Regulation of Estrogen Receptor alpha -mediated Transcription by a Direct Interaction with Protein Phosphatase 2A*

Qing LuDagger , Howard K. SurksDagger , Heather EblingDagger , Wendy E. BaurDagger , Donald Brown§, David C. Pallas, and Richard H. KarasDagger ||

From the Dagger  Department of Medicine and Molecular Cardiology Research Institute, New England Medical Center Hospitals, Inc., Tufts University School of Medicine, Boston, Massachusetts 02111, the  Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322, and the § Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536

Received for publication, October 25, 2002, and in revised form, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen receptor alpha  (ERalpha ) mediates the effects of estrogen by altering gene expression following hormone binding. It has recently been shown that kinase-mediated phosphorylation of ERalpha also transcriptionally activates the receptor in the absence of estrogen. We now report that ERalpha -dependent gene expression also is regulated by protein phosphatase 2A (PP2A). ERalpha co-immunoprecipitates with enzymatically active PP2A. ERalpha binds directly to the catalytic subunit of PP2A, which dephosphorylates serine 118 of the receptor. Amino acids 176-182 in the A/B domain of ERalpha are required for the interaction between PP2A and the receptor. Phosphatase inhibition disrupts the ERalpha -PP2A complex and induces formation of an ERalpha -activated mitogen-activated protein kinase complex, phosphorylation of ERalpha on serine 118, and transcriptional activation. These findings demonstrate that estrogen receptors exist in complexes with phosphatases as well as kinases. We propose a new model of ligand-independent activation of estrogen receptors in which the level of phosphorylation of ERalpha , and hence its transcriptional activation, is determined by the net effect of these counterregulatory pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The biological effects of estrogen are mediated by the two known estrogen receptors, ERalpha 1 and ERbeta (1-4). Estrogen receptors are transcription factors that regulate gene expression in response to hormone binding (reviewed in Refs. 5 and 6). Although this pathway mediates many of the known effects of estrogen, it has recently become apparent that estrogen receptors also can transduce signals in the absence of estrogen via hormone-independent activation pathways (reviewed in Refs. 7 and 8). Transcriptional activation of estrogen receptors in the absence of estrogen has been reported in a variety of cells (9-14). Although the mechanisms that mediate hormone-independent activation of the ER are incompletely understood, the most completely studied pathway to date involves direct phosphorylation of ERalpha on serine 118 in the N-terminal transcriptional activation (A/B) domain by MAP kinase (13). Ligand-independent activation of the ER links mitogenic stimulation of cells by growth factors with estrogen receptor-dependent regulation of gene expression, which may support a role for estrogen receptors in regulating cellular function in situations when estrogen levels are low, such as in men and postmenopausal women.

Given the importance of phosphatases in regulating the activity of kinases, we hypothesized that phosphatases may also regulate ER-dependent signaling. In the current study, ERalpha is shown to directly bind the catalytic subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase known to regulate many important cell signaling pathways. We also demonstrate that PP2A regulates the transcriptional activation of ERalpha by counteracting MAP kinase-mediated phosphorylation of the receptor. These findings thus support a new model for the regulation of ligand-independent transcriptional activation of ERalpha .

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Unless stated otherwise, all reagents were obtained from Sigma.

Cells-- Rat pulmonary vein endothelial cells were the kind gift of Una Ryan. EAhy926 cells, a human aortic endothelial cell hybridoma, were the kind gift of C. J. Edgell (15). Bovine aortic endothelial cells (BAEC) were grown from explants as described (16). The rat pulmonary vein endothelial cells are devoid of ER, whereas both of the other types of endothelial cells endogenously express ERalpha (see below, and data not shown). Rad91 cells, developed in our laboratory, are a spontaneously immortalized vascular smooth muscle cell line derived from a human radial artery. They have been characterized as vascular smooth muscle cells by immunostaining for smooth muscle actin expression and by morphologic criteria (data not shown). ERalpha expression is not detectable by immunoblotting of lysates from these cells (see below). Cells were grown in phenol red-free Dulbecco's modified Eagle's medium in 10% estrogen-deficient fetal bovine serum.

Plasmids/Adenoviruses-- Construction of the expression plasmid for full-length, wild-type human ERalpha , and ER-S118A, which contains an alanine for serine substitution at amino acid 118, was described previously (9). The plasmid pGFP-ERalpha coding for an N-terminal chimeric enhanced green fluorescent protein (GFP)-human ERalpha protein was constructed by cloning the full-length ER cDNA into the backbone vector pEGFP. The GFP-ERalpha cDNA was then cloned into the adenovirus shuttle vector pACCMV.pLpA. The adenovirus adeno-GFP-ERalpha was constructed by co-transfection of GFP-ERalpha -pACCMV.pLpA and pJM17 into HEK293 cells followed by standard selection and virus purification procedures. Correct insertion of the appropriate cDNA into the shuttle vector was confirmed by sequence analysis. The transcriptional integrity of the chimeric protein produced by the adenovirus was studied extensively in preliminary experiments using an estrogen response element reporter plasmid (as described Ref. 9), and the chimeric GFP-ERalpha behaved identically in all instances to the wild-type ERalpha , confirming previous reports with a similar construct (17). Plasmids for the GST pull-down experiments were constructed by cloning full-length human ERalpha , a mutant ER containing an alanine for serine substitution at position 118 (ER-S118A), PCR-derived ERalpha fragments, and the full-length PP2A catalytic (C)-subunit into pGEX-4T-1 (Amersham Biosciences). The plasmid pGST-PP2A A-subunit was the kind gift of Marc Mumby. The reporter plasmid pERE-Luc in which an estrogen response element drives expression of the luciferase cDNA was the kind gift of Chris Glass, and has been previously described (9, 18). The presence of the correct sequence in all vector inserts was confirmed by sequencing.

