize ER{alpha} to components of the nuclear matrix in treated cells (20, 33). Given our results on the accumulation of ERβ in the matrix compartment in response to MG132 (Fig. 5CGo), we therefore determined how inhibiting proteasome activity affects the nuclear mobility of ERβ and evaluated the possible role of disrupting AF-1 activity using FRAP on live cells. FRAP analysis was performed on cells transfected with a yellow fluorescent protein (YFP) fusion of ERβ, which was initially validated for correct size by Western blot and for activity by luciferase assay (data not shown). In absence of ligand, ERβ was found highly mobile, reaching equilibrium in the bleached region within seconds, with a half-maximal recovery time (t1/2) of 1.7 ± 0.2 sec (n = 12 nuclei) (Fig. 6Go, A and B). Although slightly longer, this half-recovery time for ERβ was comparable to unliganded ER{alpha} (t1/2 ~ 1 sec; data not shown and see Ref. 15), indicating that both receptors reside in a highly dynamic state, presumably awaiting for activation signals. As also reported for ER{alpha}, inhibiting proteasome activity with MG132 resulted in a profound immobilization of unliganded ERβ, such that the half-recovery time could not be estimated (t1/2 > 300 sec, Fig. 6BGo). Such proteasome-dependent immobilization has been suggested to result from the clustering and association to non-chromatin templates, which is consistent with the transcriptional activity we measured for ERβ in the presence of MG132. Remarkably, removal of Ser-94 and -106 in ERβ resulted in a more mobile receptor in the presence of MG132 (t1/2 = 39 ± 5 sec; n = 15 nuclei), suggesting that these sites were important to facilitate ERβ to immobilize in response to proteasome inhibition (Fig. 6BGo). This behavior was also observed with the S94,106,124A mutant (t1/2 ~ 60 sec; data not shown). These results indicate that disruption of ERβ AF-1 activity may allow the receptor to escape from associating with inactive clusters during proteasome inactivation, and therefore become available for transcriptional regulation. Consistent with these findings is the potent activation we observed for the S94,106A and S94,106,124A mutants by MG132 (Fig. 3Go, A and B).


Figure 6
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  		Fig. 6. Serines 94 and 106 Regulate ERβ Nuclear Mobility

A, Untreated and MG132-treated 293 cells transfected with wild-type or S94,106A mutant of YFP–ERβ were subjected to fluorescence recovery after photobleaching (FRAP) analysis. Images show single z-sections of whole cell nuclei obtained before and at the indicated time points after bleaching. Bleached images were obtained approximately 0.5 sec after the actual bleach corresponding to the rectangular box, which represents 10–15% of the total cell volume. Scanned images were taken at 0.1–0.5% laser intensity, and bleach was at 100%. After bleach, total nuclear fluorescence reaches equilibrium faster for untreated compared with MG132-treated cells, indicating a restrained mobility of ERβ during proteasome inhibition. Scale bar, 10 µm. B, Recovery curves from cells treated with vehicle (open squares) or 1 µM MG132 (filled squares). Fluorescence intensity values were averaged (n = 10 nuclei) and plotted over time. Values before bleach are shown to control for fluctuations of fluorescent signals. ROI, Region of interest.

 
The E6-AP Ubiquitin Ligase Regulates ERβ Degradation and Activity
E6-AP, an E3 ubiquitin-protein ligase proposed to mediate substrate specificity of the ubiquitin-proteasome degradation system (34), has been shown to participate in the degradation of ER{alpha} in response to estrogen (19, 35, 36). We tested whether E6-AP could also regulate the degradation of ERβ in transfected 293 cells. Coexpression of ERβ with E6-AP lead to a dramatic reduction in ERβ steady-state levels in absence of hormone as shown in Fig. 7AGo, an effect relieved by the addition of MG132, indicating that E6-AP is involved in ERβ degradation through the proteasome pathway. The S94,106A mutant was also subjected to E6-AP-dependent degradation but to a lesser extent than wild-type ERβ. Similarly, Fig. 7BGo shows a similar decrease in ERβ levels in response to increasing amounts of E6-AP in cells transfected with constitutive Mek1 and Erk plasmids, a condition that promotes ERβ phosphorylation and activity (25, 26). Under the same conditions, the S94,106A mutant was less affected. These effects were dependent on the E3 ligase activity of E6-AP, as demonstrated with the use of a ligase-defective C833A mutant that did not decrease ERβ levels (Fig. 7BGo).


