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INTRODUCTION |
The biological effects of estrogens are mediated by two estrogen
receptors (ERs),1 ER
and
ER
, which belong to a large superfamily of nuclear hormone receptors. These ligand-regulatable transcription factors possess six
structural domains labeled A through F (1). The A/B domain encompasses
activation function-1 (AF-1); the C and D domains correspond to the DNA
binding domain and the hinge region, respectively; the E region
encompasses a second activation function (AF-2) and an overlapping
ligand binding domain; and the F domain, located at the extreme
carboxyl terminus, is thought to play a modulatory role in ER activity.
Both ERs possess similar binding affinities for estradiol and their
cognate DNA binding site (estrogen response element; ERE), which is
likely caused by the high degree of sequence homology they share in
their ligand and DNA binding domains (2-6). The AF-2 domain of each
receptor is regulated by ligand-induced changes in receptor
conformation, but the activities of the poorly conserved AF-1 domains
are ligand-independent and can be modulated by phosphorylation (7-10).
Notable for ER, in most cases the AF-1 and the AF-2 domains interact
functionally to enhance transcription in a cooperative manner (7,
11).
In the best studied mechanism of ER and ER activation, hormone
diffuses into the cell, binds to the receptor, and induces a
conformational change in the receptor ligand binding domain (1).
Receptors, bound to their EREs either as ER or ER homodimers or
ER:ER heterodimers, can then recruit coactivators to the promoter
region of estrogen target genes via their interaction with the receptor
activation domains (2, 3, 12, 13). There, these molecules can stimulate
transcription by bridging ERs to the general transcriptional machinery
and promoting the formation of a stable preinitiation complex (12, 13).
Notably, various coactivators also possess ubiquitin ligase, arginine
methyltransferase, or histone acetyltransferase enzymatic activities
that may facilitate chromatin remodeling and gene activation
(14-17).
In addition to this relatively well characterized mode of activation,
ER can be activated via the cAMP signaling pathway in the apparent
absence of estrogens (for review, see Ref. 18). In MCF-7 cells,
endogenous ER target genes, including the progesterone receptor (PR;
Ref. 19), pS2 (20), Liv-1 (20), and cathepsin-D (21), can be stimulated
either with the cAMP analog 8-bromo-cAMP or a combination of the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and
cholera toxin, which increases cAMP production via a G protein-mediated
signal transduction pathway. Importantly, in these experiments as well
as in later studies the cAMP-induced responses are inhibited by
treatment with the pure antiestrogen ICI 164,384, signifying their
receptor dependence (19-21). Demonstrating the need to transfect ER
into cells lacking endogenous receptor further supported this receptor
dependence (22).
In addition to these ER studies a number of reports indicate that
the transcriptional activities of several other nuclear receptors can
be modulated by the cAMP signaling pathway. For example, the chicken PR
(23), androgen receptor (24), retinoic acid receptor (25), retinoid X
receptor (26), and peroxisome proliferator-activated receptor-
(27)
can be activated by cAMP signaling, demonstrating that this mode of
ligand-independent activation is not exclusive to ER. However, human
PR (28) cannot be activated ligand-independently via this mechanism,
nor can the unliganded glucocorticoid receptor, although cAMP
stimulation increases the hormone-dependent responses of
these receptors (29, 30). Collectively, these reports demonstrate the
specific nature by which the cAMP signaling pathway can cross-talk with
different nuclear receptors.
Some progress has been made in the effort to understand the mechanisms
involved in cAMP activation of nuclear receptor-dependent transcription. Although an increase in receptor phosphorylation accompanies the cAMP-mediated activation of ER (31, 32), there is no
increase in chicken PR phosphorylation associated with its activation
in cells treated with 8-bromo-cAMP (33). Rather, cAMP/protein kinase A
(PKA) signaling enhances chicken PR-dependent
transcription, in part by increasing phosphorylation of a
receptor-interacting coactivator, steroid receptor coactivator-1 (SRC-1), and thereby promotes a more stable interaction between this
coactivator and p300/CREB-binding protein (CBP)-associated factor and
facilitates functional synergism between SRC-1 and CBP (34). Although
it is still unknown how the unliganded ER is activated by cAMP/PKA,
there is evidence that the transcription factor, CREB, can interact
functionally with ER and thereby mediate synergism between the
17-estradiol (E2)-dependent and
cAMP-dependent signaling pathways (35).
The identification of ER has increased our awareness of the
diversity of potential mechanisms by which ER-dependent and
estrogenic responses may be achieved (6). Notably, the relative
magnitude of ER- and ER-mediated estrogen activation of
ERE-containing reporters typically varies depending on the cell type
and promoter context (36). In the absence of ligand, both ER- and
ER-dependent transcription can be modulated by a
mitogen-activated protein kinase (MAP kinase) signaling pathway
(8-10). It is unknown, however, whether ER can be activated by the
cAMP/PKA signaling pathway, and we therefore examined this using
transient transfection assays and synthetic agents that increase
intracellular cAMP levels. In so doing, we have defined mechanistic
differences between cAMP activation of ER and ER, particularly
with respect to phosphorylation and the influence of cross-talk with
AP-1 transcription factors. Moreover, we report that all of the p160
coactivators, as well as CBP, can coactivate ER and ER
transcriptional activity stimulated by cAMP pathways, and we
demonstrate that the importance of SRC-1 phosphorylation to cAMP
activation of gene expression is receptor-dependent.