Immunoprecipitation-- Cells grown until ~90% confluence were harvested in lysis buffer (20 mM Tris-Cl, pH 7.5, 0.137 M NaCl, 2 mm EDTA, pH 7.4, 1% Triton, 10% glycerol, 25 mM beta -glycerol phosphate, and phenylmethylsulfonyl fluoride and protease inhibitor mixture), and the lysates were incubated overnight at 4 °C with 5 µg of nonimmune mouse IgG, mouse monoclonal anti-ERalpha antibody (Ab7; Neo Markers, Freemont, CA), nonimmune goat serum IgG, or goat polyclonal anti-PP2A antibody (G-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA). Protein G beads (Amersham Biosciences) were then added and a further incubation carried out at 4 °C for 2 h. The pellets obtained after centrifugation were washed five times with wash buffer (50 mM Tris, pH 7.5, 7 mM MgCl2, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride). The washed immunopellets were then used for immunoblotting, phosphatase assays, or kinase assays as described below. In a subset of experiments a second immunoprecipitation was performed by re-suspending the beads in 0.1 ml of resolubilization buffer (50 mM Tris, pH 7.5, 5 mM dithiothreitol, and 0.5% SDS), boiling for 5 min, adding 1 ml of lysis buffer, and re-immunoprecipitating with anti-ERalpha antibody or nonimmune mouse IgG as described above.

Phosphatase Assay-- Phosphatase assays were performed essentially as described (19). Briefly, 32P-labeled myelin basic protein substrate was produced by phosphorylation with protein kinase A catalytic subunit (New England Biolabs). Radiolabeled ER substrate was produced by the kinase reaction described below. In samples where okadaic acid was included, the samples were preincubated with okadaic acid for 30 min before addition of substrate. 10 µl of 32P-myelin basic protein (0.3 mg/ml) was added and the reaction was carried out at 30 °C for 30 min. Proteins were precipitated in 10% trichloroacetic acid and the supernatant was then analyzed by scintillation counting. The activity contained in the nonimmune pellets was subtracted from all other counts. The results of the phosphatase assay are expressed as a percent of the total counts determined for the labeled substrate alone. Each determination was performed in duplicate and each experiment was independently performed three times or more.

Immunoblotting-- Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and then probed with the appropriate primary antibody. Antibodies used include anti-ERalpha antibody (Ab7 at 1:200; Neo Markers), anti-PP2A antibody (1:4000; Transduction Laboratories, Beverly, MA), anti-PP1 antibody (1:200; Santa Cruz Biotechnology), an antibody specific for ERalpha phosphorylated on serine 118 (1:2000 dilution; kind gift of Dr. Simak Ali (20)), a rabbit polyclonal anti-MAP kinase antibody (at 1:1000; Upstate Biotechnology, Lake Placid, NY), a rabbit polyclonal anti-phospho-MAP kinase antibody (at 1:1000 dilution; Cell Signaling Technology), or an anti-endothelial cell nitric-oxide synthase (eNOS) antibody (1:1000 dilution; Translab). The membranes were then incubated with the appropriate secondary antibody and developed with ECL (Amersham Biosciences).

Transient Transfections/Infections-- Quiescent cells were transfected with ER expression plasmids, and/or the ERE-Luc reporter plasmid by electroporation as previously described (9, 18). Cells were infected with adeno-GFP-ERalpha , or the control virus adeno-GFP at a multiplicity of infection of 100. Luciferase reporter assays were performed as previously described (9, 18). For the reporter assays, cells were co-transfected with a beta -galactosidase expression plasmid and all results are reported corrected for the activity of this plasmid. Each treatment was carried out in triplicate, and each experiment was performed a minimum of three different times.

GST Pull-down Experiments-- GST pull-down experiments were performed essentially as described (19). Briefly, GST fusion proteins were expressed in Escherichia coli XL10-Gold (Stratagene, La Jolla, CA). Expression was confirmed by SDS-PAGE and Coomassie Blue staining. Cell lysates were incubated with 50 µl of the GST fusion protein beads, rocked at 4 °C overnight, washed 3 times, and then boiled in SDS sample buffer. Associated proteins were resolved by SDS-PAGE and immunoblotted as described above. In a subset of experiments recombinant human ERalpha (rERalpha ; Calbiochem, San Diego, CA) was diluted 1:100, and 10 µl of the diluted rERalpha was incubated with GST fusion proteins as described above.

