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. Author manuscript; available in PMC: 2013 Jul 6.
Published in final edited form as: Mol Cell Endocrinol. 2012 Feb 19;358(1):27–35. doi: 10.1016/j.mce.2012.02.012

Phosphorylation of human estrogen receptor-beta at serine 105 inhibits breast cancer cell migration and invasion

Hung-Ming Lam a, CV Suresh Babu a, Jiang Wang b, Yong Yuan a, Ying-Wai Lam a,c, Shuk-Mei Ho a,c,d,e,*, Yuet-Kin Leung a,c,d,*
PMCID: PMC3348253  NIHMSID: NIHMS361724  PMID: 22370157

Abstract

Multiple phosphorylation sites on the human estrogen receptor (hER)α were identified and shown to influence mammary carcinogenesis. In contrast, functional phosphorylation sites of hERβ have yet to be experimentally identified and validated. Here, using mass spectrometry, we uncovered three serines (S75, S87, and S105) in the N-terminus of hERβ as targets of ERK1/2 and p38 kinases. We raised a specific antibody against phosphorylated S105 (pS105) and demonstrated that this site was endogenously phosphorylated in MDA-MB-231 and BT-474 cells. A phospho-mimetic mutant generated from hERβ1 was found to exhibit higher transactivation activity than hERβ1. Ectopic expression of this mutant inhibited cell migration and invasion, but did not affect cell growth and cell-cycle progression in these cell models. In breast cancer specimens, pS105-hERβ immunoreactivity was detected with a higher prevalence and intensity than that of hERβ1. These results underscore the functional importance of the first experimentally identified hERβ-phosphorylation site in breast cancer.

Keywords: phosphorylation, estrogen receptor-beta isoforms, phospho-mimetics, breast cancer, invasion, migration, post-translational modification, mass spectrometry, ERK1/2, p38

1. Introduction

Disruption of hormonal balance has been implicated in breast cancers (BCa), the most common malignancies among women (American Cancer Society, 2010). Estrogen was first discovered as a key factor for the growth of BCa when bilateral oophorectomy was found to result in remission of BCa in a premenopausal woman (Beatson, 1896). In women, a higher incidence of BCa has been linked to higher serum and tissue levels of estrogen (Lamar et al., 2003) and a longer lifetime exposure to estrogen (Paffenbarger, Jr. et al., 1980). Estradiol-17 beta (E2) is believed to trigger nuclear signaling primarily via two estrogen receptors (ERs), ERα and ERβ1 (traditionally referred to as ERβ), in human cancers (Leygue et al., 1998; Matthews and Gustafsson, 2003). These two receptors share many of the same ligands but have distinct and diverse cellular functions. In BCa, abnormal growth was found to be driven by ERα (Gaben et al., 2004; Lu and Serrero, 2001) but curtailed by ERβ1 (Koehler et al., 2005; Williams et al., 2008). Thus, ERβ1 appears to function as a tumor suppressor in BCa. This view is further supported by evidence reporting a loss of ERβ1 expression during BCa progression (Leygue et al., 1998).

Post-translational modifications (PTMs) are crucial events in the activation of ERs (Faus and Haendler, 2006; Lannigan, 2003; Le et al., 2011). Phosphorylation is the most extensively studied PTM, partly because of its relatively frequent occurrence and stability (Faus and Haendler, 2006). Studies of human ERα have shown that phosphorylation mediates both ligand-and growth factor-initiated genomic and non-genomic action of the receptor (Lannigan, 2003; Le et al., 2011). Specific phosphorylation sites were identified primarily in the activation function-1 (AF-1) located in the N-terminus of the receptor (Atsriku et al., 2009). Phosphorylation at serine (S) sites, in particular, has been reported to alter protein-protein interaction, subcellular localization, transactivation, and the stability of the human ERα (Lannigan, 2003; Le et al., 2011). Modulation of cancer cell proliferation due to phosphorylation of a specific serine in the ERα has recently been reported (Gburcik and Picard, 2006; Tharakan et al., 2008), and phosphorylation of ERα at various serine sites is currently being evaluated for the classification of BCas (Murphy et al., 2009; Murphy et al., 2006; Skliris et al., 2009).

Analogous information on human ERβ1 is still unavailable because, until now, no phosphorylation sites on the receptor have been experimentally identified and demonstrated to be functional in BCa cells. Our knowledge of human ERβ phosphorylation is derived primarily through studies of the mouse ERβ (Tremblay et al., 1999; Tremblay et al., 1997). For example, information from the mouse receptor was used to predict and subsequently validated that S87 on the human ERβ is a functional phosphorylation site under the regulation of stromal cell-derived factor 1 (or chemokine C-X-C motif ligand 12) in BCa cells (Sauve et al., 2009). Since the number of predicted kinase-specific motifs differs in humans and mice and the AF-1 domain in humans is shorter than that in mice, not all phosphorylation sites in human ERβ1 can be predicted from mouse ERβ studies. Therefore, de novo identification of phosphorylation sites on human ERβ is imperative for filling the data gap concerning the role of this PTM in regulating the function of the human receptor.