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EXPERIMENTAL PROCEDURES |
Chemicals--
E2, IBMX, and
N-(2-(p-bromocinnamylamino)-ethyl)-5-isoquinolinesulfonamide
(H89) were obtained from Sigma. Forskolin was obtained from Calbiochem
(San Diego).
Plasmid DNAs--
The mammalian expression vectors for human
ER, pCMV5-hER (31) and pCR3.1-hER (15), and human
ER, pCXN2-hER (37) have been described previously.
The synthetic target genes pERE-E1b-CAT (38), pE1b-CAT (38),
pERE-E1b-CAT(mTRE) (39), 17mer-E1b-CAT (40), and pS2-CAT (41) were used
in previous studies, as were pATC0, pATC1, and pATC2 (42). The
pCR3.1-hSRC-1a (43) and pCR3.1-hSRC-1a-Ala1179/1185 (34)
expression plasmids have been published, as were the pCR3.1-TIF2, pCR3.1-RAC3, and pCR3.1-CBP vectors (44). The pBind expression plasmid
encoding the Gal4 DNA binding domain (amino acids 1-147) was obtained
from Promega (Madison, WI).
The SRC-1a phosphorylation mutant pCR3.1-hSRC-1a-7Ala, which has
alanine substitutions at positions 372, 395, 517, 569, 1033, 1179, and
1185, was generated using the QuikChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA) and the appropriate mutagenic primers. The
plasmids were sequenced to ensure that errors did not occur during
mutagenesis. The constructs for hER-179C (pCR3.1-hER-179C) and
hER-143C (pCR3.1-hER-143C) were made by PCR using the primers
5'-ACCATGGCCAAGGAGACTCGCTACTGT-3' and 5'-CTCTCAGACTGTGGCAGGGAAACC-3' to amplify the segment of
pCMV5-hER encoding amino acids 179-595 and the primers
5'-ACCATGAAGAGGGATGCTCACTTCTGC-3' and
5'-GCGTCACTGAGACTGTGGGTTCTG-3' to PCR amplify the segment of
pCXN2-hER encoding residues 143-530, respectively. Each
of the resulting PCR fragments was subcloned into the pCR3.1 expression plasmid using the TA cloning kit (Invitrogen). The expression plasmids
encoding Gal-ER EF (pBind-ER EF) and Gal-ER EF (pBind-ER EF) were generated by PCR using the primers
5'-GGGATCCGTAAGAAGAACAGCCTGGCCTTGTTCC-3' and
5'-TCTAGAGACTGTGGCAGGGAAACCCTCTGCC-3' to amplify the segment of
pCMV5-hER corresponding to amino acids 302-595 and the
primers 5'-CGGGATCCGAGTGCGGGAGCTGCTGCTGG-3' and
5'-ATAGTTTAGCGGCCGCTCACTGAGACTGTGGGTTCTG-3' to amplify the
portion of pCXN2-hER encoding amino acids 254-530. Each
of the resulting fragments was subcloned into the pCR3.1 expression
plasmid via the TA cloning kit and subsequently transferred to the
pBind expression vector via a BamHI-XbaI
restriction fragment for pBind-ER EF and
BamHI-NotI fragment for pBind-ER EF. The pCR3.1-FLAG-hER construct was generated by PCR using a 5'-primer (5'-GATATTGCTAGCATGGACTACAAGGACGACGATGACAAGACCCTCCACACCAAAGCATCT-3'), which incorporated the FLAG epitope (underlined), and
5'-CGCCGCAGCCTCAGACCCGGGGCC-3' to amplify the 5'-region of a
hER cDNA within pCR3.1-hER, and the resulting PCR fragment
was substituted back into the pCR3.1-hER expression vector via
NheI and XmaI restriction sites. The
pCR3.1-FLAG-hER expression vector was constructed as follows. First,
the coding region for hER was removed from pCXN2-hER
via a partial digest with EcoRI and transferred to pCR3.1,
which yielded pCR3.1-hER. The 5'-primer
(5'-CGTGACCGTGCTAGCATGGACTACAAGGACGACGATGACAAGGATATAAAAAACTCACCATCTAGC-3'), which encompasses a coding sequence for the FLAG peptide (underlined), and 3'-primer (5'-CACAAGGCGGTACCCACATCTCTC-3') were used to PCR amplify a portion of pCR3.1-hER corresponding to the 5'-end of the
hER cDNA. The resulting PCR product was substituted into pCR3.1-hER via NheI and KpnI restriction
sites. All of the expression vectors that were made using PCR
amplification were sequenced to ensure that no errors occurred during
their synthesis. The 17mer-E1b-CAT(
TRE) reporter plasmid
was generated by an Eco0109-HindIII digest of the
17mer-E1b-CAT, which was religated after blunting the restriction ends.
