help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0148
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, J.
Right arrow Articles by Shapiro, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, J.
Right arrow Articles by Shapiro, D. J.
Endocrinology Vol. 148, No. 10 4634-4641
Copyright © 2007 by The Endocrine Society

A Functional Serine 118 Phosphorylation Site in Estrogen Receptor-{alpha} Is Required for Down-Regulation of Gene Expression by 17ß-Estradiol and 4-Hydroxytamoxifen

Jingwei Cheng, Chen Zhang and David J. Shapiro

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: David J. Shapiro, Department of Biochemistry, University of Illinois, 413 RAL, Box B4, 600 South Mathews, Urbana, Illinois 61801. E-mail: djshapir{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To evaluate the contribution of ERK1/2 phosphorylation of estrogen receptor (ER)-{alpha} to activation and repression of endogenous genes, we produced stably transfected lines of HeLa cells with functional ERK1/2 pathways that express similar levels of wild-type human ER{alpha} and ER{alpha} mutated to inactivate the well-known MAPK site at serine 118 (ER{alpha}S118A). We compared effects of the S118A mutation on 17ß-estradiol (E2)-mediated transactivation, which is heavily dependent on activation function (AF) 2 of ER{alpha} and on 4-hydroxytamoxifen (OHT)-mediated transactivation, which is heavily dependent on AF1, which includes S118. To examine whether S118 was the key ERK/MAPK phosphorylation site in ER{alpha} action, we compared the effects of the S118A mutant and the ERK inhibitor U0126 on expression of endogenous genes. In several estrogen response element-containing genes, the S118A mutation strongly reduced induction by E2, and U0126 did not further reduce expression. Expression of another group of estrogen response element-containing genes was largely unaffected by the S118A mutation. The S118A mutation had variable effects on genes induced by ER tethering or binding near specificity protein-1 and activator protein-1 sites. For five mRNAs whose expression is strongly down-regulated by E2 and partially or completely down-regulated by OHT, the S118A mutation reduced or abolished down-regulation by E2 and nearly abolished down-regulation by OHT. In contrast, for Sma and mothers against decapentaplegic-3-related, which is down-regulated by E2 and not OHT, the S118A mutation had little effect. These data suggest that there may be distinct groups of genes down-regulated by ER{alpha} and suggest a novel role for ERK phosphorylation at serine 118 in AF1 in regulating expression of the set of genes down-regulated by OHT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS, ACTING THROUGH estrogen receptors (ERs), exert pleotropic effects on diverse cells and organ systems including the reproductive system, brain, immune system, liver, bone, colon, and prostate. At the molecular level, estrogens, such as the prototypical estrogen, 17ß-estradiol (E2), bind to ER and can act via three basic pathways. The E2-ER complex regulates gene expression by direct binding to canonical DNA response elements termed estrogen response elements (EREs) and dispersed half-sites and direct repeats (1). E2-ER complex can be brought to DNA by tethering though other proteins bound at specificity protein-1 (SP1) and activator protein (AP)-1 sites and by binding at ERE half-sites near SP-1 and AP-1 sites (2, 3, 4, 5). The E2-ER complex, and probably E2 bound to a distinct membrane protein, can rapidly activate several membrane-associated protein kinase-based signaling pathways (6, 7, 8, 9). All of these mechanisms contribute to the diverse effects of estrogens. In the classical model for ER action, when bound to a potent estrogen, such as E2, the ER dimerizes, stimulating binding of ER to the ERE and the ligand binding domain of ER assumes a conformation that enables the recruitment of coactivators. The bound coactivators help assemble a dynamic multiprotein complex that facilitates both chromatin remodeling and formation of an active transcription complex (10, 11). The ER adopts different conformations when bound to different ligands and EREs. ER ligand and ERE sequence work together to regulate binding of ER to estrogen-responsive promoters and in coactivator recruitment (12, 13, 14, 15, 16, 17, 18, 19). Transcription activation by ER is mediated by two interacting activation functions. Activation function (AF)-1, in the N-terminal A/B domain is important in ligand-independent transcription and in the actions of tamoxifen and other selective estrogen receptor modulators (SERMs). Activation function 2 in the C-terminal ligand binding domain is important in ligand-dependent recruitment of coactivators (20, 21, 22).

Binding of potent estrogens and some SERMs and activation of signal transduction pathways elicit functionally significant phosphorylation of ER{alpha}. Activation of the epidermal growth factor (EGF) receptor can enhance the effects of E2 (23) and in some cases completely eliminates the requirement for exogenous estrogens (24, 25). E2-ER{alpha} induces moderate activation the ERK1/2 pathway (6), and the activated ERK1/2 pathway then enhances phosphorylation of E2-ER{alpha}, thereby enhancing transactivation by E2-ER{alpha} (25, 26, 27, 28, 29, 30, 31, 32).

Although microarray studies show that levels of at least as many mRNAs decline in response to estrogen and SERMs as increase (33), much less is known about mechanisms by which estrogens and SERMs bound to ER down-regulate gene expression than about activation of gene expression. Proposed mechanisms include recruitment of corepressors and histone deacetylases (34) and interfering with binding of activator proteins (35, 36). Little is known about the relationship between ER phosphorylation and ER-mediated down-regulation of gene expression.