To this end, in the present study, we identified three serine phosphorylation sites (S75, S87, and S105) localized in the N-terminus of the full-length human ERβ1 using high-accuracy mass spectrometry (MS). Using a newly raised in-house phospho-specific S105 antibody, we showed the PTM to be mediated by E2-induced ERK1/2 activation or osmotic stress-induced p38 activation in BCa cell-line MDA-MB-231 (ERα-negative) and BT-474 (ERα-positive). Use of the phospho-mimetic mutant S105E and the phospho-defective mutant S105A further revealed that pS105 in ERβ1 enhances its ability to inhibit cell migration and invasion in these cancer cell-line models. Immunohistochemistry (IHC) analyses demonstrated wide-spread S105-phosphorylation (pS105) positivity in BCa specimens. In total, this study identified the first functional phosphorylation site (S105) of the human ERβ1.

2. Materials and Methods

2.1. Breast cancer specimens

Twenty-five formalin-fixed, paraffin-embedded BCa sections were obtained from an archival collection in the Pathology Department at the University of Cincinnati Medical School. All specimens were graded by Dr. J. Wang and his colleagues on the basis of representative hematoxylin and eosin (H&E)-stained sections. The use of the specimens was reviewed and approved by the University’s IRB committee.

2.2. In vitro kinase assay

All kinase buffers were prepared according to the manufacturers’ instructions. Active recombinant ERK1/2 (#14-439 for ERK1; #14-550 for ERK2), p38 (#14-251), Src (#14-326) and PKA (#14-440) were purchased from Millipore (Billerica, MA). Kinases were incubated with full-length recombinant ERβ1 (Invitrogen, Carlsbad, CA) or myelin basic protein (MBP, positive control, #13-104, Millipore) along with Mg2+/adenosine triphosphate (ATP) solution and 5 µCi (γ-P32) ATP (Perkin Elmer, Waltham, MA) at 30°C for 20 min. After termination of in vitro phosphorylation reactions, phosphorylated ERβ1 was resolved by SDS/PAGE and detected by autoradiography.

2.3. Phosphorylation-site identification by mass spectrometry

Tryptic digestion was performed as described previously (Lam et al., 2008; Lam et al., 2010). Each digest was analyzed by capillary LC-MS/MS with a Finnigan LTQ-Orbitrap (Thermo Fisher Scientific, MA). Half of the digest was loaded directly onto the 75 µm × 100 mm PicoFrit capillary column (New Objective, MA) packed with MAGIC C18 (100 Å, 5 µm, Michrom Bioresources, CA) at a flow rate of 300 nL/min, and peptides were separated by a gradient comprising 2–60% acetonitrile (ACN)/0.1% formic acid (FA) in 30 min, 60–98% ACN/0.1% FA in 5 min, and held at 98% ACN/0.1% FA for 5 min. The LTQ-Orbitrap was operated in standard data-dependent ‘top-three’ mode with lock mass function activated (protonated polydimethylcyclosiloxane [Si(CH3)2O))6; m/z 445.120025)] to enable accurate mass measurement (< 2 ppm) of the precursor ions. A survey scan from m/z 300–1600 at 60,000 resolution in the Orbitrap was paralleled by 3 MS/ MS scans in the LTQ. Phosphopeptides were detected by neutral loss scanning (−H3PO4) of 98, 49, 32.7 amu for 1+, 2+ or 3+ charged precursor ions respectively and consecutive MS3 scans. Multistage activation was also utilized in parallel experiments to confirm the sites of modification. The product ion spectra were searched against the human subset of the International Protein Index (IPI) database (ipi.HUMAN.v3.19) using the SEQUEST search engine in Bioworks 3.3. The database was indexed with fully enzymatic activity and two missed cleavage sites allowed for trypsin. Searching criteria included a mass tolerance of 15 ppm and 1 amu for precursor and fragment ions and variable modifications set on methionine (oxidized methionine: +15.9949), cysteine (carboxyamidomethylated cysteine: +57.0215), and phosphorylated serine/threonine (HPO3: +79.9663). In addition, phosphorylated serines/threonines were pinpointed in the MS3 by the loss of water (−H2O, −18). Peptides were filtered according to criteria: XCorr: 1.5 (1+), 2.0 (2+), 2.5(3+)]; DelCN: > 0.1, SP: >/= 300. The MS/MS spectra of the identified phospho-peptides were evaluated manually.