Cell Culture and Transfections--
HeLa (human cervical
carcinoma) cells were routinely maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
24 h prior to transfections for transactivation assays, cells were
plated in six-well culture dishes at a density of 3 × 105 cells/well in phenol red-free DMEM with 5%
charcoal-stripped fetal bovine serum (sFBS). For transfections, medium
was replaced with serum-free medium, and DNA was introduced into cells
in the indicated amounts using LipofectAMINE (Invitrogen) according to the manufacturer's guidelines. 5 h later, serum-free medium was replaced with phenol red-free DMEM supplemented with 5% sFBS. 12 h thereafter, cells were treated with the indicated amounts of various
hormones. After 24 h of hormone treatment (12 h for inhibitor
experiments), cells were harvested, and extracts were assayed for CAT
activity, as described previously (45, 46) using butyryl-coenzyme A
(Amersham Biosciences) and [3H]chloramphenicol
(PerkinElmer Life Sciences). The quantity of resulting radiolabeled
product was determined by scintillation counting using biodegradable
counting scintillant (Amersham Biosciences) and a Beckman LS 6500 scintillation counter, and then normalized to total cellular protein
measured by Bio-Rad protein assay. Experiments were done in duplicate,
and values represent the average ± S.E. of at least three
individual experiments.
Western Blot Analysis--
To determine ER expression levels,
cells were transfected as above and harvested. Cell pellets were
resuspended in a 50 mM Tris (pH 8.0) buffer containing 5 mM EDTA, 1% Nonidet P-40, 0.2% Sarkosyl, 0.4 M NaCl, 100 µM sodium vanadate, 10 mM sodium molybdate, and 20 mM NaF and
incubated on ice for 1 h. The lysates were subsequently centrifuged at 21,000 × g for 10 min at 4 °C. The
resulting supernatants were mixed with SDS-PAGE loading buffer,
resolved on a 7.5% SDS-polyacrylamide gel, and subsequently
transferred to nitrocellulose membrane. The membranes were blocked
using 1% nonfat dried milk in 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20, and sequentially incubated
with an anti-FLAG M2 antibody (Sigma) and a horseradish peroxidase-conjugated, anti-mouse antibody. Blots were visualized using
enhanced chemiluminescence (ECL) reagents as recommended by the
manufacturer (Amersham Biosciences).
Gel Mobility Shift Assays--
HeLa cells were harvested in
phosphate-buffered saline using a cell scraper and subsequently
resuspended in ice-cold lysis buffer (10 mM Hepes (pH 7.9),
1 mM EDTA, 60 mM KCl, 1 mM
dithiothreitol, Complete Mini-Tablet protease inhibitor (Roche
Diagnostics), and 0.5% Nonidet P-40), and incubated on ice for 5-15
min to lyse the cell membranes. The resulting lysates were centrifuged
for 5 min at ~ 1,400 × g to pellet the nuclei.
Nuclei were washed in 2.5 ml of lysis buffer lacking Nonidet P-40 and
repelleted as before. Thereafter, the nuclei were resuspended in a
minimum volume of 250 mM Tris (pH 7.8) buffer containing 60 mM KCl and protease inhibitors and subjected to three
cycles of rapid freeze/thaw. The nuclear lysates were centrifuged for
15 min at 21,000 × g and 4 °C, and the protein
concentrations of the resulting supernatants were determined by
Bradford assay.
Complementary oligonucleotides corresponding to the region of the
ERE-E1b-CAT plasmids containing the
12-O-tetradecanoylphorbol-13-acetate response element (TRE)
(5'-TGAAAACCTCTGACACATGCAGCTCCC-3' and 5'-GGGAGCTGCATGTGTCAGAGGTTTTCA-3') or mTRE
(5'-TGAAAACCTCGGACTCATGCAGCTCCC-3' and
5'-GGGAGCTGCATGAGTCCGAGGTTTTCA-3') were synthesized by Invitrogen and
annealed in 10 mM Tris (pH 7.5) buffer containing 1 mM EDTA and 50 mM NaCl to create
double-stranded oligonucleotides. These DNA fragments were labeled
with [
-32P]ATP (7,000 Ci/mmol; ICN Biomedicals Inc.,
Irvine, CA) and T4 polynucleotide kinase. Unincorporated
[-32P]ATP was removed with a MicroSpin G25 column
(Amersham Biosciences) according to the manufacturer's recommendation.
The radiolabeled oligonucleotides were combined with nuclear extract in
binding reaction buffer (50 mM Tris (pH 7.5) containing 2.5 mM EDTA, 5 mM MgCl2, 2.5 mM dithiothreitol, 250 mM NaCl, 20% glycerol,
and 0.25 mg/ml poly(dI-dC)) and incubated for 15 min at room
temperature. For oligonucleotide binding competition and antibody
supershift experiments, unlabeled oligonucleotides or antibody
(anti-c-Jun (Upstate Biotechnology, Lake Placid, NY) or anti-c-Fos
(Santa Cruz Biotechnology, Santa Cruz, CA)), respectively, were
incubated with the nuclear extract for 30 min on ice before the
addition of 32P-labeled probe. Free and complexed DNAs were
resolved on a nondenaturing acrylamide gel. To avoid any possible
influence of the loading dye on complex mobility, the gel-loading
buffer containing 250 mM Tris (pH 7.5), 0.2% bromphenol
blue, and 40% glycerol was added to the minus-extract control sample
only. After electrophoresis at 350 V, the gel was transferred to 3MM
Whatman paper, vacuum dried, and exposed to X-Omat film for autoradiography.