Figure 7
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  		Fig. 7. The E6-AP Ubiquitin Ligase Regulates ERβ Degradation and Activity

A, Western analysis of wild-type and S94,106A mutated ERβ in response to E6-AP expression. After transfection, cells were treated with vehicle or 1 µM MG132 for 16 h and analyzed for ERβ content. Protein amounts were normalized relative to β-actin. B, The S94,106A mutation impairs the E6-AP-dependent decrease of ERβ levels in Erk-activated cells. Cells were transfected with ERβ or S94,106A mutant in the presence of increasing amounts of E6-AP construct, and analyzed by Western. Erk was activated by coexpressing constitutive Mek1 and Erk plasmids. Cells were also transfected with the C833A ubiquitin ligase-deficient form of E6-AP. C, The S94,106A ERβ mutant is less prone to degradation by E6-AP and Erk activation. Cells were transfected with the respective constructs as in panel B and subjected to a cycloheximide chase. Representative blots are shown for ERβ and S94,106A mutant in cells expressing E6-AP (left panel) or Mek1 and Erk (right panel). Corresponding quantitation of signal intensity of ERβ and S94,106A mutant derived from two separate experiments of cycloheximide chase is also shown. Results are normalized to β-actin content and expressed as the percentage change of time zero, which was set at 100%. D, E6-AP reduces ERβ transcriptional activity. Cells were transfected as in panel B in the presence of an EREbLuc reporter gene for luciferase assay. Erk was activated by coexpressing constitutive Mek1 and Erk plasmids and the C833A mutant of E6-AP was also tested. Values are expressed as fold response in luciferase activity relative to cells expressing ERβ, which was set at 1.0.

 
The expression of E6-AP also reduced the half-life of ERβ in cells to approximately 4 h, compared with approximately 8 h in cells not transfected with E6-AP, whereas the turnover rate of the S94,106A mutant was less affected with a half-life of approximately 11 h (compare Figs. 7CGo, left panel, and 4B). Activation of Erk in cells expressing ERβ also contributed to accelerate its degradation with a half-life of approximately 2 h, an effect shown to be highly dependent upon the integrity of Ser-94 and -106 (Fig. 7CGo, right panel). Besides its role as an E3 ubiquitin-protein ligase, E6-AP has also been described to enhance the transcriptional activity of various steroid hormone receptors such as progesterone receptor, androgen receptor and ER{alpha} in response to hormone (19, 37). We therefore determined the potential of E6-AP to regulate ERβ activity using luciferase assay. Interestingly, both basal and Erk-stimulated activities of ERβ were reduced upon expression of E6-AP in cells, whereas disruption of Ser-94 and -106 severely impaired the effects of Erk and E6-AP on ERβ and enhanced its basal activity presumably due to the increased mutant stability (Fig. 7DGo). The repression of ERβ by E6-AP was also dependent on the integrity of E6-AP E3 ligase activity, as the C833A mutant had no effect. These results indicate that the repression exerted by E6-AP on ERβ activity is linked with its ability to mediate ERβ degradation in absence of hormone, and further emphasize the role of Ser-94 and 106 as critical residues involved in ERβ degradation and activity.