2.4. Generation and characterization of the anti-pS105-ERβ antibody and its utilization for immunohistochemical (IHC) and immunoblotting studies

Rabbit polyclonal monospecific antibody against the phosphoserine at 105 on human ERβ (pS105-ERβ) was commercially generated from immunizing New Zealand White rabbits with a KLH-conjugated peptide (CAEPQK*SPW), and then affinity-purified by phosphorylated peptide-conjugated column (affinity-purified package, New England Peptide, Gardner, MA). Its specificity was tested based on our published protocols (Leung et al., 2010). Briefly, pS105-ERβ (1:300) was incubated with 10× excess (by weight) of S105 peptide or 105 peptide at 4°C overnight. Antibody-peptide complex was centrifuged at 14,000 ×g for 10 min and the supernatant was used for immunohistochemical (IHC) analyses. The antibody was used for of expression of the phosphorylated receptor in formalin-fixed and paraffin-embedded archival BCa sections (n=25), according to our previously published protocol (Leav et al., 2001). Histological evaluation and IHC scoring were performed by Dr. J. Wang, a board-certified clinical pathologist, and his associate in a blinded-manner. The Allred score (Allred et al., 1993; Leung et al., 2010) was used to quantify pS105-ERβ positivity. In parallel, the ERβ1-specific antibody, GC17 (Leav et al., 2001), was used to quantify the ERβ1 expression. Pictures were captured using AxioCam MRm camera and Axiovision 4.7 software (Carl Zeiss, Thornwood, NY, USA). For immunoblotting analysis, pS105-ERβ (1:1000) was incubated with 200× molar excess of S105 peptide or 105 peptide at 4°C overnight. Antibody-peptide complex was centrifuged at 14,000 ×g for 10 min and the supernatant was used for immunoblotting.

2.5. Cell culture and transfection

HEK293 and MDA-MB-231 were purchased from American Type Culture Collection (ATCC, Manassas, VA). BT-474 was a generous gift from Dr. Nira Ben-Jonathan (University of Cincinnati, OH). HEK293 cells was cultured in ATCC-recommended medium supplemented with 10% FBS (recognized as complete medium); MDA-MB-231 was cultured in MEMalpha medium (Invitrogen) supplemented with 10% FBS and 2 mM L-glutamine (recognized as complete medium); BT-474 was cultured in DMEM medium (ATCC) supplemented with 10% FBS. Wild-type (pDEST40-ERβ1), mutants (pDEST40-ERβ1-S105A, pDEST40-ERβ1–S105E), or control vector (pDEST40) was transfected into each cell line by the following methods. For MDA-MB-231 and BT-474 cells, 5 µg of vector was incubated with 20 µl of FuGENE HD transfection reagent (Roche Diagnostics, Indianapolis, IN). For HEK293 cells, 3 µg of vector was mixed with 20 µl of lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA). After 24 h of transfection, the cells were replated for other assays.

2.6. In cellulo induction of receptor phosphorylation in cell model systems and quantification by Western blot analysis

MDA-MB-231 cells and BT-474 cells cultured in phenol-red and serum-free DMEM for 24 h were treated for 15 min (MBA-MB-231) or 30 min (BT-474) with 10 nM E2, an ERK1/2 inducer or 0.7 M NaCl, a p38 inducer. PD 98059 (25 µM, EMD chemicals, Gibbstown, NJ) and SB 203580 (10 µM, EMD chemicals) were used to block ERK1/2- and p38-induced phosphorylation, respectively. These inhibitors were added 30 min before E2 or NaCl treatment. The treated cells were collected and 100 µg of protein extract was analyzed by immunoblotting experiments. The following antibody dilutions were used: 1:200 for in-house anti-pS105-ERβ; 1:200 for anti-ERβ (H-150, Santa Cruz Biotechnology Inc., Santa Cruz, CA), 1:1000 for anti-pERK1/2, anti-ERK1/2, anti-p38 (Cell Signaling Technology, Danvers, MA); 1:5000 for anti-β-actin (Abcam); 1:10,000 for IRD800 dye-conjugated donkey anti-mouse or donkey anti-rabbit antibodies (Rockland Immunochemicals, Gilbertsville, PA). The signals were detected by Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE). β-actin served as loading controls, and phosphorylated and unphosphorylated forms of a kinase were positive controls of the kinase action.

2.7. Site-directed mutagenesis

Site-directed mutagenesis was used to prepare a phospho-defective mutant S105A (ERβ1-S105A), in which serine was changed to an alanine, and a phospho-mimetic mutant S105E (ERβ1-S105E), in which serine was replaced with a glutamate. The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used with pENTR-ERβ1 as the DNA template and complementary pairs of primers with S105A or S105E mutation: ERβ1-S105A-F: 5’-gta tgc gga acc tca aaa ggc tcc ctg gtg tga agc-3′; ERβ1-S105A-R: 5′- gct tca cac cag gga gcc ttt tga ggt tcc gca tac -3′; ERβ1-S105E-F: 5′-ctg tat gcg gaa cct caa aag gag ccc tgg tgt gaa gca aga tc-3'; ERβ1-S105E-R: 5′-gat ctt gct tca cac cag ggc tcc ttt tga ggt tcc gca tac ag-3′. The sequence identities of mutants were verified by sequencing (Macrogen, Seoul, Korea). The mutant vectors were subcloned into a mammalian expression vector, pcDNA-DEST40, by Gateway LR Clonase II recombination reaction (Invitrogen, Carlsbad, CA). The resulting vectors were designated as pDEST40-ERβ1-S105A and pDEST40-ERβ1-S105E, respectively.