Assessment of Forskolin/IBMX-induced
Phosphorylation--
For metabolic labeling studies, 2 × 106 HeLa cells were plated onto 150-mm Petri dishes, and
24 h thereafter they were transfected with pBind-ER EF or
pBind-ER EF using a nonrecombinant adenovirus DNA transfer procedure
(47). Briefly, plasmid DNA (200 ng/plate) was mixed with
polylysine-coupled adenovirus and incubated for 30 min at room
temperature. Before the addition of the adenovirus-plasmid particles to
the cells, the medium was aspirated and replaced with DMEM. The
adenovirus-plasmid particles were added to the cells at a 400:1
adenovirus:cell ratio. After incubation with the cells for 2 h at
37 °C, an equal volume of DMEM with 5% sFBS was added to each dish
resulting in a final concentration of 2.5% sFBS. 24 h after
transfection, the medium was aspirated from the dishes and replaced
with phosphate-free DMEM, and the cells were incubated for 1 h at
37 °C. The medium was removed and replaced with phosphate-free DMEM
containing 1% dialyzed FBS before the addition of 0.14 mCi/ml
[32P]H3PO4. After a 14-16-h
incubation, the plates were incubated for 90 min with either 0.1%
dimethyl sulfoxide (vehicle) or 10 µM forskolin + 100 µM IBMX.
After treatments, cells were harvested and incubated with lysis buffer
(10 mM Tris (pH 8.0), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM
-mercaptoethanol, 50 mM potassium phosphate, 50 mM sodium fluoride, 0.1% Triton X-100, 0.1 mM
phenylmethylsulfonyl fluoride, and a protease inhibitor mixture of 1 µg/ml each leupeptin, antipain, aprotinin, benzamidine HCl,
chymostatin, and pepstatin), vortexed, and centrifuged at 20,000 × g for 10 min at 4 °C. Protein A-Sepharose beads
(Amersham Biosciences) were incubated with 2 µg of antibody to the
Gal4 DNA binding domain (sc-577; Santa Cruz Biotechnology) for 1 h
at room temperature and then incubated with the cell lysate supernatant
for 2 h while rotating at 4 °C. The beads were washed with 100 volumes of phosphate-buffered saline. After the addition of Laemmli
sample buffer and boiling for 5 min, the samples were electrophoresed
on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose
membrane for autoradiography. The same membrane was subsequently used
for Western blotting to detect pBind-ER EF and pBind-ER EF using
a horseradish peroxidase-conjugated antibody to the Gal4 DNA binding
domain (sc-510HRP; Santa Cruz Biotechnology) and ECL. Autoradiography
signals were quantitated by densitometric scanning of films or
quantitation of the ECL signal using a Kodak Image Station 440CF.
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RESULTS |
Activation of ER and ER by a cAMP Signaling
Pathway--
Several studies have demonstrated that agents that
stimulate increases in cAMP can promote ER-dependent
gene expression in the apparent absence of hormone (19-22, 32, 48). To
determine whether ER could be activated in a similar manner, HeLa
cells were transiently transfected with expression vectors for either human ER or ER along with an ERE-E1b-CAT synthetic target
construct that possesses an ERE from the Xenopus
vitellogenin A2 promoter linked to the adenoviral E1b TATA box and CAT
reporter gene. Cells were subsequently stimulated with either
E2 or a combination of forskolin and IBMX (forskolin/IBMX),
which increases cAMP production by activating adenylate cyclase and
inhibiting phosphodiesterases, respectively, and CAT activity was
measured. As expected, E2 stimulated both ER- and
ER-dependent transcription of this reporter (Fig. 1). Consistent with previous reports (22,
32), stimulation with forskolin/IBMX also resulted in a robust
stimulation of ER transcriptional activity, although a longer (24 h)
hormone treatment greatly enhanced the E2-stimulated
relative to forskolin/IBMX-stimulated response (compare Figs. 1 and
2). Importantly, under the same conditions ER was also activated upon stimulation of cells with forskolin/IBMX (Fig. 1). Pretreatment with a PKA-selective inhibitor, H89 (49), blocked forskolin/IBMX-induced gene expression by both
receptor subtypes, thus demonstrating that forskolin/IBMX induction of
ER and ER activity is mediated by a cAMP/PKA signaling pathway in
these cells. Moreover, this inhibition was specific for forskolin/IBMX
induction because H89 treatment did not significantly alter basal or
E2-stimulated responses for either ER or ER.
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Fig. 1.
Forskolin and IBMX-induced
ER and ER
transcriptional activity are dependent upon PKA signaling.
HeLa cells were transiently transfected with 10 ng of expression
plasmid for ER (pCMV5-ER) or ER
(pCXN2-ER) and 1 µg of ERE-E1b-CAT reporter plasmid.
Cells were subsequently treated for 12 h with vehicle (0.1%
ethanol), 1 nM E2, or 10 µM
forskolin + 100 µM IBMX (F/I) after
a 1-h pretreatment with either 10 µM H89 (+) or dimethyl
sulfoxide ( ). Values are normalized to the activity of ER in the
absence of hormone and represent the average ± S.E. of three
independent experiments.