The E6-AP Ubiquitin Ligase Is Recruited to ERβ in a Phosphorylation-Dependent Manner through Serines 94 and 106
Based on the potential of E6-AP to regulate ERβ turnover, we then addressed whether E6-AP can be recruited to ERβ using coimmunoprecipitation assays. Figure 8AGo shows that E6-AP was detected in the ERβ immunoprecipitates, indicating that both proteins can interact. However, the extent by which E6-AP can be recruited to ERβ was severely diminished upon disruption of Ser-94 and -106, suggesting that these sites behave as important determinants in the interaction. More interestingly, the recruitment of E6-AP to ERβ was more pronounced in response to MAPK activation as shown in cells transfected with Mek1 and Erk compared with control cells (Fig. 8BGo). The S94,106A mutation severely impaired the effect of Erk activation on the ability of ERβ to efficiently recruit E6-AP (Fig. 8BGo). These results suggest that the interaction of ERβ with E6-AP is regulated in a phosphorylation-dependent manner and involves Ser-94 and -106 of ERβ AF-1 domain.


Figure 8
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  		Fig. 8. Phosphorylation-Dependent Recruitment of E6-AP to ERβ through Serines 94 and 106

A, E6-AP coimmunoprecipitates with ERβ. 293 cells were tranfected with myc-tagged ERβ or S94,106A mutant in absence or presence of HA-E6-AP, and then treated with 1 µM MG132 for 16 h. Immunoprecipitation (IP) was carried out with an anti-myc antibody, and E6-AP was detected by Western analysis using an anti HA antibody. ERβ was also monitored in each sample using an ERβ antibody. B, E6-AP is recruited in a phosphorylation-dependent manner involving Ser-94 and -106. Coimmunoprecipitation assay as described in panel A except that Mek1/Erk plasmids were used in transfection to promote Erk activation and no MG132 was added to cells. IB, Immunoblot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we identify an important and unrecognized function of the AF-1 domain in targeting ERβ to the degradation process mediated by the ubiquitin-proteasome system. The current model of ER degradation has mostly been described for ER{alpha} in response to estrogen. Studies using proteasome inhibitors have shown that the hormonal activation of ER{alpha} was dependent upon the ubiquitin-proteasome pathway, therefore establishing a correlation between receptor degradation and activity, an observation also made for other nuclear receptors (12, 38). Similarly to ER{alpha}, we report that the response of ERβ to estrogen also required the proteasome function to achieve optimal receptor activation. However, in absence of hormone, whereas both ERs accumulated in cells treated for proteasome inhibition, the role of the proteasome in maintaining basal ER activity seemed to be different between the two receptor isoforms. Such dependence resides within the N-terminal region of each receptor, which were both stabilized upon proteasome inhibition but conferred a different responsiveness in terms of activity. Using a truncated ER{alpha}, Lonard et al. (14) have also reported an increased stability of ER{alpha} N-terminal region during proteasome inhibition. The significance of an apparent correlation between AF-1 activity and ER degradation is not completely understood, but it becomes clear that AF-1 activity could impact ER turnover and that this may occur in an isoform-dependent manner. Consistent with this, whereas the AF-1 region was shown to regulate the ligand-induced degradation of ER{alpha} (39), our results indicate that the role of ERβ AF-1 on receptor degradation can be uncoupled from the hormonal response.