Although several early reports suggested that tyrosine residues in ER{alpha} can be phosphorylated (37, 38, 39, 40, 41), considerable research suggests that in response to estradiol binding, ER{alpha} is phosphorylated only on serine residues (28, 29, 42, 43, 44, 45, 46, 47). Among the several potential phosphorylation sites in human ER{alpha}, phosphorylation of the consensus MAPK site at serine 118 has received a great deal of attention. Serine 118 (S118) is phosphorylated in response to cyclin-dependent kinase-7 (48) and after EGF activation of the ERK1/2 pathway (20, 49). Mutating S118 to Glu, which may mimic phosphorylation, enhances transcription by ER{alpha} (29). Several laboratories report that mutating serine 118 to alanine reduces transactivation by ER{alpha} (20, 25, 29, 49), whereas others find that this mutation has little effect (28). The considerable disagreement on the importance of the S118 phosphorylation site in ER{alpha} may stem in large part from the widespread use of transient transfections and assays of synthetic ERE-containing reporter genes to study its role. The use of transient transfections suffers from several problems. The EREs in reporter genes are usually not in their endogenous gene backgrounds, the transiently transfected genes are not fully assembled into chromatin, the level of ER{alpha} protein expressed in the transiently cells is usually not known and can be quite high, and these assays usually do not address ER{alpha}-mediated transactivation that is based on tethering of ER{alpha} to proteins bound at AP1 and SP1 sites or binding to ERE half-sites near SP1 and AP1 sites. Little is known about the effect of S118A phosphorylation on down-regulation of gene expression by ER{alpha}.

Our objective was to evaluate the contribution of ERK1/2 phosphorylation of ER{alpha} at serine 118 to the activation and repression of well-studied endogenous genes. To examine the importance of ERK1/2 phosphorylation of S118 in ER{alpha} action, we compared the effect of the S118A mutant and the global ERK inhibitor U0126 on expression of endogenous genes. Because S118 is located in the AF1 region of ER{alpha}, and transactivation by 4-hydroxytamoxifen (OHT; the active metabolite of tamoxifen) is largely AF1 dependent, whereas transactivation by E2 largely relies on AF2, we compared the effect of the S118 mutation on regulation of gene expression by E2 and OHT. To carry out these studies in a more biologically relevant background than most previous studies, we produced stably transfected lines of HeLa cells expressing wild-type human ER{alpha} at levels similar to ER{alpha} levels in widely used MCF-7, human breast cancer cells. We then identified a pair of cells lines expressing nearly identical levels of wild-type human ER{alpha} and ER{alpha}S118A. In addition to important effects on ER-mediated transactivation, we identified a previously unsuspected role for serine118 in down-regulation of gene expression by ER{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
Full-length, wild-type human ER{alpha} cDNA with a FLAG epitope tag on its N terminus (50) and the Ser 118 -> Ala ER{alpha} mutant (produced by quick change mutagenesis; Stratagene, La Jolla, CA) were subcloned into the EcoRI/BamHI site of pIREsIneo vector (CLONTECH, Palo Alto, CA). We previously reported that the FLAG epitope tag had no detectable effect on the properties of ER{alpha} (51).

Production of stably transfected cell lines
A clone of HeLa cells exhibiting robust growth and high transfection efficiency with liposomes was identified in our laboratory and used in these studies (63). Five million HeLa cells were resuspended in 0.5 ml DMEM, mixed with 10 µg Ssp-I linearized plasmid DNA, incubated on ice for 10 min, and then electroporated at 200 V, 1180 µF with an electroporator (Bio-Rad Laboratories, Hercules, CA). The cells were then transferred to selection media (50% DMEM/10% charcoal-dextran stripped fetal bovine serum, 50% HeLa cell conditioned medium). After 24 h, G418 (600–1000 µM in different selections) was added to the medium. The medium was changed every 2–3 d. After 2–3 wk, colonies were picked, transferred to a 96-well plate, and grown out. More than two dozen individual clonal lines were produced and evaluated to produce the matched cell lines used in this study.

Western blots
Cells were resuspended in ice-cold lysis buffer [(adapted from Ref. 52): 20 mM HEPES (pH 7.9); 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; and 10 mM dithiothreitol, 100 µg/ml phenylmethylsulfonyl fluoride, protease inhibitor cocktail (P8340; Sigma, St. Louis, MO) was added right before use]. After sonication on ice, the extracts were centrifuged for 5 min at 4 C and protein concentration determined with Coomassie Blue (Bio-Rad). Samples were combined with 6x sodium dodecyl sulfate loading buffer, boiled for 5 min, and loaded onto a 12.5% SDS-PAGE. After electrophoresis, the gel samples were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Primary antibodies were antibody against phosphorylated ER{alpha} at S118 (Cell Signaling Technology, Beverly, MA), ER{alpha} antibody (clone 6F11; Biocare Medical, Walnut Creek, CA), and phosphorylated ERK antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were visualized with ECL plus reagents (Amersham Biosciences, Piscataway, NJ), and band intensity was quantitated using a Molecular Dynamics Storm PhosphorImager (Amersham).

Real-time quantitative PCR
RNAs from HeLa-ER cells were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), purified with the RNeasy minikit (QIAGEN, Valencia, CA), and 1 µg of RNA from HeLa-ER cells was reverse transcribed using Muloney murine leukemia virus reverse transcriptase (Invitrogen), and 1% of the cDNA product was used in quantitative RT-PCR. The forward and reverse primers mix (1 µl of 10 µM) and 12.5 µl SYBR green 2x PCR master mix (Applied Biosystems, Carlsbad, CA) in a total volume of 25 µl were put into each well of a 96-well iCycler iQ PCR plate and assayed using a Bio-Rad iCycler optical system (Bio-Rad). PCR was as follows: 95 C for 2 min; 45 repeats (95 C for 20 sec, 55 C for 20 sec, 72 C for 30 sec); melting from 55 to 95 C with 0.5 C increase per 10 sec in each cycle for 80 cycles. The internal standard was 36B4 mRNA.