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Fig. 2.
cAMP/PKA stimulation of
ER-dependent transcription requires ER binding to its
cognate hormone response element. HeLa cells were transiently
transfected with either 10 ng of expression plasmid for ER
(pCMV5-ER) or ER (pCXN2-ER) along with
1 µg of ERE-E1b-CAT or E1b-CAT. Cells were subsequently treated with
vehicle, 1 nM E2, or 10 µM
forskolin + 100 µM IBMX (F/I) for
24 h. Values are normalized to ERE-E1b-CAT reporter activity for
ER in the absence of hormone and represent the average ± S.E.
of three experiments.
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An ERE Is Required for ER and ER Activation of ERE-E1b-CAT
Expression by the cAMP/PKA Signaling Pathway--
To test
whether the cAMP signaling response was mediated through an ER genomic
mechanism in which the ER binds directly to the promoter, the effects
of forskolin/IBMX on ER and ER was assessed on the E1b-CAT
reporter, which lacks an ERE. The expected ER- and
ER-dependent responses were observed for the ERE-E1b-CAT reporter. As anticipated, basal transcription of the E1b-CAT promoter was minimal, and E2 did not further increase CAT gene
expression in cells transfected with either ER or ER (Fig. 2).
Moreover, forskolin/IBMX did not stimulate E1b-CAT reporter gene
activity in the presence of transfected ER, and only a very weak
forskolin/IBMX-dependent E1b-CAT expression was observed in
the presence of ER. These data indicate that ER- and
ER-dependent transcription in response to forskolin/IBMX
requires the presence of an ERE.
CBP and p160/SRC Coactivators Enhance cAMP-stimulated
ER and ER Responses--
ER- and ER-dependent
transcriptional differences are partly attributable to the selectivity
these receptors possess for the different coactivators in the presence
of various ligands (50, 51). To extend this concept, we overexpressed
the p160 coactivators (SRC-1, TIF2, and RAC3) as well as the general
coactivator/cointegrator, CBP, in cells to determine whether any of
them might distinguish between ER and ER in their ability to
potentiate activation by cAMP-dependent signaling. Each of
these coactivators strongly enhanced ER- and
ER-dependent transcription compared with reporter activity in the absence of exogenous coactivator (Fig.
3). However, none of the coactivators
selectively enhanced the activity of estrogen-activated receptor over
cAMP-activated receptor, nor of one receptor subtype over the other.
These results suggest that each of the p160 coactivators and CBP can
contribute significantly to the forskolin/IBMX-induced activation of
both ER and ER.
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Fig. 3.
The p160 and CBP coactivators enhance
cAMP/PKA-mediated ER- and
ER-dependent transcription.
HeLa cells were transiently transfected with 10 ng of expression
plasmid for ER (pCMV5-ER) or ER
(pCXN2-ER) along with 250 ng of expression plasmid for
SRC-1e, TIF2, RAC3, or the empty vector (pCR3.1) and 1 µg of
ERE-E1b-CAT reporter. Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). CAT measurements
were standardized to total protein, and the results are the
average ± S.E. of three experiments.
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Activation of ER and ER by cAMP Depends on Promoter
Context--
In general, ER-mediated transcription depends on the
promoter context in which an ERE is found (11, 52, 53). We therefore tested the extent to which forskolin/IBMX could stimulate
ER-dependent gene expression in the context of various
synthetic and natural ERE-containing promoters. The pS2 construct
contains the 1100 to +10 region of the E2-responsive
human pS2 promoter subcloned into a CAT reporter plasmid (41). The
pATC0, pATC1, and pATC2 are synthetic reporters that possess 0, 1, or 2 EREs, as indicated in their nomenclature. The ERE-E1b-CAT reporter
construct was included in these experiments as a control. There was no
E2-induced response for pATC0 because it had no ERE, a weak
response for pATC1, and a synergistic
E2-dependent response for pATC2, for both ER
subtypes (Fig. 4, A and
B). Notably, there was no forskolin/IBMX-induced response on
either of these promoters, demonstrating that the number of EREs, in
and of itself, had no effect on the forskolin/IBMX-induced activation.
Moreover, although the expected E2-dependent
increases were present for both ER and ER on the pS2 promoter,
there was no significant increase in reporter activity in response to
forskolin/IBMX. Thus, the cAMP/PKA signaling pathway mediates ER-
and ER-dependent gene expression in a
promoter-dependent fashion.
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Fig. 4.
cAMP activation of ER
and ER depends on promoter context.
HeLa cells were transiently transfected with 10 ng of expression
plasmid for ER (pCMV5-ER) (A) or ER
(pCXN2-ER) (B) along with 1 µg of the
indicated CAT reporter plasmids. Cells were subsequently treated with
vehicle, 1 nM E2, or 10 µM
forskolin + 100 µM IBMX (F/I).
Values for ERE-E1b-CAT and pS2-CAT are normalized to their respective
vehicle-treated samples, which were arbitrarily set as 100. Values for
pATC0, pATC1, and pATC2 are normalized to vehicle treatment for pATC2,
which was set as 100. The results are the averages ± S.E. of
three experiments.