Kinase Reactions-- In vitro labeling using ERalpha immunopellets, or nonimmune controls, from Rad91 cells were washed and then suspended in kinase buffer (20 mM Tris, pH 7.5, 10 mM magnesium). 10 µCi of [gamma -32P]ATP was added and the solutions were incubated for 20 min at 30 °C. The reaction was terminated by adding SDS sample buffer and boiling for 5 min. In vivo labeling using quiescent cells were cultured in phosphate- and serum-free Dulbecco's modified Eagle's medium with 0.5 mCi of 32P (PerkinElmer Life Sciences) in 10-cm dishes. Cells were then treated with vehicle, 17beta -estradiol (E2; 10 nM), or okadaic acid (OA; 1 µM) for 2 h. In a subset of experiments, the cells were also preincubated with the MEK1 inhibitor PD98059 for 30 min (50 µM; New England Biolabs). The cells were then rinsed with cold phosphate-buffered saline, lysed, and ERalpha was immunoprecipitated as described above. In a subset of experiments, recombinant ERalpha or GST-ER fusion proteins were used as a substrate for the kinase reaction using 10 ng of recombinant MAP kinase (Upstate Biotechnology). In vitro labeled ER was also used as a substrate for phosphatase reactions using PP2A immunopellets or purified PP2A complexes (Upstate Biotechnology). After either in vitro or in vivo labeling, the proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and exposed to a PhosphorImager overnight. The membranes were then subsequently immunoblotted as described above.

Statistical Analyses-- All experiments shown were performed a minimum of three independent times. Where quantitative comparisons were made, groups were compared using the Student's t test, and a p value <0.05 was considered statistically significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Increased Phosphorylation of ERalpha Is Induced by Inhibition of Phosphatase Activity-- As a first step to investigate the possible role of phosphatases in regulating estrogen receptor function, in vivo 32P labeling experiments were conducted in Rad91 cells infected with adeno-GFP-ER and cultured in the absence or presence of the phosphatase inhibitor okadaic acid. Okadaic acid treatment resulted in a substantial increase in phosphorylation of the ER (Fig. 1A). As expected, incubation of the cells with estrogen also induced an increase in ER phosphorylation (Fig. 1A).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   The phosphatase inhibitor okadaic acid increases phosphorylation of ERalpha . A, ERalpha immunopellets were prepared from Rad91 cells cultured in 32P-containing serum-free medium (SFM) for 2 h in the presence or absence of 10 nM E2 or 1 µM OA. OA increased phosphorylation of the ER. B, ERalpha immunopellets or nonimmune IgG pellets (NI) prepared from Rad91 cells following a 2-h exposure to 1 µM OA or vehicle were used in an in vitro kinase reaction. OA increased phosphorylation of a predominant band that migrated at the expected size for GFP-ERalpha , and immunoblot analysis identified this band as ERalpha . C, Rad91 cells were transfected with either wild-type ERalpha (WT) or a point mutant containing an alanine substitution for serine 118 (S118A). ERalpha immunopellets were obtained from cells treated with 1 µM OA or vehicle-treated cells, and an in vitro kinase reaction was performed. Autoradiography demonstrated that OA enhanced the phosphorylation of the wild-type ER, but not the S118A mutant. Serine 118 was also identified as the OA-induced phosphorylation site in wild-type ER by immunoblotting with an antibody specific for the 118 phospho-form of the ER (118-P).

The effect of okadaic acid on ERalpha phosphorylation was also examined in vitro. In vitro kinase reactions were performed on ERalpha immunopellets obtained from lysates of Rad91 cells infected with adeno-GFP-ER. Okadaic acid pretreatment also enhanced phosphorylation of ERalpha in these experiments (Fig. 1B). Additional studies using Rad91 cells transfected with ERalpha containing a different epitope tag confirmed the identity of this phosphoband as ERalpha (data not shown).

To identify the site(s) on ERalpha that are phosphorylated following exposure to okadaic acid, wild-type and mutant ERalpha s were expressed and then immunoprecipitated from Rad91 cells treated with vehicle or okadaic acid and subjected to the in vitro kinase reaction. In contrast to the wild-type ER, ERalpha containing a serine to alanine mutation at position 118 (ER-S118A) was only minimally phosphorylated in the presence of okadaic acid (Fig. 1C). These data demonstrate that okadaic acid-mediated phosphatase inhibition results in enhanced phosphorylation of ERalpha on serine 118.