2.8. ERE Reporter assay

ERβ1 transactivation activity was performed according to our previously published method (Leung et al., 2006). Cells were transfected with 300 ng of control vector or ERβ1 plasmids, together with 150 ng of ERE-luciferase reporter plasmid and 100 ng of β-galactosidase plasmid. E2 (1nM) or MPP dihydrochloride (Tocris Bioscience, Ellisville, MO, 10 µM) was added after 24 h, and luciferase activity was measure after another 24 h. Control was added with 0.01% DMSO. Transactivation activity was performed with Bright Glo Luciferase assay kit (Promega) by using Wallac 1420 Vector2 Multilabel counter (PerkinElmer), and normalized by β-galactosidase activity (β-galactosidase enzyme assay kit; Promega). Results were presented as ‘relative light units’.

2.9. Cell viability assay and cell-cycle analysis

ERβ1, S105A, or S105E vectors transfected cells were allowed to grow for 3 days in the complete medium and cell viability was assessed by CellTiter 96 AQueous Non-Radioactive Cell Proliferation MTS assay (Promega) according to the manufacturer’s recommended protocol. Cell cycle analysis was carried out by transfecting cells with plasmids and harvesting them after 72 h. Cells were fixed, stained with PI, and subjected to flow cytometry analysis of DNA content as described previously (Chan et al., 2010).

2.10. Cell migration and invasion assays

For cell migration, MDA-MB-231 and BT-474 cells were transfected with ERβ1-, ERβ1-S105A-, and ERβ1-S105E-transfected cells as mentioned before. A scratch was made on a confluent layer of cells in the complete medium. Distance of migration was calculated by half of the difference between the width of the wound 0 h and 48 h after scratching. Cell growth was monitored by MTS assay stated in section 2.9 above. For cell invasion, transfected cells were seeded in serum-free medium in the upper chamber and cells invaded to the complete medium in the lower chamber were fixed, stained, and counted after 24 h as described previously (Leung et al., 2010).

2.11. Statistical Analysis

One-way ANOVA was used to compare means among experimental groups.

3. Results

3.1. Identification of S75, S87, and S105 as ERK1/2- and p38-susceptible phosphorylation sites of human ER β1

In in vitro assays, in the presence of phosphate donor ATP, recombinant human ERβ1 was shown to be phosphorylated by ERK1/2 (phosphorylation results are similar for ERK1 and ERK2, thus ERK1 was presented and used in subsequent experiments) and p38, and weakly phosphorylated by PKA and Src (Fig. 1A). MS analysis and site mapping (sequence coverage of ERβ1: 44%) following in vitro phosphorylation of the recombinant human ERβ1 by the various kinases revealed S75 (QTTpSPNVLWPTPGHLSPLVVHR), S87 (QTTSPNVLWPTPGHLpSPLVVHR), and S105 (QLSHLYAEPQKpSPWCEAR; Fig. 1B) as common ERK1 and p38 phosphorylation sites. All three sites are in the N-terminus of the receptor. Accurate mass measurement of the phosphopeptide containing S105 demonstrated a mass deviation of 1.5 ppm of the theoretical mass (Fig. 1B, upper panel). In addition, MS2 exhibits a continuous b- and y- ion series covering the pS105. Because MS2 analyses of phosphopeptides are often accompanied by a loss of phospho-group (neutral loss), leading to the limited fragmentation, we used a consecutive neutral loss-triggered MS3 to confirm the identity of the peptide (Fig. 1B, lower panel). To our satisfaction, database searching of both MS2 and MS3 spectra revealed the same match (QLSHLYAEPQKpSPWCEAR). MS2 (with or without multistage activation) gave enough fragmentation to identify both S75 and S87 as phosphorylation sites, thus a MS3 analysis was deemed unnecessary. All peptide masses measured were within 10 ppm of the theoretical masses. Owing to the fact that both PKA and Src did not effectively phosphorylate ERβ, the present study did not follow up with these two kinases.

Figure 1. Discovery of ERβ phosphorylation sites from in vitro phosphorylation by mass spectrometry.