Statistical analysis
The statistical significance in the data was assessed by ANOVA, and P values were calculated using the unpaired, two-tailed Student’s t test. Data are presented as mean ± SEM. Significance was established when P < 0.05. Because the data in most figures allow for multiple comparisons such as wild-type and S118A and E2 and OHT, these comparisons are described in the figure legends and are not shown in the body of the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of stably transfected cell lines expressing similar levels of ER{alpha} and ER{alpha}S118A
To analyze the role of serine 118 in ER{alpha} action without using transient transfections, we produced lines of stably transfected HeLa cells expressing wild-type ER{alpha} and ER{alpha}S118A. Because HeLa cells have been passaged for many years, HeLa cell populations can be quite heterogeneous. To use cells that were as similar as possible except for the two different ERs, we used HeLa cells recently recloned in our laboratory. To increase the efficiency with which stable cells lines grow out, we used a bicistronic vector in which the ER and the selectable marker (neomycin phosphotransferase) are transcribed as a bicistronic mRNA in which the mRNA encoding the selectable marker is transcribed from an internal ribosome entry site (51). The ER{alpha} and ER{alpha}S118A in the stably transfected cell lines are expressed from a constitutive promoter. To identify cell lines expressing levels of wild-type ER{alpha} and ER{alpha}S118A that were similar to levels in MCF-7 cells and were nearly identical with each other and contained a functional ERK1/2 pathway, we tested more than 25 stably transfected cell lines (data not shown). Quantitation using PhosphorImager analysis showed that ER{alpha} levels in the wild-type ER{alpha} and ER{alpha}S118A cell lines in the absence of ligand were within 10% of each other. The two cell lines also expressed similar levels of ER after down-regulation of ER{alpha} by the antagonist ICI 182,780/Faslodex (Fig. 1AGo). The cell line that expresses wild-type ER{alpha} is designated HeLaER{alpha}, and the cell line that expresses ER{alpha}S118A is HeLaER{alpha}s118a. These cell lines contain levels of ER{alpha} protein that are similar to those expressed in MCF-7 cells (data not shown). Transient transfection with ERE-luciferase-containing reporter genes showed that the ER in the two cell lines was functional (data not shown).


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 1. The HeLaER{alpha} and HeLaER{alpha}S118A cell lines express similar levels of ER{alpha} and contain functional ERK1/2 pathways. A, Western blot of the levels of ER{alpha} expressed in the cell lines. Western blots were performed as described in Materials and Methods. The cells were maintained for 24 h in medium containing ethanol (EtOH) or 100 nM of the pure antagonist ICI 182,780/Faslodex (ICI). Actin was used as the internal standard. The data are representative of multiple Western blots. B, The two cell lines were maintained in medium containing EGF for 20 min and phosphorylated ERK determined with a phospho-ERK-specific antibody.

 
Because we were studying phosphorylation mediated by the ERK1/2 pathway, it was important to show that cell lines contained a functional ERK1/2 pathway. When the HeLaER{alpha} and HeLaER{alpha}S118A cell lines were treated with the ERK1/2 activator, EGF, we observed the expected increase in the level of phospho-ERK (Fig. 1BGo).

Ligand-dependent phosphorylation of serine 118 of ER{alpha}
To measure the level of phosphorylation of serine 118 in HeLaER{alpha} cells subjected to different treatments, antibody specific to p-ER{alpha}S118 was used. As a control for antibody specificity, we tested the HeLaER{alpha}S118A cell line. As expected, no phosphorylation signal on S118 was detected (Fig. 2Go, HeLaER{alpha}S118A). The ER ligands E2 and OHT and the ERK1/2 inhibitor U0126, plus E2 and OHT and EtOH vehicle, were separately added to the medium. Compared with EtOH, both E2 and OHT enhance the phosphorylation of serine 118 in HeLaER{alpha} cells (Fig. 2Go, HeLa ER, E2, and OHT). OHT stabilizes ER{alpha} and increases levels of ER{alpha} (53, 54). This contributes to the strong phosphorylation signal seen on S118 of ER{alpha} in the OHT-treated cells. The ERK/1/2 inhibitor U0126 largely blocked phosphorylation of S118 (Fig. 2Go, HeLaER, E2+U0126, and OHT+U0126). To test the effectiveness of the ERK1/2 inhibitor U0126, we also examined phospho-ERK. ERK phosphorylation was nearly abolished when U0126 was present (Fig. 2Go, p-ERK). Levels of ER{alpha} and ERK proteins were used as controls (Fig. 2Go).


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		FIG. 2. E2 and OHT induce phosphorylation of serine 118 of ER{alpha}. HeLaER{alpha} and HeLaER{alpha}S118A cells were maintained for 24 h in medium containing ethanol vehicle (EtOH), 10 nM E2, or 10 nM OHT. In some samples treatment with 10 µM U0126 was included with the 10 nM E2 or 10 nM OHT. Phosphorylation of ER{alpha} and of ERK was measured using phospho-specific antibodies. ER{alpha} and ERK protein levels were also measured as controls. The data in Fig. 2Go are representative of a total of three similar experiments.