ERalpha Associates with and Is a Substrate for PP2A-- To determine whether ERalpha immunopellets contain active phosphatases, we immunoprecipitated ERalpha from EAhy926 cells and BAEC and tested for phosphatase activity. As shown in Fig. 2A, phosphatase assays using ER immunopellets derived from BAEC released 31.5 ± 16.0% of total counts (p < 0.01 versus nonimmune) and ERalpha immunopellets from Eahy926 cells released 35.2 ± 13% of total counts (p < 0.01 versus nonimmune). Phosphatase activity also was detected in ERalpha immunopellets derived from Rad91 cells infected with adeno-GFP-ER (40.5 ± 3.7% of the total counts; Fig. 2A; p < 0.001; n = five independent experiments). Phosphatase activity of nonimmune pellets was minimal (1.4 ± 0.3% of total counts; Fig. 2A), as was that in immunopellets from Rad91 cells not infected with ERalpha (data not shown). The phosphatase activity recovered with ERalpha immunoprecipitated from Rad91 was inhibited by co-incubation with 10 nM okadaic acid (40.5 ± 4.2%; Fig. 2A). 1 µM okadaic acid inhibited 76.1 ± 4.3% of the phosphatase activity in the Rad91 ER immunopellet (Fig. 2A). 10 nM okadaic acid inhibited the phosphatase activity associated with PP2A immunopellets by 99.0 ± 0.2% but that of immunoprecipitated PP1 by <10% (data not shown), consistent with previous reports demonstrating relative specificity for inhibition of PP2A at low concentrations of okadaic acid (21).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   ERalpha associates with and is a substrate for PP2A. A, phosphatase assays were performed using 32P-myelin basic protein as a substrate in the presence of nonimmune IgG, or ERalpha immunopellets derived from BAEC, human aortic endothelial cells (EAhy926 cells), and Rad91 cells infected with adeno-GFP-ERalpha . Left panel, significant phosphatase activity was detected in ER immunopellets derived from all three cell types. Right panel, the phosphatase activity associated with the Rad91 cell ER immunopellet was partially inhibited by 10 nM OA, and more completely inhibited by 1 µM okadaic acid. Bars represent the mean ± S.E. of three independent experiments. *, p < 0.05 versus no okadaic acid. B, protein lysates were immunoprecipitated with an anti-ERalpha antibody (IP-ER), or with nonimmune IgG (NI) and the resulting immunopellets were resolved by SDS-PAGE and immunoblotted for PP2A, PP1, and ER. Both PP2A and PP1 were detected in ERalpha immunopellets, but not in nonimmune IgG pellets derived from BAEC. PP2A and PP1 were also detected in ERalpha immunopellets, but not in nonimmune IgG pellets, and derived Rad91 cells were infected with adeno-GFP-ER (+ER). No phosphatases were detected in immunopellets prepared from Rad91 cells infected with the control virus adeno-GFP (-ER). C, ERalpha immunopellets from GFP-ERalpha -infected, okadaic acid-treated Rad91 cells were phosphorylated in vitro and the reaction products were then used as substrate in phosphatase reactions. Autoradiography demonstrated diminished labeling of ERalpha following incubation with 0.1-0.2 units of highly purified PP2A (pPP2A), and this was blocked by co-incubation with 10 nM OA.

These data support that the phosphatase activity detected in the ER immunopellet is most likely attributable to the presence of members of the PPP family of phosphatases (22), which is predominantly comprised of (23, 24) PP1, PP2A, and PP2B (22, 24). The partial inhibition of the ER-associated phosphatase activity observed with low dose okadaic acid is consistent with PP2A being a member of the complex (21). The additional inhibition observed at 1 µM okadaic acid supports that PP1 also may be in the complex (21). Immunoblotting studies confirmed that both PP2A and PP1 were present in ER immunopellets derived from BAEC (Fig. 2B). PP2A and PP1 also were both detected in ER immunopellets derived from Rad91 cells infected with adeno-GFP-ER, but not those infected with only the backbone adenoviral vector (Fig. 2B). PP2B and PP5, a phosphatase previously reported to complex with glucocorticoid receptors (26), were absent or only barely detectable, respectively, in these immunopellets (data not shown).

Ligand-independent activation of the ER is known to occur by both MAP and Akt kinases (reviewed in Ref. 24), and these enzymes are in turn known to be regulated by PP2A. Therefore, we sought to characterize further the interaction between ERalpha and PP2A. To determine whether ERalpha is a substrate for PP2A, ER immunoprecipitated from Rad91 cells was radiolabeled in vitro and used as substrate in phosphatase assays. Addition of PP2A immunoprecipitated from Rad91 cells significantly reduced the degree of ERalpha phosphorylation in mixing experiments (p < 0.05; data not shown). Incubation of radiolabeled ER with highly purified PP2A reduced the degree of ER phosphorylation in a dose-dependent manner, and this was blocked completely by co-incubation with okadaic acid (Fig. 2C).