The mechanistic activation of AF-2 function by ligand has been well detailed for nuclear receptors and in particular, crystallographic studies of ER{alpha} and ERβ ligand binding domains have helped to identify the structural determinants involved (40, 41, 42, 43). In contrast, modulation of AF-1 activity of estrogen receptors is intricate and variable and mostly depends on posttranslational modifications including phosphorylation. It is known that in response to growth factors, such as EGF, the activity of ER{alpha} and ERβ is associated with phosphorylation of their respective AF-1 (2, 22, 44). The AF-1 domain of ERβ contains many putative phosphorylation sites of which Ser-106 and -124 were described to be directly phosphorylated by MAPK resulting in AF-1 activation of the receptor in response to EGF or ras (24, 25). Here, we show that these clustered serines along with Ser-94 are also involved in ubiquitination and turnover of ERβ, therefore providing evidence that the region within AF-1 that signals ERβ to degradation overlaps with the one involved in its activation by growth factor signaling pathways. This apparent paradoxical roles shared by the same domain within ERβ raised the possibility that both events, i.e. ubiquitination and transactivation, are required to mediate optimal AF-1 response of ERβ to nonhormonal stimuli. Such dual role for an activation domain to also signal to ubiquitination was reported to be important in limiting the activation potential of transcription factors (45). As opposed to other steroid receptors such as ER{alpha} and progesterone receptor, for which phosphorylation of their AF-1 was linked to hormone-dependent degradation and activity (39, 46), our results indicate that the AF-1 of ERβ appears to be sufficient in mediating such response. Supporting this hypothesis, we have observed that the N-terminal region of ERβ can be ubiquitinated in a manner similar to the full-length receptor and that such ubiquitination was also dependent on the integrity of Ser-94 and -106 (data not shown). In addition, we found that ERβ missing the AF-1 domain (CDEFβ construct) did not accumulate in response to MG132, an effect also observed by others (47). Our interpretation from these findings is that, in absence of hormone, Ser-94 and Ser-106 within the AF-1 domain provide the signal(s) that triggers ERβ ubiquitination. Recently, Tateishi et al. (47) have reported that the N-terminal domain of human ERβ corresponding to amino acids 1–37 was necessary for its degradation by estrogen. Whether the immediate N-terminal region of ERβ regulates ligand-dependent ubiquitination and/or receptor degradation in a manner similar to the region containing Ser-94 and -106 in absence of hormone is not known, but it can be predicted that although different stimuli, either hormonal or kinase-derived, regulate ERβ activity, they may also converge toward a common mechanism that dictates receptor turnover. In addition, given the significant impact of the N-terminal region of ERβ to regulate receptor ubiquitination, the exact site(s) of such ubiquitination remains, however, unknown. Ubiquitin are typically conjuguated to lysine residues in targeted proteins and many conserved lysines reside in the N-terminal region of ERβ from several species. However, attempts to delineate the ubiquitination sites in the N-terminal domain of ER{alpha} and ERβ in response to hormone have not resulted in the identification of the targeted lysines (39, 47). So clearly, the exact mapping of the ubiquitination sites in the context of AF-1 activity of ERβ remains to be determined.

Recent studies based on fluorescent-based approaches have revealed the dynamic nature of ER{alpha} movement within the nucleus and its association with active chromatin templates to engage transcription in response to ligands, or its immobilization to inactive clusters referred to as the nuclear matrix during proteasome inhibition (10, 15, 32, 33, 48). We have therefore performed FRAP analysis to evaluate the mobility of ERβ in conditions in which proteasome activity was inhibited and on the possible role of disrupting AF-1 activity on ERβ nuclear dynamics. In absence of ligand, ERβ was found highly mobile, reaching equilibrium with a half-maximal recovery time comparable with ER{alpha}, indicating that both ERs reside in a highly dynamic state, presumably awaiting activation signals. Remarkably, whereas addition of MG132 resulted in a profound immobilization of ERβ, likely reflecting its clustering to non-chromatin templates, removal of Ser-94 and Ser-124, concomitantly with Ser-106, resulted in a more mobile receptor. These results suggest that disruption of ERβ AF-1 activity may allow the receptor to escape from associating to inactive clusters during proteasome inhibition, and to become or remain available for transcriptional regulation. Consistent with this hypothesis is the potent activation we observed for the S94,106A mutant in the presence of MG132, and its reduced ubiquitination and capacity to cluster to nuclear matrix, indicating that disruption of these residues leads to a deregulated ERβ less prone to proteasome-mediated degradation. The apparent inability of the AF-1 mutants to behave such as the wild-type ERβ and remained clustered and immobilized during proteasome inhibition, raised the interesting question as to whether AF-1 phosphorylation could signal ERβ to transit from active to inactive chromatin compartments. Our observations indicate that this process occurs in a ligand-independent manner, thereby providing a unique potential of AF-1 to target ERβ to the nuclear matrix in kinase-activated cells. Because serine 106 and other residues that participate in the response of ERβ to growth factor signaling are conserved among nuclear receptors (2, 25), it would be expected that the ERβ intranuclear behavior may be shared with other nuclear receptors described to also be regulated by kinase-activated pathways. Consistent with this, we recently reported that activation of Akt modulated the intranuclear behavior and activity of ERβ, and its segregation with coactivator CBP through a conserved Akt site shared with other nuclear receptors (49).