 
An intact S118 site is critical for estrogen induction of some ERE-containing genes and unnecessary for others
To evaluate the role of the S118 site in ER action, we began with genes that were estrogen inducible in MCF-7 human breast cancer cells or other cells and had been shown to contain functional EREs (1). We studied ERE-containing genes that were strongly induced by estrogen in the HeLaER{alpha} cells. We compared the ability of E2 and OHT to induce the ERE-containing genes in the HeLaER{alpha} cells and HeLaER{alpha}S118A cells. The importance of the serine118 ERK1/2 phosphorylation site relative to other ERK/12 phosphorylation sites in ER{alpha} and coregulators is not well established. We therefore also analyzed the effects of the ERK1/2 inhibitor U0126 on gene expression in the presence of E2 and OHT in the two cell lines. We tested the two cell lines, two ligands, E2 and OHT, and the ERK1/2 inhibitor U0126. Our primary grouping of the data was based on the effect of the S118A mutation on E2-ER mediated induction of the mRNAs.

The robust E2 induction of the ERE-containing genes Ap-2{gamma}, PDZK1, and complement component 3 (Fig. 3AGo) was significantly reduced in the HeLaER{alpha}S118A cells and was not further reduced when the HeLaER{alpha}S118A cells were treated with E2 + U0126 (Fig. 3AGo). For these genes, serine 118 therefore appears to represent the major site at which ERK/12 phosphorylation potentiates ER{alpha}-mediated transcription. The pS2 gene is intermediate in its response. In the HeLaER{alpha}S118A cells, induction of pS2 is reduced by approximately half.


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 3. The S118A mutation has diverse effects on ERE-containing genes. A and B, The symbols and ligand concentrations used in Figs. 2–4GoGoGo are: HeLaER{alpha} and HeLaER{alpha}S118A cells were maintained in medium containing ethanol (cross-hatched light lines) 10 nM E2 (black bars), 10 nM E2 + 10 µM U0126 (right diagonal dark lines), 10 nM OHT (open bars), 10 nM OHT + 10 µM U0126 (left diagonal dark lines). U0126 was added 1 h before E2 or OHT. After 24 h RNA was isolated and expression of the indicated mRNAs was determined by quantitative RT-PCR as described in Materials and Methods. A and B, Expression of each mRNA in the presence of ethanol was set equal to 1. The data represent the mean ± SEM for at least three independent experiments. For the four ERE-containing genes in A, comparing E2-stimulated mRNA levels in the HeLaER{alpha} and HeLaER{alpha}S118A cells, two-sided P values are less than 0.05 for Ap-2{gamma} and PDZK1 and less than 0.01 for pS2 and complement component 3. Comparing OHT-simulated mRNA levels for the mRNAs shown in A, two-sided P values are less than 0.05 for complement component 3 and pS2 and were greater than 0.05 for the other mRNAs. For all of the mRNAs in B, comparing mRNA levels in the HeLaER{alpha} and HeLaER{alpha}S118A cells in the presence of E2 or OHT, there was no statistically significant difference in mRNA induction between the HeLaER{alpha} and HeLaER{alpha}S118A cells (P > 0.1).

 
Keratin 13 and 19, prolactin, agiotensinogen, and ABCA3 are ERE-containing genes that were robustly induced by E2 in both HeLaER{alpha} cells and the HeLaER{alpha}S118A cells. The angiotensinogen and ABCA3 genes were unaffected by the S118A mutation or the presence of the ERK1/2 inhibitor U0126. Therefore, expression of these genes is not linked to the activation state of the ERK1/2 pathway. At the other extreme, E2 induction of prolactin mRNA was nearly identical in HeLaER{alpha} cells and HeLaER{alpha}S118A cells but was nearly abolished in cells treated with E2 and U0126. ERK1/2-mediated phosphorylation at sites other than S118 of ER{alpha} therefore plays an important role in E2 induction of prolactin mRNA. The U0126 data suggest that phosphorylation sites other than S118 may also play a role in induction of the two keratin mRNAs.

OHT partially induced keratin 13 and 19, prolactin, and angiotensinogen. The presence of the S118A mutation also had no effect on OHT induction (Fig. 3BGo). Because several studies suggest that tamoxifen and OHT enhance ER-mediated transcription primarily through AF-1 (21, 55), the absence of an effect of the S118A mutation in the AF-1 region on expression of these genes was surprising.

Genes activated by tethering of liganded ER to DNA show variable requirements for S118
In some genes, ER is brought to the DNA not by direct binding to DNA but by tethering through proteins bound SP1 and AP-1 sites or binding at ERE half-sites near SP1 sites. We identified only a few inducible genes known to use this mechanism, and the fold induction of these genes was small. CKB contains a Sp1 site in its promoter. Although cyclinD1 reportedly contains an AP-1 site, recent studies suggest a different region distant from the transcription start site is the site at which ER is tethered to the DNA (56). The S118A mutation did not interfere with E2 induction of CKB and abolished E2 induction of cyclinD1 (Fig. 4Go). The small set of test genes, the modest level of induction, and the fact that many of the DNA elements involved in tethering are not yet well defined all dictate caution in making general conclusions about the role of S118 in tethering.


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 4. The S118A mutation abolishes E2 induction of cyclin D1 and has no effect on the induction of CKB. The cells were treated as described in the legend to Fig. 2Go. The data represent the mean ± SEM for three separate experiments. Comparing E2 and OHT-stimulated mRNA levels in the HeLaER{alpha} and HeLaER{alpha}S118A cells, two sided P values were: cyclin D1, E2, P < 0.05; OHT, P < 0.05; for CKB there was no significant difference between the two cell lines (P > 0.1).