Figure 1

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(A) Phosphorylation of recombinant ERβ by recombinant kinases (ERK1/2, p38, Src and PKA in the presence of ATP in vitro. Myelin basic protein (MBP) was used as a positive control. P-ERβ: phospho-ERβ, p-MBP: phospho-MBP. (B) Upper panel, MS2 spectrum of the tryptic peptide containing pS105 (QLSHLYAEPQKpSPWCEAR). A continuous b- and y-ion series was observed in the MS2. Accurate mass measurement of the precursor (FT-MS) demonstrated a small mass deviation to the theoretical mass (<2 ppm). Lower panel, Confirmation of ERβ peptide identity in a consecutive neutral loss-triggered MS3. Database searching of MS3 spectrum revealed the same match (QLSHLYAEPQKpSPWCEAR), with a loss of H2O on S105.

3.2. Antibody validation and induction of pS105 in ERβ by ERK1/2 and p38 signaling

We successfully raised a specific antibody to pS105-ERβ. We validated its specificity by immunohistochemistry (IHC, Fig. 2A) and immunoblotting (IB, Fig. 2B), in which the positive signal was abrogated by phosphorylated peptide but not unphosphorylated peptide against serine 105 site of human ERβ. Immunoblotting result further showed that recombinant ERβ was not able to be phosphorylated in the absence of the substrate ATP, despite the presence of an active recombinant ERK1 (Fig. 2B). The availability of this new antibody allows us to study the function of S105.

Figure 2. Validation of pS105-ERβ antibody and detection of human ERβ S105 phosphorylation in vitro and in cellulo.

Figure 2

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(A) Evaluation of pS105 antibody specificity in a breast specimen by immunohistochemistry. Immunopositive staining of pS105-ERβ in the nuclei and cytoplasm of epithelial cells can be abolished by overnight incubation with a phosphorylated peptide (p105 peptide, CAEPQK*SPW) but not with an unphosphorylated peptide (105 peptide, CAEPQKSPW) flanking the serine 105 site of human ERβ. (B) Evaluation of pS105 antibody specificity by immunoblotting assay. The pS105-specific band was only detected upon addition of ERK1 and ATP (+ATP) in the in vitro phosphorylation experiment using human recombinant ERβ1. The band was abolished when the antibody was pre-incubated with the phosphorylated 105 peptide but not with the unphosphorylated 105 peptide. Signals from panERβ antibody were used as a protein loading control. (C) In vitro phosphorylation of a recombinant ERβ1 with ERK1 and p38 in the absence (−ATP) or presence (+ATP) of ATP. Only the reaction mixture containing ATP yielded a single band with the correct size in immunoblotting analyses. (D) Detection of endogenous pS105 in (left) MDA-MB-231 and (right) BT-474 cells upon ERK1/2 activation by 10nM E2, and p38 activation by 0.7M NaCl. PD: PD 98059, a MEK inhibitor. SB: SB 203580, a p38 inhibitor. β-actin is a protein loading control. Representative pictures are shown from two to three independent experiments of similar results.

To confirm that S105 is a target site for ERK1/2 and p38, we treated a recombinant ERβ1 with the respective kinase and observed a single band with the correct size only when the phosphate donor ATP was present (Fig. 2C). To test whether pS105 occurs in cellulo (Fig. 2D), we stimulated MDA-MB-231 (ERα-negative) and BT-474 (ERα-positive) cells, both express endogenous ERβ, with E2 to activate ERK1/2 (Henson and Gibson, 2006; Kelly and Levin, 2001) or with 0.7 M NaCl to activate p38 (Meriin et al., 1999). Both E2 and NaCl did not change the protein level of basal ERβ but increased the level of phosphorylated ERβ at S105 (pS105-ERβ) in both cell lines. Such phosphorylation events were abolished by a MEK1 inhibitor, PD 98059 (Fig. 2D, upper panel), or by a p38 inhibitor, SB 203580 (Fig. 2D, lower panel). Collectively, these data indicate that S105 on human ERβ is a target of ERK1/2 and p38.

3.3. Phospho-mimetic ERβ1-S105E enhanced ligand-dependent transactivation

To determine if pS105 affects the transactivation of ERβ1, we first compared transcriptional activities of the wild-type receptor and its phospho-mimetic (ERβ1-S105E) or phospho-defective (ERβ1-S105A) mutants in the HEK293 cell background using an ERE-luciferase reporter. In the presence of E2, ERβ1-S105E exhibited significantly higher transactivation activity than did ERβ1 and ERβ1-S105A (Fig. 3A). ERβ1-S105E inhibited ERα transactivation in a dose-dependent manner (Supplementary Fig. S1). In the absence of ligand, the mutants exhibited transactivation activities that were very low but similar to those of the wild-type ERβ1 (Fig. 3A). In a BCa-cell background, the phospho-mimetic mutant ERβ1-S105E showed an enhanced E2-induced transactivation in both MDA-MB-231 (ERα-negative) and BT-474 (ERα-positive) cells when compared with wild-type ERβ1 (Fig. 3B and C). Expectedly, the phospho-defective mutant ERβ1-S105A induced a significantly weaker transactivation when compared with that of wild-type ERβ1 following E2-stimulation (Fig. 3B). Similar result was obtained when the cells were treated with DPN (Diarylpropionitrile, an ERβ-specific agonist), except that DPN did not trigger the transactivation in the control vector-transfected cells when compared with the results treated with E2 (Supplementary Fig. S2). The E2-induced transactivation in control vector-transfected BT-474 cells was due to endogenous ERα, which can be blocked by MPP dihydrochloride (an ERα-specific antagonist, data not shown). Blockade of ERα caused cell death, so it was not applied to subsequent cell studies. These data indicate that pS105 can augment the ligand-induced transactivation activity of ERβ1 in both ERα-positive or negative BCa cell background and inhibit the transactivation of ERα.