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The Upstream TRE Enhancer in the ERE-E1b-CAT Reporter Is Required
but Not Sufficient for Activation of ER-dependent Gene
Activation by Forskolin/IBMX--
Promoter differences in
forskolin/IBMX-induced responses suggested that cis-acting
factor(s) in addition to EREs contributed to cAMP-stimulated ER and
ER transcriptional activities. It has been reported that ER and
ER can mediate ligand-dependent responses at TREs,
independent of EREs, when they are tethered to the promoter via AP-1
transcription factor complexes (54, 55). Previous sequence analysis
(56) revealed that a putative TRE (TGACACA) that differs from the
consensus TRE sequence (TGAGTCA) by two nucleotides resides in the
backbone of many plasmid vectors. In ERE-E1b-CAT such a putative TRE is
located ~255 bp upstream from the ERE. To determine whether this TRE
played any role in the ability of forskolin/IBMX to stimulate the
activity of either ER, we examined the ability of forskolin/IBMX to
increase CAT gene expression from a reporter plasmid
(ERE-E1b-CAT(mTRE)) in which the TRE was mutated to GGACTCA, a mutation
previously demonstrated to abolish AP-1 binding (39, 57). As shown in
Fig. 5A, the TRE mutation
decreased overall reporter gene activity in the presence of both ER
and ER whether cells were treated with vehicle, E2, or
forskolin/IBMX. However, E2 was still able to increase
ERE-E1b-CAT(mTRE) activity above basal for both ERs, and forskolin/IBMX
stimulated this reporter activity in the presence of ER. In
contrast, forskolin/IBMX was unable to stimulate
ER-dependent transcription of ERE-E1b-CAT(mTRE), suggesting that this putative TRE was necessary for cAMP-induced ER-mediated increases in ERE-E1b-CAT reporter gene expression. Similar results were obtained when this TRE was removed by an NdeI to Eco0109I deletion of a 195-bp region
surrounding the site (39) as opposed to mutating it (data not shown).
Western blot analysis indicated that cAMP activation of ER on the
ERE-E1b-CAT reporter (with or without the TRE) was not caused by an
increase ER protein levels (Fig. 5B). In contrast to
ER, ER protein expression is elevated modestly by forskolin/IBMX.
However, this does not permit cAMP-stimulated ERE-E1b-CAT(mTRE)
activity by ER. Thus, cAMP activation of ER is dependent on a
putative TRE site, but loss of this cis element only
partially attenuates ER activity and indicates that activation of
ER and ER by cAMP signaling is distinct. Taken together with the
above data that demonstrated a requirement for an ERE, these data
suggest that forskolin/IBMX promotes synergism between either ER or
ER and a factor(s) that is bound to a neighboring DNA response
element (i.e. the putative TRE).
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Fig. 5.
A putative AP-1 response element in the
target gene promoter is essential for cAMP/PKA-mediated transcription
by ER. A, HeLa cells were
transiently transfected with 10 ng of expression plasmid for ER
(pCMV5-ER) or ER (pCXN2-ER) along with
1 µg ERE-E1b-CAT or ERE-E1b-CAT(mTRE). Cells were subsequently
treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX
(F/I). Values are normalized to ERE-E1b-CAT
reporter activity for ER in the absence of hormone and represent the
average ± S.E. of three experiments. B, HeLa cells
were transfected with 1 µg of either FLAG-ER or 3xFLAG-ER
expression plasmid, and receptor expression was detected with anti-FLAG
(M2) antibody. The blot shown is representative of three
experiments.
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To determine whether c-Jun and/or c-Fos expressed in HeLa cells could
bind to the putative TRE site, an electrophoretic mobility shift assay
was performed. As shown in Fig. 6, a
27-bp oligonucleotide corresponding to the TRE-containing region of the
target gene bound to factors present in a HeLa cell nuclear extract,
and this binding could be competed with an excess of cold
oligonucleotide. The addition of antibodies against c-Jun or c-Fos to
the binding reaction resulted in the supershift of the
oligonucleotide-protein complex, indicating that both of these proteins
associate with this DNA fragment. Notably, equivalent levels of
oligonucleotide-bound material were observed regardless of whether the
nuclear extract was prepared from vehicle- or forskolin/IBMX-treated
cells, indicating that the forskolin/IBMX-induced activation of
ER-dependent gene expression was not a result of increased
Jun/Fos occupancy of the promoter region. This is consistent with the
comparable levels of c-Jun and the small increase in c-Fos expression
detected by Western blot analyses of extracts prepared from vehicle-
and forskolin/IBMX-treated cells (data not shown).
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Fig. 6.
c-Jun and c-Fos binding to the putative TRE
is similar in vehicle- and forskolin/IBMX-treated HeLa cells.
Nuclear extracts prepared from vehicle () or 10 µM
forskolin/100 µM IBMX-treated cells (+) were subjected to
electrophoretic mobility shift assays using a 32P-labeled
TRE as probe. Competitions with a 100-fold excess of unlabeled probe
(TRE) or a DNA fragment containing a mutation in the TRE
site (mTRE) are shown in lanes 3 and
6, and 4 and 7, respectively.
Supershift assays are shown with c-Jun (lanes 9 and
11) or c-Fos (lanes 8 and 10)
antibodies. Lane 1 contains no nuclear extract. A
representative experiment is shown.