The Catalytic Subunit of PP2A Directly Binds to Amino Acids 176-182 of ERalpha -- Cell lysates from Rad91 cells infected with adeno-GFP-ER were incubated with GST-PP2A fusion proteins to localize which domain(s) of ERalpha and PP2A mediate their binding. ERalpha bound to the catalytic (C)-subunit of PP2A, but not the A-subunit of the phosphatase (Fig. 3). Incubation of the catalytic subunit of PP2A with recombinant ERalpha gave similar results (Fig. 3), confirming that the catalytic subunit of PP2A binds directly to ERalpha . To localize the ERalpha domain that interacts with PP2A C subunit, GST pull-down experiments were performed with lysates of COS 1 cells transiently transfected with cDNAs for specific domains of ERalpha (Fig. 4A). The truncation mutant ER-(1-271) and the deletion mutant ER-(Delta 1-176) both interacted with GST-PP2A C-subunit, as did fragments ER-(176-271), ER-(Delta 254-370), and ERalpha -(176-253), but not the control fragment ERalpha -(254-370) (Fig. 4B). None of these ERalpha fragments were pulled down in parallel experiments using GST-PP2A A-subunit (data not shown). Complementary experiments using GST fusion proteins containing ERalpha fragments confirmed amino acids 176-253 of ERalpha as the domain mediating binding to PP2A (Fig. 4C). Co-immunoprecipitation experiments in Rad91 cells transfected with ERalpha fragment 176-253 supported that this interaction also occurs in vivo (Fig. 4D).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   ERalpha directly binds PP2A. GST alone, GST-PP2A A-subunit (GST-PP2A-A), or GST-PP2A C-subunit (GST-PP2A-C) were incubated with cell lysates from Rad91 cells infected with adeno-GFP-ERalpha , or with recombinant ERalpha (rERalpha ). GFP-ERalpha from cell lysates bound to GST-PP2A-C, but not to GST-PP2A-A or to GST alone. Recombinant ERalpha also bound to GST-PP2A-C, but not to GST alone.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Definition of the PP2A binding domain of ERalpha . A, schematic representation of ERalpha fragments used in GST pull-down and coimmunoprecipitation experiments. B, GST-PP2A-C was incubated with cell lysates from COS 1 cells, untransfected (Ctrl), or transfected with ERalpha fragments shown in panel A. Immunoblotting for ERalpha fragments localized the interacting domain to amino acids 176-253 of the ER. C, GST fusion proteins containing full-length ERalpha (WT) or PCR-derived ERalpha fragments were incubated with lysates of Rad91 cells. Immunoblotting for PP2A confirmed the interaction between the ERalpha fragment containing amino acids 176-253 and PP2A. D, ERalpha was immunoprecipitated from Rad91 cells transfected with ERalpha -(176-253). Immunoblotting revealed PP2A in the ERalpha fragment immunopellet, but not in the nonimmune pellet.

Although many PP2A-interacting proteins have been identified, few have been shown definitively to directly bind with the catalytic subunit of PP2A (27, 28). Sequence comparison of the PP2A binding region of one such protein, the alpha 4 protein (amino acids 94-202), and full-length ERalpha identified an 8-amino acid sequence within ERalpha , beginning with amino acid 175, that contains 6 amino acids that are identical or similar to the alpha 4 sequence (Fig. 5). Additional GST-PP2A-C subunit pull-down experiments demonstrated that ERalpha fragment 183-253 that lacks these amino acids no longer interacted with PP2A, identifying the sequence between amino acids 176 and 182 as critical for PP2A binding with ERalpha (Fig. 5).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Amino acids 176-182 of ERalpha mediate binding to PP2A. Sequence analysis detected an 8-amino acid sequence of high homology between ERalpha (amino acids 175-182) and the previously identified PP2A C-subunit binding domain of the alpha 4 protein (amino acids 174-181). The sequence alignment is shown. In contrast to ERalpha -(176-253), ERalpha -(182-253) expressed in COS 1 cells was not detected in GST-PP2A C-subunit pellets.

Estrogen-Receptor Complexes Are Dynamically Regulated, Containing Either PP2A or MAP Kinase-- As shown above, in quiescent Rad91 cells infected with GFP-ERalpha , PP2A co-immunoprecipitated with the ER (Fig. 6A, lane 1). Pretreatment of the cells in vivo with 1 µM okadaic acid for 2 h disrupted the interaction between these two proteins (Fig. 6A, lane 2). Identical results were obtained by immunoprecipitating PP2A and immunoblotting for ERalpha (data not shown). Because okadaic acid treatment induces phosphorylation of ERalpha on serine 118 (cf. Fig. 1), and previous reports have identified this serine as the MAP kinase phosphorylation site in ERalpha , we hypothesized that ERalpha might also bind MAP kinase. Okadaic acid treatment, which markedly reduces the amount of PP2A recovered in ERalpha immunopellets, markedly increased the abundance of MAP kinase in ERalpha immunopellets (Fig. 6A). Preincubation with PD98059, an inhibitor of MEK1-dependent activation of the MAP kinase, reversed the association of ER with phospho-MAP kinase induced by okadaic acid (Fig. 6B). PD98059 also inhibited okadaic acid-induced hyperphosphorylation of the ER. Immunoblotting using an antibody that specifically detects ERalpha phosphorylated on serine 118 confirmed this as the site of phosphorylation of the ER under these conditions (Fig. 6B). The okadaic acid-induced increase in the serine 118 phospho-form of the ER also was blocked by pretreatment with PD98059 (Fig. 6B).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Estrogen receptor complexes are dynamically regulated and can contain either PP2A or MAP kinase. A, ERalpha was immunoprecipitated from lysates of Rad91 cells infected with adeno-GFP-ER and treated with either vehicle alone (SFM) or 1 µM okadaic acid in vivo (OA). Okadaic acid treatment in vivo disrupted the ERalpha /PP2A interaction and induced complex formation between ERalpha and MAP kinase. B, quiescent Rad91 cells infected with adeno-GFP-ER were cultured in serum-free medium (SFM) and then stimulated with 1 µM OA in the presence or absence of 50 µM PD98059 (PD + OA). ERalpha immunopellets were prepared and phosphorylated in vitro. Autoradiography demonstrated that PD98059 inhibited OA-induced phosphorylation of the ERalpha on serine 118. The association between ERalpha and phospho-MAP kinase, and phosphorylation of the ER on serine 118 was markedly enhanced by OA, and this was inhibited by PD98059. C, in vitro kinase reactions were carried out using recombinant MAP kinase and GST-ER, GST-S118A, or recombinant ERalpha (rERalpha ) as substrate. Autoradiography demonstrated that MAP kinase phosphorylated the wild-type ER, but not the S118A mutant. MAP kinase also phosphorylated rERalpha and this was inhibited by co-incubation with highly purified PP2A (pPP2A; 0.2 unit). The inhibitory effect of PP2A on MAP kinase-mediated phosphorylation of ERalpha was abolished by addition of 10 nM OA.