Figure 3. Ligand-dependent ERE transactivation of ERβ.

Figure 3

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ERβ1 wild-type or mutants (S105A and S105E) were transfected in (A) HEK-293, (B) MDA-MB-231, and (C) BT-474 cells and S105E enhanced the ligand-dependent transactivation of ERE in both cell lines. Ctl: 0.1% DMSO; E2: Estradiol-17 beta. Values are the average of triplicate samples from an independent experiment that was repeated thrice with similar results. Mean ± SD, *p<0.05 compared with ERβ1 with E2 treatment.

3.4. Activation of ERβ1-S105E inhibited cell migration and invasion but not cell growth/cycle in MDA-MB-231 and BT-474 cells

To elucidate the functional roles of S105 in ERβ, we transfected MDA-MB-231 and BT-474 cells with wild-type ERβ1, ERβ1-S105E, or ERβ1-S105A plasmid. Ectopic expression of the wild-type ERβ1 and its mutants in MDA-MB-231 and BT-474 cells did not alter cell growth (Fig. 4A) or cell-cycle progression (Fig. 4B). Ectopic expression of ERβ1 inhibited cell invasion in MDA-MB-231 but not BT-474 cells, whereas transgenic expression of phospho-mimetic mutant ERβ1-S105E induced a drastic suppression of both cell migration and cell invasion in both cell lines (Fig. 4C and D, p<0.01 compared with the control vector-transfected cells). Transgenic expression of ERβ1-S105A did not inhibit migration or invasion when compared with the control vector-transfected cells.

Figure 4. S105E inhibited cell migration and invasion but not growth or cell cycle in MDA-MB-231 and BT-474 cells.

Figure 4

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(A) S105E did not affect the cell growth by MTS assay. Control vector (Ctl vec) was used as a negative control. Cell viability was compared with that of Ctl vec. Values are mean ± SD of three independent experiments, triplicate for each experiment. (B) S105E did not affect the cell cycle in flow-cytometry analysis. Values are mean ± SD of three independent experiments. (C) S105E inhibited cell migration in the wound-healing assay. (D) S105E inhibited cell invasion in the Matrigel transwell assay. Values are mean ± SD of three independent experiments for (C) and (D), * p<0.05, **p< 0.01 compared with Ctl vec.

3.5. Detection of endogenous Serine-105 phosphorylation of ERβ in human BCa specimens

Since S105 resides in the N-terminus of ERβ, our anti-pS105 antibody recognizes wild-type ERβ1 as well as its isoforms, which only differ from ERβ1 at their C-termini (Leung et al., 2006). To gain insight of the potential role of pS105 in BCa when compared with ERβ1 that has been widely shown to be anti-proliferative (Warner and Gustafsson, 2010), we performed IHC with GC17 (an ERβ1-specific antibody) and our pS105-antibody.

In 25 BCa specimens, pS105 signal was detected in benign duct (myoepithelial and luminal secretory cells, Fig. 5A), benign lobule (Fig. 5B), ductal carcinoma in situ (DCIS, Fig. 5C) and invasive cancer (Fig. 5D). Nuclear staining was stronger than cytoplasmic staining in both benign luminal epithelial (p=0.1) and cancer cells (p<0.01, Table 1A). Occasional negative staining was detected regardless of the clinical classifications (Fig. 5A–D, red dotted arrows). Nuclear and cytoplasmic pS105-staining in cancer (Grade I/II and III) and their adjacent benign glands showed similar positivity (Table 1A). Importantly, pS105-staining was not correlated with ERα positivity or TNM staging (data not shown). Unlike the wide-spread pS105 positivity in invasive BCas (Fig. 5D), both nuclear and cytoplasmic ERβ1 signals were significantly diminished in invasive BCas as compared with signals in benign glands regardless of cancer grade, ERα positivity, and TNM staging (Table 1B, p<0.01, Fig. 5I and J).

Figure 5. Representative immunohistochemical staining of pS105-ERβ in human breast specimens.

Figure 5

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Positive pS105-ERβ staining was detected in both nuclei (stronger) and cytoplasm (weaker) of (A) luminal and myoepithelial cells in the benign duct, (B) epithelial cells in the benign lobule, cancer cells in (C) ductal carcinoma in situ (DCIS) and (D) invasive cancer. Red dotted arrow indicated unstained cells. ERβ1 was found in the nuclei and cytoplasm of cells in (E) normal lobules and weak ERβ1 staining was detected in (F) invasive cancer. Magnification: 200×, scale: 100 µm.