 
The S118A mutation interferes with down-regulation of gene expression by ER{alpha}
IL-6 is a well-characterized gene in which E2-ER strongly down-regulates gene expression. We therefore began these studies with IL-6. In HeLaER{alpha} cells, 24 h after addition of E2 or OHT to the medium, IL-6 mRNA levels are reduced by more than 10-fold. The ability of E2 and OHT to down-regulate IL-6 mRNA is completely lost in the HeLaER{alpha}S118A cells (Fig. 5AGo). These data led us to identify and analyze five additional mRNAs whose levels were down-regulated at least 2-fold by E2 in HeLaER{alpha} cells. E2 and OHT exhibit similar abilities to down-regulate DECR1 mRNA and this down-regulation was abolished in the HeLaER{alpha}S118A cells (Fig. 5AGo).


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		FIG. 5. The S118A mutation interferes with down-regulation of gene expression by ER{alpha}. A and B, The cells were maintained as described in the legend to Fig. 2Go. For each mRNA, the mRNA level in the ethanol control was set equal to 1 and the decline in mRNA level is plotted as fold change. After down-regulation, the PCR cycle number for each of the mRNAs was less than 40 (45 is the maximum with this instrument), enabling accurate measurement of the mRNA level. The data represent the mean ± SEM for three separate experiments. Comparing E2 and OHT-down-regulated mRNA levels in the HeLaER{alpha} and HeLaER{alpha}S118A cells, two sided P values were: IL-6, E2, P < 0.01, OHT, P < 0.01; DECR1, E2, P < 0.01, OHT, P = 0.1; EPOR, E2, P < 0.01, OHT, P < 0.01; DDIT4, E2, P < 0.05, OHT, P < 0.01; NOTCH3, E2, P < 0.05, OHT, P < 0.01; SMAD3 E2, P < 0.05, OHT, P > 0.1 (no significant difference).

 
A second subgroup of mRNAs, EPOR, DDIT4, and NOTCH3, is strongly down-regulated by E2 and partially down-regulated by OHT. For these genes, the S118A mutation interfered with the ability of E2 to down-regulate and nearly abolished down-regulation by OHT. Mothers against decapentaplegic (SMAD)-3 is down-regulated by E2 but not OHT. The S118A mutation had only a modest effect on SMAD3 mRNA levels. For four of the five genes whose expression is strongly down-regulated by E2, mutation of serine118 to alanine reduced the ability of E2-ER{alpha} to down-regulate gene expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To investigate the role of serine118 in ER action, we produced multiple stably transfected cell lines using a constitutive promoter and identified cell lines that expressed nearly identical levels of wild-type ER{alpha} and ER{alpha}S118A. These levels of ER{alpha} are similar to the level of ER{alpha} in MCF-7 cells. Because we were concerned about possible effects of the tet-VP16 protein on transcription, we used this more laborious approach rather than producing stable cell lines expressing tetracycline-regulated promoters.

Our studies and those of others establish that the DNA sequence to which E2-ER{alpha} is bound is important both for in vivo binding of E2-ER{alpha} to ERE-containing genes and for recruitment of proteins to the E2-ER{alpha} complex (12, 13, 14, 15, 16, 17, 18, 19). To evaluate the roles of serine118 and ERK activation in E2-ER{alpha}-mediated transcription, we used genes that contained both well-characterized EREs and exhibited robust induction by E2-ER{alpha} in our stably transfected HeLa cell lines. Ap-2{gamma}, PDZK1, complement component 3, and pS2 were strongly induced by E2-ER{alpha}. E2 induction of these genes was reduced by the S118A mutation and was not further reduced by the ERK1/2 inhibitor U0126. These data indicate that the S118 site represents the major site at which ERK1/2 enhances expression of these genes. However, the ERK1/2 inhibitor U0126 was less effective in reducing induction of these genes by E2 than the S118A mutation. It is possible that the basal level of phosphorylation of serine 118 is sufficient for activation of these genes and that U0126-sensitive activation of the ERK1/2 pathway is not required. Alternatively, other kinases including cyclin-dependent kinases that are not sensitive to inhibition by U0126 may participate in the phosphorylation of serine 118 (48). U0126 was quite effective in blocking the E2 induction of pS2 mRNA in the HeLaER{alpha} cells. This suggests that pS2 is either more sensitive to the level of S118 phosphorylation than the other genes or requires phosphorylation of both S118 and other sites for full induction.

E2 induction of a second group of ERE-containing genes including keratin 13 and 19, prolactin, angiotensinogen, and ABCA3 was unaffected by the S118A mutation. For prolactin and keratin 19, the S118A mutation had no effect on E2 induction, whereas the Erk/1/2 inhibitor strongly reduced induction. For these genes activation of the ERK pathway plays a significant role in expression, but serine 118 is not an important phosphorylation target of activated ERK1/2. E2 induction of angiotensinogen and ABCA3 was unaffected by the S118A mutation or the ERK1/2 inhibitor. The EREs in these genes must induce an ER conformation that allows full activation independent of S118 and of Erk/12 activation. For these genes other kinases and phosphorylation sites, such as serine167, may play important roles in gene expression.