The effect of PP2A on MAP kinase-mediated ER phosphorylation was examined next. In vitro kinase reactions were carried out with recombinant MAP kinase using either recombinant ERalpha or GST-ERalpha as substrate. Recombinant MAP kinase phosphorylated GST-ER, but not GST-ER-S118A (Fig. 6C). Recombinant MAP kinase also phosphorylated recombinant ERalpha , and this was abolished by co-incubation with highly purified PP2A (Fig. 6C). PP2A-mediated inhibition of MAP kinase-induced ER phosphorylation was blocked by okadaic acid (Fig. 6C).

Inhibition of PP2A by Okadaic Acid Activates ERalpha -mediated Gene Transcription-- To investigate the possible role of PP2A in regulating transcriptional activation of ERalpha , pulmonary vein endothelial cells were infected with adeno-GFP-ER and co-transfected with an estrogen response element-driven luciferase reporter plasmid. Exposure of quiescent cells to okadaic acid dose dependently activated the reporter plasmid (maximal activation = 3.7 ± 1.4-fold at 100 nM okadaic acid; Fig. 7A; p < 0.05 versus control; n = 4 independent experiments). Exposure of the cells to 10 nM 17beta -estradiol increased activity of the reporter plasmid 6.1 ± 1.6-fold, and this was further enhanced to 15.9 ± 6.3 to 18.3 ± 5.5-fold by okadaic acid (Fig. 7A; p < 0.05 versus estradiol alone at both 50 and 100 nM okadaic acid; n = 4 independent experiments). In control experiments, okadaic acid had no effect on reporter plasmid activity in cells that were not infected with adeno-GFP-ER, and okadaic acid-induced activation of the reporter was blocked completely by co-incubation with the estrogen receptor antagonist ICI 182780 (data not shown).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   Okadaic acid treatment transcriptionally activates ERalpha . A, rat pulmonary vein endothelial cells were infected with adeno-GFP-ER and transfected with an estrogen response element-driven luciferase reporter assay. Treatment of the cells with OA for 48 h dose dependently increased transcriptional activity of the ER in the absence and presence of 17beta -estradiol (E2). Bars represent the mean ± S.E. of 4 independent experiments. *, p < 0.05 versus no okadaic acid. B, untransfected, quiescent EAhy926 cells were maintained in serum-free medium (SFM) in the presence or absence of 10-7 M ICI 182780 (ICI), 50 nM OA, or 10-8 M E2 for 24 h. Top panel, total cell lysates were immunoblotted for eNOS and beta -actin. Bottom panel, densitometric analysis of three independent experiments normalized for beta -actin levels demonstrates that OA increases eNOS protein abundance and that this is inhibited by ICI. Bars represent the mean ± S.E. *, p < 0.05.

To determine whether okadaic acid-induced transcriptional activation of ERalpha alters the abundance of endogenously expressed proteins, we next examined the effect of okadaic acid on expression of eNOS, a protein previously shown to be regulated by estrogen in an ER-dependent manner (29). Okadaic acid increased eNOS protein abundance in untransfected Eahy926 cells (Fig. 7B). Furthermore, the increase in eNOS protein abundance following okadaic acid exposure was blocked by co-incubation with ICI 182780 (Fig. 7B).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen receptors are transcription factors that regulate gene expression by both estrogen-dependent and estrogen-independent pathways (reviewed in Refs. 5-7). Kinase-mediated ER phosphorylation contributes to transcriptional activation of the ER by both of these pathways. The data presented above demonstrate for the first time that ERalpha directly binds phosphatases that are important for regulating the degree of ER phosphorylation. ERalpha -associated phosphatase activity was partially inhibited by low dose okadaic acid, and more completely inhibited by high dose okadaic acid, suggesting that multiple phosphatases exist in this complex. Indeed, immunoprecipitation experiments identified both PP1 and PP2A as ERalpha -associated proteins. In the current series of studies we focused specifically on the interaction of ERalpha with PP2A because PP2A regulates signaling by kinases such as MAP kinase that are known to regulate ligand-independent transcriptional activation of the ER (13, 30). GST fusion protein pull-down experiments confirmed the direct interaction between PP2A and ERalpha and demonstrated further that this interaction occurs via the catalytic subunit of PP2A. This identifies ERalpha as one of only a small number of proteins known to interact directly with the catalytic subunit rather than a structural subunit of PP2A. The interacting domain of ERalpha was localized to amino acids 176-253, which contains an 8-amino acid sequence that shares a high degree of homology with a portion of the alpha 4 protein, another protein known to bind the C-subunit of PP2A. Deletion of this sequence from the ERalpha fragment abolished binding with PP2A, supporting that this sequence mediates the direct interaction between these two proteins and suggesting further that this sequence (AMESAKET) may represent a previously unidentified binding motif for the catalytic subunit of PP2A.

Because of the technical difficulties involved in attempting to overexpress PP2A (31, 32), we turned instead to inhibitor studies to explore the functional significance of the interaction between PP2A and ERalpha . In vitro and in vivo experiments demonstrated that okadaic acid, at concentrations that preferentially inhibit PP2A over PP1 (21, 23), increased phosphorylation of the ER on serine 118 and resulted in transcriptional activation of the ER. Additional studies showed further that okadaic acid treatment disrupted the ER/PP2A interaction and induced an interaction between ERalpha and phospho-MAP kinase. Although ERalpha has previously been reported to be a substrate for MAP kinase, a physical interaction between these two proteins has not previously been demonstrated.

These studies show that estrogen receptors exist in complexes with both kinases and phosphatases that exert opposing effects on the phosphorylation and transcriptional activation of the ER (cf. Fig. 4D). The data presented above suggest that PP2A can regulate the degree of phosphorylation of the ER by two pathways: (i) indirectly by regulating activation of kinase cascades that in turn regulate transcriptional activation of the ER, and (ii) directly by dephosphorylating the ER. Based on this model, the overall degree of transcriptional activation of ERalpha is likely determined by the reciprocal formation of complexes between ERalpha and either PP2A or MAP kinase (Fig. 8). The current findings, demonstrating a specific interaction between PP2A and ERalpha extend previous reports of stable interactions between PP2A and other proteins. For example, PP2A has previously been shown to regulate Wnt- and beta -catenin-dependent downstream transcriptional events via a specific interaction between the B56 regulatory subunit of PP2A and the adenomatous polyposis coli protein (33, 34).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8.   Proposed model of pathways by which PP2A regulates transcriptional activation of ERalpha . Schematic representation summarizing the current findings demonstrating that ERalpha can exist in its transcriptionally inactive state complexed with PP2A, and in its transcriptionally active state complexed with MAP kinase. Reciprocal formation of each of these complexes is dynamically regulated and depends on the degree of activity of PP2A and MAP kinase.

Although the findings presented above clearly demonstrate that PP2A directly binds to ERalpha and regulates its degree of phosphorylation, they also leave open many unanswered questions. For example, despite using okadaic acid at concentrations that preferentially inhibit PP2A, the effects of okadaic acid clearly are not restricted to PP2A alone. Thus the specificity of the observed effects of okadaic acid for PP2A requires further investigation. Similarly, we have shown that ERalpha also complexes with PP1, but the importance of this interaction has not been explored. The observation that 1 µM okadaic acid did not fully inhibit the phosphatase activity contained in the ER immunopellet (cf. Fig. 1) suggests that additional phosphatases may also complex to the ER, and these have yet to be identified. Although we have identified MAP kinase as an ER-associated protein, previous reports have shown that Akt kinase also can transcriptionally activate the ER (25), making it likely that other kinases also can form complexes with ERalpha . It also remains to be seen whether PP2A and ERbeta bind one another.

In summary, the data presented above support that estrogen receptors can exist in complexes with both kinases and phosphatases that regulate its transcriptional activation. The importance of kinase-mediated phosphorylation of the ER in regulating its transcriptional activity has previously been recognized. The present findings extend these observations by demonstrating for the first time, a counterregulatory role for the phosphatase PP2A in determining the state of estrogen receptor phosphorylation and its transcriptional activation.

    ACKNOWLEDGEMENTS

We thank Sharon Lynch and Patti Griffiths for preparing the manuscript. We also thank Michael E. Mendelsohn for many helpful discussions and review of the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HL61290 (to R. H. K.). Under agreements between Upstate Biotechnology Inc. and Emory University and Calbiochem and Emory University, D. Pallas is entitled to a share of sales royalty received by the University from these companies. In addition, this same author serves as a consultant to Upstate Biotechnology Inc. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Established Investigator of the American Heart Association. To whom correspondence should be addressed: Molecular Cardiology Research Center, Tufts-New England Medical Center, 750 Washington St., Box 80, Boston, MA 02111. Tel.: 617-636-8776; Fax: 617-636-1444; E-mail: rkaras@lifespan.org.

Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M210949200

    ABBREVIATIONS

The abbreviations used are: ERalpha , estrogen receptor alpha ; MAP, mitogen-activated protein; PP2A, protein phosphatase 2A; BAEC, bovine aortic endothelial cells; GFP, green fluorescent protein; eNOS, endothelial nitric-oxide synthase; GST, glutathione S-transferase; E2, 17beta -estradiol; OA, okadaic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Walter, P., Green, S., Krust, A., Bornert, J. M., Jeltsch, J. M., Staub, A., Jensen, E., Scrace, G., Waterfield, M., and Chambon, P. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7889-7893[Abstract]
2. Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. Å. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925-5930[Abstract/Free Full Text]
3. Mosselman, S., Polman, J., and Dijkema, R. (1996) FEBS Lett. 392, 49-53[CrossRef][Medline] [Order article via Infotrieve]
4. Tremblay, G. B., Tremblay, A., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Labrie, F., and Giguère, V. (1997) Mol. Endocrinol. 11, 353-365[Abstract/Free Full Text]
5. Carson-Jurica, M. A., Schrader, W. T., and O'Malley, B. W. (1990) Endocr. Rev. 11, 201-220[Abstract]
6. Greene, G. L., and Press, M. F. (1986) J. Steroid Biochem. Mol. Biol. 24, 1-7
7. Bruno, C., and Picard, D. (1999) Trends Endocrinol. Metab. 10, 41-46[CrossRef][Medline] [Order article via Infotrieve]
8. Mendelsohn, M. E., and Karas, R. H. (1999) N. Engl. J. Med. 340, 1801-1811[Free Full Text]
9. Karas, R. H., Gauer, E. A., Bieber, H. E., Baur, W. E., and Mendelsohn, M. E. (1998) J. Clin. Invest. 101, 2851-2861[Abstract/Free Full Text]
10. Ignar-Trowbridge, D. M., Nelson, K. G., Bidwell, M. C., Curtis, S. W., Washburn, T. F., McLachlan, J. A., and Korach, K. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4658-4662[Abstract]
11. Ignar-Trowbridge, D. M., Teng, C. T., Ross, K. A., Parker, M. G., Korach, K. S., and McLachlan, J. A. (1993) Mol. Endocrinol. 7, 992-998[Abstract]
12. Ignar-Trowbridge, D. M., Pimentel, M., Parker, M. G., McLachlan, J. A., and Korach, K. S. (1996) Endocrinology 137, 1735-1744[Abstract]
13. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract]
14. Power, R. F., Mani, S. K., Codina, J., Conneely, O. M., and O'Malley, B. W. (1991) Science 254, 1636-1639[Medline] [Order article via Infotrieve]
15. Edgell, C. J., McDonald, C. C., and Graham, J. B. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3734-3737[Abstract]
16. Chen, Z., Yuhanna, I. S., Galcheva-Gargova, Z. I., Karas, R. H., Mendelsohn, M. E., and Shaul, P. W. (1999) J. Clin. Invest. 103, 401-406[Abstract/Free Full Text]
17. Htun, H., Holth, L., Walder, D., Davie, J. R., and Hager, G. L. (1999) Mol. Biol. Cell 10, 471-486[Abstract/Free Full Text]
18. Karas, R. H., Patterson, B. L., and Mendelsohn, M. E. (1994) Circulation 89, 1943-1950[Abstract]
19. Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587[Abstract/Free Full Text]
20. Ali, S., Lutz, Y., Bellocq, J.-P., Chenard-Neu, M.-P., Rouyer, N., and Metzger, D. (1993) Hybridoma 12, 391-405[Medline] [Order article via Infotrieve]
21. Favre, B., Turowski, P., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 13856-13863[Abstract/Free Full Text]
22. Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999) Trends Biochem. Sci. 24, 186-191[CrossRef][Medline] [Order article via Infotrieve]
23. Mumby, M. C., and Walter, G. (1993) Physiol. Rev. 73, 673[Abstract/Free Full Text]
24. Cohen, P. T. W. (1997) Trends Biochem. Sci. 22, 245-251[CrossRef][Medline] [Order article via Infotrieve]
25. Campbell, R. A., Bhat-Nakshatri, P., Patel, N. M., Constantinidou, D., Ali, S., and Nakshatri, H. (2001) J. Biol. Chem. 276, 9817-9824[Abstract/Free Full Text]
26. Zuo, Z., Urban, G., Scammell, J. G., Dean, N. M., McLean, T. K., Aragon, I., and Honkanen, R. E. (1999) Biochemistry 38, 8849-8857[CrossRef][Medline] [Order article via Infotrieve]
27. Inui, S., Sanjo, H., Maeda, K., Yamamoto, H., Miyamoto, E., and Sakaguchi, N. (1998) Blood 92, 539-546[Abstract/Free Full Text]
28. Muarata, K., Wu, J., and Brautigan, D. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10624-10629[Abstract/Free Full Text]
29. Weiner, C. P., Lizasoain, I., Baylis, S. A., Knowles, R. G., Charles, I. G., and Moncada, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5212-5216[Abstract]
30. Ugi, S., Takeshi, I., Ricketts, W., and Olefsky, M. (2002) Mol. Cell. Biol. 22, 2375-2387[Abstract/Free Full Text]
31. Baharians, Z., and Schonthal, A. H. (1998) J. Biol. Chem. 273, 19019-19024[Abstract/Free Full Text]
32. Chung, H. Y., and Brautigan, D. L. (1999) Cell. Signalling 11, 575-580[CrossRef][Medline] [Order article via Infotrieve]
33. Li, X., Yost, H., Virshup, D. M., and Seeling, J. M. (2002) EMBO 20, 4122-4131[Abstract/Free Full Text]
34. Seeling, J. M., Miller, J. R., Gil, R., Moon, R. T., White, R., and Virshup, D. M. (2002) Science 283, 2089-2091[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.