The transcriptional deregulation of ERβ characterized by its enhanced activity during proteasome inhibition upon AF-1 disruption was rather intriguing and suggested a mechanism by which the AF-1 may participate in restraining or at least maintaining an adequate and regulated response of ERβ to cellular pathways involving MAPK activation. In an attempt to partly elucidate such mechanism, the E6-AP ubiquitin ligase was found to be favorably recruited to ERβ in response to signals that promote ERβ phosphorylation. Notably, this interaction was strongly dependent upon the presence of Ser-94 and -106, which resulted in a more rapid degradation of ERβ in response to Erk activation. This suggests that phosphorylation at these sites regulates ERβ turnover through the functional recruitment of components of the proteasome degradation pathway. Many studies, mostly involving ER{alpha}, have depicted a role for estrogen to modulate ER cellular levels through the ubiquitin-proteasome pathway, thereby providing a functional link between receptor turnover and activity. As such, several proteins that exhibit E3 ubiquitin-protein ligase activity, such as Mdm2 (18, 50), CHIP (carboxyl terminus of Hsc70-interacting protein) (51, 52), BRCA1 (breast cancer gene 1) (53) and E6-AP (19), have been shown to modulate the hormonal response of ER{alpha}. In that respect, E6-AP was described as a dual-function protein that associates with ER{alpha} to promote its degradation and coactivation by estrogen (19, 35, 36). This study extends the role of E6-AP to regulate ERβ degradation through the proteasome pathway and, with the increased ability of ERβ to recruit E6-AP compared with the S94,106A mutant in response to Erk activation, it further identifies a novel function for serines 94 and 106 in maintaining suboptimal ERβ levels. These observations support the model shown in Fig. 9Go in which the AF-1 domain provides the necessary signals through phosphorylation of critical residues that serve to recruit E6-AP to dictate ERβ turnover under ligand-independent mechanisms. The requirement of estrogen to achieve such ERβ regulation to kinase activation by E6-AP is not necessary because it was also the case for the recruitment of coactivators SRC-1 and CBP in response to MAPK activation (Refs. 25 and 26 and Fig. 9Go). However, we found that in contrast to ER{alpha}, the response of ERβ to estrogen was reduced by E6-AP (Picard, N., and A. Tremblay, unpublished observations), indicating that the potential of E6-AP to regulate the response to hormone differs for each ER. This raises the interesting possibility that a selective recruitment and use of specific E3 ligases may depend on how ER{alpha} and ERβ integrate various cellular signals to regulate their turnover and activity. Consistent with this, the recruitment of Mdm2 and E6-AP to ER{alpha}-responsive genes in response to estrogen was recently shown to be dependent on the presence of Ser-118, a phosphorylation site within ER{alpha} AF-1 (39). With the increasing number of E3 ligases reported in ER degradation by estrogen, it is likely that the cellular context and/or activation signals, whether it is hormonal or kinase-derived, might dictate selectivity of each ER in recruiting and use components of the proteasome pathway. Our studies demonstrate the ability of AF-1 domain to provide signals in regulating receptor mobility, ubiquitination, and turnover and to add to the intricate regulatory mechanisms involved in the control of nuclear receptor function by kinase signaling pathways.


Figure 9
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  		Fig. 9. A Proposed Model for the Role of AF-1 in the Response of ERβ to Erk Activation