Our data are consistent with the idea that the ERE sequence and the unique set of proteins associated with each gene elicits diverse conformations of E2-ER{alpha} that exhibit dramatically divergent requirements for phosphorylation of serine 118 and for Erk/12 activation.

An unexpected finding of these studies was the absence of a strong effect of the S118A mutation on OHT induction of mRNAs in ERE-containing genes. Tamoxifen and OHT are thought to activate gene transcription largely through the ligand-independent AF1 activation domain in the N-terminal A/B region of ER{alpha} (21). Whereas this might suggest a larger role for S118A in tamoxifen-ER activation of gene expression, the opposite was true. The Ap-2{gamma}, PDZK1, and complement component 3 were induced by E2 and to a lesser extent OHT. For each of these genes, the S118A mutation elicited a major reduction in the fold induction by E2 but had less effect on induction by OHT. The S118A mutation had little effect on OHT induction of any of the six genes induced at least 3-fold by OHT. The diverse effects of the S118A mutation on E2-ER{alpha} induction of different ERE-containing genes and the lack of effect of the S118A mutation on OHT-ER induction supports the view that there are important differences in the way that activating E2-ER{alpha} and OHT-ER{alpha} complexes are interpreted at the same DNA sites (19).

Although the number of genes whose expression is repressed by E2-ER{alpha} is roughly similar to the number of genes whose expression is induced (33), the sequence elements responsible for E2-ER{alpha} down-regulation of most genes remain largely undefined. Whereas repression of some genes by glucocorticoid receptor involves specific DNA elements termed negative glucocorticoid response elements that are distinct from activating glucocorticoid response elements (57), the limited data available to date suggest that ERE half-sites and SP1, AP-1, and nuclear factor-{kappa}B sites not obviously different from activating sites are important in down-regulation. Although a role for S118A in induction of gene expression by estrogens has been suggested, our observation that the S118A mutation nearly abolishes down-regulation of several genes by both E2 and OHT was unexpected. The much larger effect of the S118A mutation on E2-ER{alpha}-mediated repression of gene expression than on induction of gene expression suggests that a basal level of phosphorylation of serine 118 is required for most E2-ER{alpha} repression of gene expression. The diverse effects of the ERK inhibitor on the down-regulated genes must be interpreted with caution. ERK activation is known to stabilize IL-6 mRNA (58), and it is therefore not surprising that inhibiting the ERK pathway reduced the level of IL-6 mRNA in the S118A mutant cell line.

In striking contrast to the lack of an effect of the S118A mutation on ERE-dependent activation by OHT-ER, down-regulation of gene expression by OHT was nearly abolished by the S118A mutation. It is known that OHT-ER recruits corepressors to several genes (59, 60, 61, 62). Whereas the mechanisms responsible for repression of gene expression by E2 and OHT are only beginning to be identified, very recent studies of a few down-regulated genes indicate that recruitment of corepressors and histone deacetylases plays a central role (34). Our data suggest that phosphorylation of serine 118 may play a much more important role in the ability of OHT-ER to recruit corepressors than in its ability to recruit coactivators. Of course, mechanisms other than coregulator recruitment may well be important for down-regulation of some genes by ER.

Our data highlight the critical role of DNA sequence in interpreting information in the ligand-ER{alpha} complex and integrating signals based on ER phosphorylation by activated signal transduction pathways to achieve the diverse gene-specific outcomes important to execution of the ER transcription program. Although distinct repressor complexes are likely recruited at different genes down-regulated by ER{alpha}, phosphorylation of serine 118 of ER{alpha} appears to play an important role in the assembly of functional repression complexes.


   Footnotes
 
This work was supported by National Institutes of Health Grants CA90371, HD16720, and DKO71909.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 5, 2007

Abbreviations: AF-1, Activation function 1; AP, activator protein; CKB, creatine kinase B; E2, 17ß-estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; OHT, 4-hydroxytamoxifen; S118, serine 118; SERM, selective estrogen receptor modulator; SMAD, SMA and mothers against decapentaplegic; SP1, specificity protein-1.

Received February 1, 2007.