Table 1.

Quantification of (A) pS105-ERβ and (B) ERβ1 (GC17) immunostaining in the human breast specimens.

A. pS105-ERβ staining
Breast (n=25)
Benign Cancer Gr I/II Gr III
Nucleus 7.0±0.2 7.0±0.1a 7.0±0.1 7.1±0.1
Cytoplasm 6.5±0.3 6.6±0.1 7.0±0.1 6.3±0.1
B. ERβ1 (GC17) staining
Breast (n=25)
Benign Cancer Gr I/II Gr III
Nucleus 6.7±0.2 4.5±0.5b 4.3±0.7c 4.6±0.6b
Cytoplasm 6.4±0.2 5.0±0.3b 5.0±0.5c 5.0±0.3b
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Allred score (0–8) represents the sum of the staining intensity (0–3) and coverage of positive stain (0–5).

GS, Gleason Score; Gr, grade.

a

p<0.01 compared to cytoplasmic staining.

b

p<0.01 compared to normal tissue.

c

p<0.05 compared to normal tissue.

Collectively, the prevalence of pS105-ERβ staining was higher in the nucleus than in the cytosol and did not mirror the ERβ1 positivity in BCa specimens.

4. Discussion

Protein phosphorylation is one of the most common PTMs in mammalian cells, playing crucial roles in regulating the functions of the target proteins and their downstream cellular processes (Lannigan, 2003). Such phosphorylation can occur at serine, threonine, and tyrosine residues, with serine phosphorylation being the most common modification. However, because of the low stoichiometry of phosphorylation and the transient nature of this PTM in vivo (Jorgensen and Linding, 2008; Mayya and Han, 2009), localizing phosphorylation sites in a protein still presents great challenges. In the past, progress has been hindered by the limited availability of high throughput, accurate methods. The recent advent of high-resolution tandem MS has allowed rapid developments in the field. The mapping of the human ERβ has not been reported until now. In this study, we have taken advantage of this new technology and successfully uncovered three phosphorylation sites (S75, S87, and S105) in ERK1/2- or p38-treated recombinant human ERβ1 protein (wild-type). Worthy of mention is that all three sites are in the N-terminus of the protein and likely regulate the function of the AF-1 domain, a relatively understudied region of the receptor. However, since the substrate specificity of a kinase is generally broader in vitro than in vivo (Lannigan, 2003), the existence of these phosphorylated sites in tissue samples and in cell models must be further validated. We thus have developed and validated a highly specific antibody to pS105 that can be used for IHC and IB analyses to uncover the physiological roles of pS105.

Most nuclear receptors are known to be regulated by unique kinase systems, hence dictating the molecular functions of the receptors (Faus and Haendler, 2006). In this study, we found that both ERK1/2 and p38 can phosphorylate S105 of ERβ in vitro and in cellulo in ERα-negative (MDA-MB-231) and ERα-positive (BT-474) BCa cells. Using S105 phospho-mimetic (ERβ1-S105E) and -defective (ERβ1-S105A) mutants of ERβ1 and the specific ERβ agonist DPN, we further demonstrated that phosphorylation of ERβ at this site may promote the receptor’s transactivation activity in the cancer cells. Of interest, Tremblay and co-workers (1999) found that phosphorylation of the analogous site (S124) in the mouse ERβ was also ERK1/2-sensitive and associated with increased transactivation of the receptor. Thus, phosphorylation of S105/S124 in ERβ by ERK1/2 appears to be a conserved feature in humans and mice. Tremblay and his colleagues further demonstrated that the ERK1/2-mediated phosphorylation of the mouse ERβ increased recruitment of a co-activator SRC1 and a co-mediator CBP to the transcriptional complex (Tremblay and Giguere, 2001; Tremblay et al., 1999). However, p38 showed opposite effects on ERβ phosphorylation and receptor transactivation activity in humans and mice. First, in mice, p38 represses ERβ transactivation via ErbB2/ErbB3 pathway (St-Laurent et al., 2005), whereas in humans, p38 stimulates ERβ-mediated transcription (Driggers et al., 2001; Frigo et al., 2006), acting through phosphorylation of a co-activator, glucocorticoid receptor-interacting protein 1 (Frigo et al., 2006). Second, no p38-mediated phosphorylation site has been identified in the mouse or human ERβ, prior to this study. Therefore, it remains to be determined if the phosphorylation of S124 of the mouse receptor is a direct target of p38. This is in stark contrast to findings from our study of the human ERβ in MDA-MB-231 and BT-474 BCa cell lines clearly demonstrating that S105 is a bona fide phosphorylation target of p38. Here, by comparing human and mouse data, we discovered conserved and divergent roles of phosphorylation of an analogous site (human S105/mouse S124) that may be involved in regulating the functions of ERβ in the two species. Furthermore, it is not surprising to find that ERK1/2 and p38 are specific inducers of pS105 because both MAP kinases are known to regulate proliferation, survival, apoptosis, and invasion in most cancers (Atanaskova et al., 2002; Frigo et al., 2006). What remain to be deciphered are the pathophysiological conditions that would trigger pS105 in ERβ through either or both signaling cascades.