Activation of Erk in cells promotes ERβ phosphorylation at the AF-1 domain which mediates a favored recruitment of transcriptional coactivators SRC-1 and CBP, and subsequent transactivation (25 26 ). The phosphorylation of AF-1 also promotes the recruitment of the E3 ubiquitin ligase E6-AP to ERβ which results in receptor ubiquitination, transcriptional repression, clustering into inactive nuclear compartments, and degradation by the ubiquitin-proteasome system. This activation-degradation pathway is highly dependent upon the phosphorylation of the AF-1 domain of ERβ at distinct sites and does not require the presence of hormone, thereby providing an exclusive and functional role for the AF-1 to integrate receptor activity and turnover to kinase signaling pathways.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Expression pCMX plasmids coding for mouse ER{alpha}, mouse ERβ, and related truncated (ABC, CDEF, and chimeric constructs) and serine (positions 106 and 124) mutants have been described previously (25, 26, 28). Other ERβ serine to alanine mutants (positions 69 and 94) were generated by PCR mutagenesis using Pfu polymerase (Stratagene, La Jolla, CA) and confirmed by automated sequencing. Plasmids coding for constitutively active Mek1 and Erk2 (28), and hemagglutinin (HA)-tagged ubiquitin (54) have been described. The coding region of E6-AP has been inserted into pCMX-HA to produce a N-terminal tagged protein and the C833A ligase-deficient mutant was generated by mutagenesis as above.

Cell Culture, Transfection, and Luciferase Assay
Human embryonic kidney 293 cells were routinely maintained in DMEM (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum, in a humidified atmosphere of 5% CO2 at 37 C. For transient transfection, cells were seeded in phenol red-free DMEM supplemented with charcoal dextran-treated serum and plasmid constructs were introduced into cells using the calcium phosphate precipitation method as described (28). Typically, for luciferase assay, cells were transfected with 500 ng EREtkLuc reporter construct, 100 ng ER expression plasmids, 50 ng each of Mek1 and Erk2 expression plasmids, and 250 ng pCMX-β gal in a total of 1.5 µg DNA per well. After 5–6 h, the medium was changed and cells were treated for 16 h with 10 nM 17β-estradiol (E2) and/or 1 µM proteasome inhibitor MG132 (Sigma) or clasto-lactacystin β-lactone (BioMol, Plymouth Meeting, PA), unless otherwise stated. Cells were then harvested in potassium phosphate buffer containing 1% Triton X-100 and lysates analyzed for luciferase activity using a luminometer (Wallac, Turku, Finland). Luciferase values were normalized for transfection efficiency to β-galactosidase activity and expressed as relative fold response compared with controls. Luciferase assays are performed in duplicates from at least three independent experiments.

Cell Lysates, Immunoprecipitation, and Immunoblotting
Determination of ER{alpha} and ERβ cellular content by Western analysis has been described (28). Briefly, transfected cells were treated with 10 nM E2 or 1 µM MG132 for 16 h, washed with ice-cold PBS and lysed in PBS containing 1% Triton X-100, 0.5% deoxycholate acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Roche, Laval, Quebec, Canada). 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) in Tris-buffered saline, probed with antibodies against ER{alpha} or ERβ, and signals revealed by enhanced chemiluminescence using appropriate horseradish peroxidase-conjuguated secondary antibodies. For cells transfected with HA-tagged ERβ constructs (truncated forms and serine mutants), an anti-HA antibody (12CA5) was used for immunoblotting. In each experiment, total protein content was normalized using an anti-β-actin antibody (Abcam, Cambridge, MA). To detect ubiquitinated forms of ERβ, cells were transfected with ERβ (wild type or mutated) in the presence or absence (control) of HA-tagged ubiquitin plasmid. Cells were treated with vehicle or 1 µM MG132 for 16 h, washed with ice-cold PBS and lysed with 1% Nonidet P-40, 0.5% deoxycholate acid, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, and protease inhibitors in PBS. Cell lysates were precleared before incubation with 1–2 µg of anti ERβ antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C with gentle agitation. Immune complexes were recovered with protein A/G-PLUS agarose, washed three times in lysis buffer, and subjected to SDS-PAGE and immunoblotting as described above using anti-HA antibody. Coimmunoprecipitation analysis was performed to detect ERβ /E6-AP interaction in cells transfected with wild-type or mutated ERβ (myc-tagged) in the presence of HA-tagged E6-AP. Immunoprecipitation of ERβ was performed as above with an anti-myc antibody (9E10), except that salt concentration was raised to 0.7 M and no SDS was added in the lysis buffer. The anti-HA antibody (12CA5) was used for immunoblotting.