Accepted for publication June 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. O’Lone R, Frith MC, Karlsson EK, Hansen U 2004 Genomic targets of nuclear estrogen receptors. Mol Endocrinol 18:1859–1875[Abstract/Free Full Text]
  2. Qin C, Singh P, Safe S 1999 Transcriptional activation of insulin-like growth factor-binding protein-4 by 17ß-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes. Endocrinology 140:2501–2508[Abstract/Free Full Text]
  3. Safe S 2001 Transcriptional activation of genes by 17ß-estradiol through estrogen receptor-Sp1 interactions. Vitam Horm 62:231–252[Medline]
  4. Jakacka M, Ito M, Weiss J, Chien PY, Gehm BD, Jameson JL 2001 Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway. J Biol Chem 276:13615–13621[Abstract/Free Full Text]
  5. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P 2000 Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311–317[CrossRef][Medline]
  6. Pedram A, Razandi M, Levin ER 2006 Nature of functional estrogen receptors at the plasma membrane. Mol Endocrinol 20:1996–2009[Abstract/Free Full Text]
  7. Levin ER 2005 Integration of the extranuclear and nuclear actions of estrogen. Mol Endocrinol 19:1951–1959[Abstract/Free Full Text]
  8. Boonyaratanakornkit V, Edwards DP 2004 Receptor mechanisms of rapid extranuclear signalling initiated by steroid hormones. Essays Biochem 40:105–120[Medline]
  9. Wong CW, McNally C, Nickbarg E, Komm BS, Cheskis BJ 2002 Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. Proc Natl Acad Sci USA 99:14783–14788[Abstract/Free Full Text]
  10. Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141[Free Full Text]
  11. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474[CrossRef][Medline]
  12. Klinge CM, Jernigan SC, Mattingly KA, Risinger KE, Zhang J 2004 Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors {alpha} and ß by coactivators and corepressors. J Mol Endocrinol 33:387–410[Abstract/Free Full Text]
  13. Hall JM, McDonnell DP, Korach KS 2002 Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements. Mol Endocrinol 16:469–486[Abstract/Free Full Text]
  14. Klinge CM, Jernigan SC, Smith SL, Tyulmenkov VV, Kulakosky PC 2001 Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor {alpha}. Mol Cell Endocrinol 174:151–166[CrossRef][Medline]
  15. Loven MA, Likhite VS, Choi I, Nardulli AM 2001 Estrogen response elements alter coactivator recruitment through allosteric modulation of estrogen receptor ß conformation. J Biol Chem 276:45282–45288[Abstract/Free Full Text]
  16. Loven MA, Wood JR, Nardulli AM 2001 Interaction of estrogen receptors {alpha} and ß with estrogen response elements. Mol Cell Endocrinol 181:151–163[CrossRef][Medline]
  17. Wood JR, Greene GL, Nardulli AM 1998 Estrogen response elements function as allosteric modulators of estrogen receptor conformation. Mol Cell Biol 18:1927–1934[Abstract/Free Full Text]
  18. Wood JR, Likhite VS, Loven MA, Nardulli AM 2001 Allosteric modulation of estrogen receptor conformation by different estrogen response elements. Mol Endocrinol 15:1114–1126[Abstract/Free Full Text]
  19. Krieg AJ, Krieg SA, Ahn BS, Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. J Biol Chem 279:5025–5034[Abstract/Free Full Text]
  20. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract/Free Full Text]
  21. Berry M, Metzger D, Chambon P 1990 Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9:2811–2818[Medline]
  22. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
  23. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662[Abstract/Free Full Text]
  24. Ignar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLachlan JA 1993 Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element. Mol Endocrinol 7:992–998[Abstract/Free Full Text]
  25. Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174–2183[Medline]
  26. Jessop HL, Sjoberg M, Cheng MZ, Zaman G, Wheeler-Jones CP, Lanyon LE 2001 Mechanical strain and estrogen activate estrogen receptor {alpha} in bone cells. J Bone Miner Res 16:1045–1055[CrossRef][Medline]
  27. Ahamed S, Foster JS, Bukovsky A, Wimalasena J 2001 Signal transduction through the Ras/Erk pathway is essential for the mycoestrogen zearalenone-induced cell-cycle progression in MCF-7 cells. Mol Carcinog 30:88–98[CrossRef][Medline]
  28. Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 269:4458–4466[Abstract/Free Full Text]
  29. Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160[Medline]
  30. Joel PB, Traish AM, Lannigan DA 1995 Estradiol and phorbol ester cause phosphorylation of serine 118 in the human estrogen receptor. Mol Endocrinol 9:1041–1052[Abstract/Free Full Text]
  31. Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  32. Feng W, Webb P, Nguyen P, Liu X, Li J, Karin M, Kushner PJ 2001 Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through a serine 118-independent pathway. Mol Endocrinol 15:32–45[Abstract/Free Full Text]
  33. Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR, Katzenellenbogen BS 2003 Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144:4562–4574[CrossRef][Medline]
  34. Stossi F, Likhite VS, Katzenellenbogen JA, Katzenellenbogen BS 2006 Estrogen-occupied estrogen receptor represses cyclin G2 gene expression and recruits a repressor complex at the cyclin G2 promoter. J Biol Chem 281:16272–16278[Abstract/Free Full Text]
  35. Kalaitzidis D, Gilmore TD 2005 Transcription factor cross-talk: the estrogen receptor and NF-{kappa}B. Trends Endocrinol Metab 16:46–52[CrossRef][Medline]
  36. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, Brodsky AS, Keeton EK, Fertuck KC, Hall GF, Wang Q, Bekiranov S, Sementchenko V, Fox EA, Silver PA, Gingeras TR, Liu XS, Brown M 2006 Genome-wide analysis of estrogen receptor binding sites. Nat Genet 38:1289–1297[CrossRef][Medline]
  37. Arnold SF, Melamed M, Vorojeikina DP, Notides AC, Sasson S 1997 Estradiol-binding mechanism and binding capacity of the human estrogen receptor is regulated by tyrosine phosphorylation. Mol Endocrinol 11:48–53[Abstract/Free Full Text]