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The Carboxyl Terminus of ER and ER Mediates
Forskolin/IBMX-induced Transcription, Which Can Be Enhanced
Further by the Amino Terminus of ER but Not ER--
Two previous
reports have characterized the physical interactions between ER and
the AP-1 transcription factor family member, c-Jun (55, 58). Although
one of these studies demonstrated that ER interaction with c-Jun is
predominantly mediated by the centrally located hinge region (domain D)
of the receptor (58), both indicated that an interaction with the amino
terminus of ER is also possible. Therefore, to assess the potential
contribution of the amino-terminal A/B domain in mediating
forskolin/IBMX-induced responses, ER and ER deletion mutants
lacking their A/B domains (ER-179C and ER-143C) were constructed,
and the ability of the resulting receptors to stimulate ERE-E1b-CAT
reporter activity in response to forskolin/IBMX stimulation was
examined. In the absence of transfected ER (Fig.
7A, Empty), there
is very weak CAT gene expression in response to forskolin/IBMX
treatment. This minimal promoter activity is substantially less
compared with activity in the presence of transfected ERs. As shown by
comparison of ER-179C (ER amino acids 179-595) with wild type
ER, deletion of the amino terminus of ER reduced forskolin/IBMX
as well as basal and E2-stimulated activities, which is
consistent with the constitutively active amino-terminal AF-1 domain of
this receptor contributing to E2-dependent
ER responses (7, 11). In contrast, removing the A/B region of ER
to generate ER-143C (ER amino acids 143-530) did not reduce
forskolin/IBMX induction of ER-dependent target gene
expression. Notably, the E2-stimulated activity of ER-143C is much higher than that of wild type ER, which is in agreement with a previously reported inhibitory function for the amino
terminus of ER (59).
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Fig. 7.
Different ER and
ER domains mediate promoter-specific gene
expression in response to the cAMP/PKA signaling pathway. HeLa
cells were transiently transfected with 10 ng of expression plasmid for
ER (pCR3.1-ER), ER-179C (pCR3.1-ER-179C), ER
(pCR3.1-ER), ER-143C (pCR3.1-ER-143C), or empty vector
(pCR3.1) along with 1 µg of either ERE-E1b-CAT (A) or
ERE-E1b-CAT(mTRE) (B). Cells were subsequently treated with
vehicle, 1 nM E2, or 10 µM
forskolin + 100 µM IBMX (F/I).
Values are the average ± S.E. of three experiments.
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As shown in Fig. 7B, ER but not ER retained the
ability to stimulate CAT expression from the TRE-minus reporter
(ERE-E1b-CAT(mTRE)) in response to forskolin/IBMX (see also
Fig. 5). Interestingly, much of the E2-induced and all of
the remaining forskolin/IBMX-induced ER activity are lost when the
A/B domain is removed, as shown for ER-179C, thus supporting the
above data that this domain can mediate cAMP-dependent
activation of ER. Taken together, these results demonstrate that
domains C through F of ER and ER are sufficient for cAMP/PKA
signaling pathway activation of either receptor provided that an AP-1
DNA binding site is present on the promoter; these results also
indicate that this functional interaction is enhanced by the A/B domain
of ER but not ER.
Several studies have focused on the ability of the MAP kinase signaling
pathway to stimulate the AF-1 activity of ER (8, 10). Because it is
known that the cAMP/PKA signaling pathway can cross-talk with the MAP
kinase signaling pathway (34, 60), we examined whether the MAP
kinase-directed phosphorylation site in the amino terminus of ER
(Ser118) might be important for the forskolin/IBMX-induced
activity of the ER AF-1 domain. However, mutating this serine to an
alanine (ER-S118A) did not alter the ability of ER to stimulate
transcription of the ERE-E1b-CAT(mTRE) reporter in response to
forskolin/IBMX (data not shown). Similarly, alanine mutation of the
other known amino-terminal phosphorylated residues in ER
(Ser104/106/118 or Ser167) did not inhibit
forskolin/IBMX induction of reporter gene expression (data not shown),
indicating that these phosphoserine residues did not account for the
forskolin/IBMX-dependent activity of the ER AF-1 domain.
Thus, ER AF-1 activity in response to forskolin/IBMX is likely to be
mostly the result of cAMP/PKA signaling to a factor(s) that can
interact with the A/B domain rather than altering the phosphorylation
of the ER amino terminus itself.
Functional Interactions with the TRE-bound Factor(s) Can Be
Mediated by the EF Region of ER but Not ER--
It has been
demonstrated that amino acids 259-302 of ER constitute a major
interaction site with c-Jun (58), and because the ability of ER-179C
to mediate forskolin-induced activation of CAT expression was dependent
on a TRE site within the reporter gene that has been shown to support
c-Jun physical and functional interactions (39, 56), we investigated
the possibility that cAMP activation of ER-179C was caused by a
direct interaction between the hinge region of the receptor and c-Jun.
To test this hypothesis, the EF domain of ER (amino acids 302-595)
and the corresponding ER fragment (amino acids 254-530) were fused
to the Gal4 DNA binding domain (Gal-ER EF and Gal-ER EF) and
examined for their abilities to stimulate expression of 17mer-E1b-CAT, which contains four Gal4 binding sites upstream from the TATA box and
CAT gene. This reporter also possesses the TRE site upstream of the
Gal4 binding sites. As expected, both Gal-ER EF and Gal-ER EF
were stimulated by E2 (Fig.