Cycloheximide Chase
The 293 cells were transiently transfected with plasmids expressing HA-tagged wild-type or S94,106AERβ in absence or presence of E6-AP or Mek1/Erk2 plasmids. At 12 h after transfection, cycloheximide (Sigma) was added at a concentration of 50 µM, and cells were lysed for Western blot analysis at the indicated time points. Each signal intensity derived from two separate experiments was quantitated using an image analyzer (Alpha Innotech, San Leandro, CA) and expressed relative to β-actin levels.

Preparation of Nuclear and Cytoplasmic Extracts
To prepare nuclear and cytoplasmic extracts, cells were pelleted by centrifugation, resuspended in hypotonic buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 0.5 mM PMSF and protease inhibitors, and lysed by three freeze-thaw cycles. Nuclei were separated from the cytosolic fraction by centrifugation at 12,000 x g for 5 min at 4 C. The nuclear pellet was resuspended in the same hypotonic buffer except that salt concentration was raised to 420 mM KCl and 25% (vol/vol) glycerol was added. The matrix fraction was isolated from total nuclear extracts by centrifugation and supernatants corresponded to the soluble nuclear fractions. Equivalent amounts of cytoplasmic and nuclear fractions were then subjected to SDS-PAGE for Western analysis. The content of selective markers for nuclear (anti-PARP; Santa Cruz Biotechnology) and cytoplasmic (anti-GAPDH; Santa Cruz Biotechnology) compartments was tested by immunoblotting to ensure for the qualitative purity of the prepared fractions.

FRAP
FRAP analysis was carried out on live 293 cells transfected with YFP fusions of wild-type or S94,106A mutant of ERβ. Cells were grown on LabTek chambered slides (Nunc, Rochester, NY) in phenol red-free DMEM containing 5% charcoal dextran-treated serum, and transfected by the calcium-phosphate procedure as described above. FRAP experiments were performed on a Zeiss (Jena, Germany) LSM510 confocal microscope equipped with an Argon 514-nm laser and a 530-nm low-pass filter. A single z-section image of whole cell nuclei was captured before the bleach and at the indicated time points after. The bleached regions correspond to less than 10% of total nucleus using 100% laser intensity, and scanned images were taken at 0.1% with an open pinhole and a numerical aperture of 0.8 to ensure that diffusion in the z dimension is avoided. Fluorescence intensities were analyzed using Zeiss Physiology software 3.2 and averaged from at least 10 nuclei.


   ACKNOWLEDGMENTS
 
We thank Dirk Bohmann and Martin Schefner for the generous gifts of respective plasmids for ubiquitin and E6-AP. We acknowledge the technical assistance of Simon Blouin and Julie Gabbay. We thank members of both labs for critical reading and useful comments.


   FOOTNOTES
 
N.P. is supported by a doctoral award from the FRSQ (Fonds de la Recherche en Santé du Québec) and from the FHSJ (Fondation de l’Hôpital Ste-Justine), C.C. holds an award from the Natural Sciences and Engineering Research Council of Canada, M.S. is supported by the FHSJ, A.L. was supported by the Ligue Nationale Contre le Cancer (LNCC), and M.B. by the Fondation pour la Recherche Médicale. A.T. is a New Investigator of the Canadian Institutes of Health Research (CIHR). This work was supported by grants from the CIHR, the Cancer Research Society Inc., and the Canadian Foundation for Innovation (to A.T.), and from ARC (Association pour la Recherche contre le Cancer, Grant No. 3582) and LNCC (to G.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 25, 2007

1 N.P. and C.C. have contributed equally. Back

Abbreviations: AF, Activation function; CBP, cAMP response element binding protein-binding protein; E2, 17β-estradiol; E6-AP, E6-associated protein; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; FRAP, fluorescence recovery after photobleaching; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; PARP, poly-ADP-ribose polymerase; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; YFP, yellow fluorescent protein.

Received for publication June 4, 2007. Accepted for publication October 18, 2007.


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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ
Coregulators:   E6AP
Ligands:   17β-Estradiol



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