  38. Arnold SF, Vorojeikina DP, Notides AC 1995 Phosphorylation of tyrosine 537 on the human estrogen receptor is required for binding to an estrogen response element. J Biol Chem 270:30205–30212[Abstract/Free Full Text]
  39. Arnold SF, Obourn JD, Jaffe H, Notides AC 1995 Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine kinases in vitro. Mol Endocrinol 9:24–33[Abstract/Free Full Text]
  40. Auricchio F, Migliaccio A, Castoria G, Di Domenico M, Pagano M 1990 Phosphorylation of uterus estradiol receptor on tyrosine. Prog Clin Biol Res 322:133–155[Medline]
  41. Auricchio F, Migliaccio A, Castoria G, Rotondi A, Di Domenico M, Pagano M, Nola E 1988 Phosphorylation of estradiol receptor on tyrosine and interaction of estradiol and glucocorticoid receptors with antiphosphotyrosine antibodies. Adv Exp Med Biol 231:519–540[Medline]
  42. Lannigan DA 2003 Estrogen receptor phosphorylation. Steroids 68:1–9[CrossRef][Medline]
  43. Denton RR, Koszewski NJ, Notides AC 1992 Estrogen receptor phosphorylation. Hormonal dependence and consequence on specific DNA binding. J Biol Chem 267:7263–7268[Abstract/Free Full Text]
  44. Aronica SM, Katzenellenbogen BS 1993 Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I. Mol Endocrinol 7:743–752[Abstract/Free Full Text]
  45. Washburn T, Hocutt A, Brautigan DL, Korach KS 1991 Uterine estrogen receptor in vivo: phosphorylation of nuclear specific forms on serine residues. Mol Endocrinol 5:235–242[Abstract/Free Full Text]
  46. Lahooti H, White R, Danielian PS, Parker MG 1994 Characterization of ligand-dependent phosphorylation of the estrogen receptor. Mol Endocrinol 8:182–188[Abstract/Free Full Text]
  47. Lahooti H, White R, Hoare SA, Rahman D, Pappin DJ, Parker MG 1995 Identification of phosphorylation sites in the mouse oestrogen receptor. J Steroid Biochem Mol Biol 55:305–313[CrossRef][Medline]
  48. Chen D, Washbrook E, Sarwar N, Bates GJ, Pace PE, Thirunuvakkarasu V, Taylor J, Epstein RJ, Fuller-Pace FV, Egly JM, Coombes RC, Ali S 2002 Phosphorylation of human estrogen receptor {alpha} at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene 21:4921–4931[CrossRef][Medline]
  49. Joel PB, Smith J, Sturgill TW, Fisher TL, Blenis J, Lannigan DA 1998 pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167. Mol Cell Biol 18:1978–1984[Abstract/Free Full Text]
  50. Kuntz MA, Shapiro DJ 1997 Dimerizing the estrogen receptor DNA binding domain enhances binding to estrogen response elements. J Biol Chem 272:27949–27956[Abstract/Free Full Text]
  51. Zhang CC, Krieg S, Shapiro DJ 1999 HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells. Mol Endocrinol 13:632–643[Abstract/Free Full Text]
  52. Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid detection of octamer binding proteins with ‘mini-extracts,’ prepared from a small number of cells. Nucleic Acids Res 17:6419[Free Full Text]
  53. Dauvois S, Danielian PS, White R, Parker MG 1992 Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89:4037–4041[Abstract/Free Full Text]
  54. Devin-Leclerc J, Meng X, Delahaye F, Leclerc P, Baulieu EE, Catelli MG 1998 Interaction and dissociation by ligands of estrogen receptor and Hsp90: the antiestrogen RU 58668 induces a protein synthesis-dependent clustering of the receptor in the cytoplasm. Mol Endocrinol 12:842–854[Abstract/Free Full Text]
  55. Liu Z, Shi HY, Nawaz Z, Zhang M 2004 Tamoxifen induces the expression of maspin through estrogen receptor-{alpha}. Cancer Lett 209:55–65[CrossRef][Medline]
  56. Eeckhoute J, Carroll JS, Geistlinger TR, Torres-Arzayus MI, Brown M 2006 A cell-type-specific transcriptional network required for estrogen regulation of cyclin D1 and cell cycle progression in breast cancer. Genes Dev 20:2513–2526[Abstract/Free Full Text]
  57. Schoneveld OJ, Gaemers IC, Lamers WH 2004 Mechanisms of glucocorticoid signalling. Biochim Biophys Acta 1680:114–128[Medline]
  58. Andoh A, Shimada M, Bamba S, Okuno T, Araki Y, Fujiyama Y, Bamba T 2002 Extracellular signal-regulated kinases 1 and 2 participate in interleukin-17 plus tumor necrosis factor-{alpha}-induced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts. Biochim Biophys Acta 1591:69–74[Medline]
  59. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657–666[Abstract/Free Full Text]
  60. Shang Y, Brown M 2002 Molecular determinants for the tissue specificity of SERMs. Science 295:2465–2468[Abstract/Free Full Text]
  61. Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz KB 1997 The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11:693–705[Abstract/Free Full Text]
  62. Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol Cell Biol 18:1369–1378[Abstract/Free Full Text]
  63. Flavin KM 2005 Functional analysis of the 15 KH domain human nucleic acid binding protein vigilin. Ph.D. Thesis, University of Illinois, Urbana, IL



This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
G. Giamas, L. Castellano, Q. Feng, U. Knippschild, J. Jacob, R. S. Thomas, R. C. Coombes, C. L. Smith, L. R. Jiao, and J. Stebbing
CK1{delta} modulates the transcriptional activity of ER{alpha} via AIB1 in an estrogen-dependent manner and regulates ER{alpha}-AIB1 interactions
Nucleic Acids Res., May 1, 2009; 37(9): 3110 - 3123.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
M. Zoubir, M. C. Mathieu, C. Mazouni, C. Liedtke, L. Corley, S. Geha, J. Bouaziz, M. Spielmann, F. Drusche, W. F. Symmans, et al.
Modulation of ER phosphorylation on serine 118 by endocrine therapy: a new surrogate marker for efficacy
Ann. Onc., August 1, 2008; 19(8): 1402 - 1406.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, J.
Right arrow Articles by Shapiro, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, J.
Right arrow Articles by Shapiro, D. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals