From the 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
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ABSTRACT |
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Estrogen receptor The biological effects of estrogen are mediated by the two known
estrogen receptors, ER 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, ER 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 ER Plasmids/Adenoviruses--
Construction of the
expression plasmid for full-length, wild-type human ER 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 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-ER 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-ER 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 ER Kinase Reactions--
In vitro labeling using ER 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.
Increased Phosphorylation of ER
The effect of okadaic acid on ER
To identify the site(s) on ER ER
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 ER The Catalytic Subunit of PP2A Directly Binds to Amino Acids
176-182 of ER
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 Estrogen-Receptor Complexes Are Dynamically Regulated, Containing
Either PP2A or MAP Kinase--
As shown above, in quiescent Rad91
cells infected with GFP-ER
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 ER Inhibition of PP2A by Okadaic Acid Activates ER
To determine whether okadaic acid-induced transcriptional activation of
ER 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 ER 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
ER 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 ER (ER
) mediates the
effects of estrogen by altering gene expression following hormone
binding. It has recently been shown that kinase-mediated
phosphorylation of ER
also transcriptionally activates the receptor
in the absence of estrogen. We now report that
ER
-dependent gene expression also is regulated by
protein phosphatase 2A (PP2A). ER
co-immunoprecipitates with
enzymatically active PP2A. ER
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 ER
are required for the
interaction between PP2A and the receptor. Phosphatase inhibition
disrupts the ER
-PP2A complex and induces formation of an
ER
-activated mitogen-activated protein kinase complex,
phosphorylation of ER
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 ER
, 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
1
and ER
(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 ER
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.
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 ER
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 ER
.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(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). ER
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.
, and
ER-S118A, which contains an alanine for serine substitution at amino
acid 118, was described previously (9). The plasmid pGFP-ER
coding
for an N-terminal chimeric enhanced green fluorescent protein
(GFP)-human ER
protein was constructed by cloning the full-length ER
cDNA into the backbone vector pEGFP. The GFP-ER
cDNA was
then cloned into the adenovirus shuttle vector pACCMV.pLpA. The
adenovirus adeno-GFP-ER
was constructed by co-transfection of
GFP-ER
-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-ER
behaved identically in
all instances to the wild-type ER
, confirming previous reports with
a similar construct (17). Plasmids for the GST pull-down experiments
were constructed by cloning full-length human ER
, a mutant ER
containing an alanine for serine substitution at position 118 (ER-S118A), PCR-derived ER
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.
-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-ER
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-ER
antibody or nonimmune mouse IgG as described above.
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
ER
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).
, 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
-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.
(rER
; Calbiochem, San Diego, CA)
was diluted 1:100, and 10 µl of the diluted rER
was incubated with
GST fusion proteins as described above.
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 [
-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, 17
-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 ER
was immunoprecipitated as described above. In
a subset of experiments, recombinant ER
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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).
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Fig. 1.
The phosphatase inhibitor okadaic acid
increases phosphorylation of ER . A, ER
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, ER
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-ER
,
and immunoblot analysis identified this band as ER
. C,
Rad91 cells were transfected with either wild-type ER
(WT) or a point mutant containing an alanine substitution
for serine 118 (S118A). ER
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).
phosphorylation was also examined
in vitro. In vitro kinase reactions were
performed on ER
immunopellets obtained from lysates of Rad91 cells
infected with adeno-GFP-ER. Okadaic acid pretreatment also enhanced
phosphorylation of ER
in these experiments (Fig. 1B).
Additional studies using Rad91 cells transfected with ER
containing
a different epitope tag confirmed the identity of this phosphoband as
ER
(data not shown).
that are phosphorylated following
exposure to okadaic acid, wild-type and mutant ER
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, ER
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 ER
on serine 118.
Associates with and Is a Substrate for PP2A--
To
determine whether ER
immunopellets contain active phosphatases, we
immunoprecipitated ER
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 ER
immunopellets from Eahy926 cells released 35.2 ± 13%
of total counts (p < 0.01 versus
nonimmune). Phosphatase activity also was detected in ER
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 ER
(data not
shown). The phosphatase activity recovered with ER
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).
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Fig. 2.
ER 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 ER
immunopellets derived from BAEC, human aortic
endothelial cells (EAhy926 cells), and Rad91 cells infected with
adeno-GFP-ER
. 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-ER
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 ER
immunopellets, but not in nonimmune IgG pellets
derived from BAEC. PP2A and PP1 were also detected in ER
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, ER
immunopellets from GFP-ER
-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 ER
following incubation with
0.1-0.2 units of highly purified PP2A (pPP2A), and this was
blocked by co-incubation with 10 nM OA.
and PP2A. To determine whether
ER
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 ER
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).
--
Cell lysates from Rad91 cells infected with
adeno-GFP-ER were incubated with GST-PP2A fusion proteins to localize
which domain(s) of ER
and PP2A mediate their binding. ER
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 ER
gave similar results
(Fig. 3), confirming that the catalytic subunit of PP2A binds directly
to ER
. To localize the ER
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
ER
(Fig. 4A). The
truncation mutant ER-(1-271) and the deletion mutant
ER-(
1-176) both interacted with GST-PP2A C-subunit, as did
fragments ER-(176-271), ER-(
254-370), and ER
-(176-253), but
not the control fragment ER
-(254-370) (Fig. 4B). None of
these ER
fragments were pulled down in parallel experiments using
GST-PP2A A-subunit (data not shown). Complementary experiments using
GST fusion proteins containing ER
fragments confirmed amino acids
176-253 of ER
as the domain mediating binding to PP2A (Fig.