Information on how ERβ phosphorylation influences cellular functions, especially in cancers, is sparse. Our major objective in this study was to understand the pathophysiological role pS105 in BCa. Most published reports have demonstrated that ERβ participates in growth inhibition of benign and malignant breast epithelial cells (Cheng et al., 2004; Lazennec et al., 2001; Martineti et al., 2005; Pravettoni et al., 2007) and/or induces apoptosis (Cheng et al., 2004; Hodges-Gallagher et al., 2008; Huang et al., 2006), but no study relates these functions to ERβ phosphorylation. In addition, only a few studies on ERβ have focused on BCa progression (Lazennec et al., 2001; Lindberg et al., 2010). Thus, an important finding of our study is the observation that transient expression of the phosphomimetic mutant ERβ1-S105E in MDA-MB-231 or BT-474 cells induced significant inhibition of cell migration and invasion without affecting cell proliferation or cell cycle progression. The data suggest that phosphorylation of ERβ at one or more specific serine sites (including S105) confers a previously overlooked property to the receptor, i.e. one that is related to the blockade of BCa invasion. Our findings are in concordance with a recent report that ERβ increased BCa cell adhesion to different extracellular matrix proteins (mostly laminin) and inhibited cell migration. The clinical relevance of these observations warrants further investigation.

Our results show pS105 immunopositivity was widely distributed in BCa specimens in both nuclear and cytoplasmic compartment. However, it did not mirror the ERβ1 positivity in BCa specimens, which in general diminished in high grade cancer (Leygue et al., 1998; Shaw et al., 2002; Zhao et al., 2003). A disparity in pS105 positivity and ERβ1 immunostaining is expected because S105 is in the N-terminus of the receptor, a region that is common among all reported isoforms (Leung et al., 2006). Thus, a pS105 antibody should recognize any isoforms whose S105 is susceptible to similar kinase regulation. Our data therefore support the hypothesis that other ERβ isoforms can be phosphorylated at this site. It is worth mentioning that ERβ isoforms may have functions different from those of ERβ1 (Leung et al., 2006). Because of the small sample size of the current IHC study, it has a limited capacity to reveal the clinical relevance of pS105 in BCa. Fortunately, two recent large-scale studies have helped addressed this important question. Expression of ERβ2 and ERβ5 isoforms predicts better survival in a cohort of 880 BCa patients (Shaaban et al., 2008). A report on 459 cases of BCa demonstrated that nuclear pS105-ERβ-positivity is associated with better survival and correlates with immunopositivity of ERβ1 and ERβ2 (Hamilton-Burke et al., 2010). Collectively, these data uncover a significant degree of complexity in how phosphorylation regulates the function of ERβ in breast carcinogenesis.

In this study, we reported, using an unbiased MS method, the identification and characterization of the first functional phosphorylation site, S105, located in the N-terminus of the human ERβ. We also described the development of a specific antibody conducive for studying this PTM in IHC and IB analyses. Both in vitro and in cellulo experiments demonstrated that pS105 is a direct target of ERK1/2 and p38 MAPK. In ERα-positive (BT-474) and −negative (MDA-MB-231) BCa cell lines, pS105 promotes E2-induced ERβ1 transactivation and enhances the ability of ERβ1 to inhibit cell migration and invasion without affecting cell growth and cell-cycle progression. Our data suggest that S105 phosphorylation confers anti-cell invasion property to ERβ1. Further investigations are necessary to resolve the potential role of phosphorylation in regulating the function of ERβ1 and its isoforms.

  • >

    First identified direct phosphorylation targets of human ERβ at serine (S) 75, S87, and S105

  • >

    ERβ phosphorylation at S105 is activated by ERK1/2 and p38 signaling in MDA-MB-231 and BT-474 breast cancer cells.

  • >

    Phospho-mimetic mutant (ERβ1-S105E) enhanced human ERβ transactivation.

  • >

    ERβ1-S105E inhibited cellular migration and invasion, but did not affect cell growth and cell-cycle progression.

Supplementary Material

01

Acknowledgements

We thanked Dan Song for her technical support in immunohistochemistry studies and Nancy Voynow for her professional editing of this manuscript. Research was in part supported by a VA Merit Award (BX000675 to SMH) and grants from NIH (ES019480, ES006096, CA112570, CA015776 to SMH), and a Prostate Cancer Foundation Young Investigator Award (to HML).

Footnotes

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