4C). Co-immunoprecipitation experiments in Rad91 cells
transfected with ER
fragment 176-253 supported that this interaction also occurs in vivo (Fig. 4D).
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Fig. 3.
ER 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-ER
, or with
recombinant ER
(rER
). GFP-ER
from cell lysates
bound to GST-PP2A-C, but not to GST-PP2A-A or to GST alone. Recombinant
ER
also bound to GST-PP2A-C, but not to GST alone.
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Fig. 4.
Definition of the PP2A binding domain of
ER . A, schematic representation of ER
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 ER
fragments
shown in panel A. Immunoblotting for ER
fragments
localized the interacting domain to amino acids 176-253 of the ER.
C, GST fusion proteins containing full-length ER
(WT) or PCR-derived ER
fragments were incubated with
lysates of Rad91 cells. Immunoblotting for PP2A confirmed the
interaction between the ER
fragment containing amino acids 176-253
and PP2A. D, ER
was immunoprecipitated from Rad91 cells
transfected with ER
-(176-253). Immunoblotting revealed PP2A in the
ER
fragment immunopellet, but not in the nonimmune pellet.
4 protein (amino acids 94-202), and full-length
ER
identified an 8-amino acid sequence within ER
, beginning with
amino acid 175, that contains 6 amino acids that are identical or
similar to the
4 sequence (Fig. 5). Additional GST-PP2A-C subunit pull-down experiments demonstrated that
ER
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 ER
(Fig. 5).
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Fig. 5.
Amino acids 176-182 of ER
mediate binding to PP2A. Sequence analysis detected an
8-amino acid sequence of high homology between ER
(amino acids
175-182) and the previously identified PP2A C-subunit binding domain
of the
4 protein (amino acids 174-181). The sequence alignment is
shown. In contrast to ER
-(176-253), ER
-(182-253) expressed in
COS 1 cells was not detected in GST-PP2A C-subunit pellets.
, 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 ER
(data not shown). Because okadaic acid treatment induces
phosphorylation of ER
on serine 118 (cf. Fig. 1), and
previous reports have identified this serine as the MAP kinase
phosphorylation site in ER
, we hypothesized that ER
might also
bind MAP kinase. Okadaic acid treatment, which markedly reduces the
amount of PP2A recovered in ER
immunopellets, markedly increased the
abundance of MAP kinase in ER
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 ER
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).
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Fig. 6.
Estrogen receptor complexes are dynamically
regulated and can contain either PP2A or MAP kinase. A,
ER 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 ER
/PP2A
interaction and induced complex formation between ER
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). ER
immunopellets were
prepared and phosphorylated in vitro. Autoradiography
demonstrated that PD98059 inhibited OA-induced phosphorylation of the
ER
on serine 118. The association between ER
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 ER
(rER
) as substrate. Autoradiography demonstrated that MAP
kinase phosphorylated the wild-type ER, but not the S118A mutant. MAP
kinase also phosphorylated rER
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
ER
was abolished by addition of 10 nM OA.
or GST-ER
as substrate. Recombinant MAP kinase phosphorylated GST-ER, but not
GST-ER-S118A (Fig. 6C). Recombinant MAP kinase also
phosphorylated recombinant ER
, 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).
-mediated Gene
Transcription--
To investigate the possible role of PP2A in
regulating transcriptional activation of ER
, 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
17
-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).
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Fig. 7.
Okadaic acid treatment transcriptionally
activates ER . 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 17
-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
-actin. Bottom
panel, densitometric analysis of three independent experiments
normalized for
-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.
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
directly binds phosphatases that are important
for regulating the degree of ER phosphorylation. ER
-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
ER
-associated proteins. In the current series of studies we focused
specifically on the interaction of ER
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 ER
and demonstrated further that this
interaction occurs via the catalytic subunit of PP2A. This identifies
ER
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 ER
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
4 protein, another protein
known to bind the C-subunit of PP2A. Deletion of this sequence from the
ER
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.
. 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 ER
and
phospho-MAP kinase. Although ER
has previously been reported to be a
substrate for MAP kinase, a physical interaction between these two
proteins has not previously been demonstrated.
is likely determined by
the reciprocal formation of complexes between ER
and either PP2A or
MAP kinase (Fig. 8). The current
findings, demonstrating a specific interaction between PP2A and ER
extend previous reports of stable interactions between PP2A and other proteins. For example, PP2A has previously been shown to regulate Wnt-
and
-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 a new window]
Fig. 8.
Proposed model of pathways by which PP2A
regulates transcriptional activation of ER .
Schematic representation summarizing the current findings demonstrating
that ER
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 ER 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 ER
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 ER
. It also remains to be seen
whether PP2A and ER
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.
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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:
ER, estrogen receptor
;
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, 17
-estradiol;
OA, okadaic
acid.
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