8). Importantly, forskolin/IBMX stimulated Gal-ER EF-dependent expression of the
17mer-E1b-CAT but not the TRE-minus reporter,
17mer-E1b-CAT(TRE). This demonstrates that an ER
construct lacking all of the c-Jun binding sites can still be
activated; this indicates that the interaction between the TRE-binding
factor and ER could be indirect and may be mediated via a
trans-acting factor that can bind to both the EF domain of
ER and a TRE-binding factor such as c-Jun. In contrast, the EF
region of ER is insufficient to activate reporter gene expression regardless of the presence or absence of a TRE site, indicating that
regions within the DNA binding domain and/or hinge of ER are
important for activation of target gene expression in response to cAMP
signaling. Taken together, these results indicate that the EF domains
of ER and ER differ in their abilities to mediate ER cooperation
with a TRE-bound factor in response to cAMP.
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Fig. 8.
Mapped c-Jun interaction sites in the
ER A/B domain and hinge are not required for
forskolin/IBMX stimulation. HeLa cells were transiently
transfected with 100 ng of expression plasmid for Gal-ER EF
(pBind-ER EF), Gal-ER EF (pBind-ER EF), or Gal4 DNA binding
domain (pBind) along with 1 µg of either 17mer-E1b-CAT or
17mer-E1b-CAT(TRE). Cells were subsequently treated with
vehicle, 1 nM E2, or 10 µM
forskolin + 100 µM IBMX (F/I).
Values are normalized to Gal-ER EF activity for 17mer-E1b-CAT in the
presence of vehicle and represent the average ± S.E. of three
experiments.
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To assess the effect of forskolin/IBMX treatment on Gal-ER EF and
Gal-ER EF phosphorylation, expression vectors for Gal-ER EF or
Gal-ER EF were transfected into HeLa cells using an
adenovirus-mediated DNA transfer technique to increase protein
expression levels (47) followed by in vivo labeling with
[32P]H3PO4 and incubation with
forskolin/IBMX or vehicle for 90 min. The results of a representative
experiment are shown in Fig. 9, A and B, and demonstrate that when
32P signals were normalized to protein levels that there
was an increase in the overall level of Gal-ER EF phosphorylation
after incubation with forskolin/IBMX, whereas the same treatment
induced a decrease in Gal-ER EF phosphorylation. This experiment was repeated three times, and the averaged results reveal that
forskolin/IBMX increased Gal-ER EF phosphorylation by 44 ± 6%
but decreased Gal-ER EF phosphorylation by 45 ± 17% (Fig.
9C).
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Fig. 9.
Effect of forskolin/IBMX treatment on
Gal-ER EF and Gal-ER
EF phosphorylation. HeLa cells were transfected with
expression vectors for Gal-ER EF
(A) or Gal-ER EF
(B), incubated with 0.14 mCi/ml
[32P]H3PO4 followed by incubation
for 90 min with either vehicle (veh) or 10 µM
forskolin + 100 µM IBMX (F/I).
Gal-ER EF and Gal-ER EF were immunopurified and then
electrophoresed on a 10% SDS-polyacrylamide gel. The proteins were
transferred to a nitrocellulose membrane, and the membrane was exposed
to X-AR film for autoradiography (left panels). After
autoradiography, the membrane was subjected to Western blotting with
anti-Gal-horseradish peroxidase antibody and detection using enhanced
chemiluminescence (right panels). C,
autoradiography signals were quantitated by densitometric scanning of
films, and Western blot signals were quantitated by either
densitometric scanning of films or quantitation of the ECL signal using
a Kodak Image Station 440CF. Normalized phosphorylation values for
Gal-ER EF and Gal-ER EF were calculated by dividing the value for
the 32P signal by the value for the protein signal. Data
are plotted as change in phosphorylation relative to vehicle treatment
(set at a value of 1). The experiment was repeated three times with the
mean ± S.E. reported.
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Forskolin/IBMX-stimulated ER Activity Is Not Caused
by cAMP/PKA-dependent SRC-1
Phosphorylation--
It had been demonstrated previously that the
chicken PR is not phosphorylated in response to cell treatment with
8-bromo-cAMP, but rather cAMP activation of transcription seems to be
mediated by an increase in SRC-1 phosphorylation (34). Moreover, SRC-1 binds to both the EF domain of ER as well as c-Jun (61, 62) and is
therefore a good candidate for mediating indirect interactions between
these two transcription factors, as described above. Therefore, we
examined whether the ability of cAMP/PKA to modulate SRC-1 phosphorylation might also contribute to
forskolin/IBMX-dependent activation of ER. SRC-1
expression vectors containing substitutions for the two cAMP-induced
phosphorylatable residues (T1179/S1185A) or substitutions for all of
the seven previously mapped phosphorylation sites (at positions 372, 395, 517, 569, 1033, 1179, and 1185; Ref. 63) were introduced into
cells, and the abilities of these mutant coactivators to enhance
ER-dependent reporter activity was assessed. Compared
with wild type SRC-1 there is an ~20 and ~35% decrease in the
ability of the SRC-1T1179/S1185A and the seven-alanine
mutant (SRC-17Ala), respectively, to potentiate
forskolin/IBMX-induced ER activity (Fig.
10, A and B).
Nonetheless, decreases in ER coactivation by both the
SRC-1T1179/S1185A and the SRC-17Ala mutants
were equal for basal as well as for E2-stimulated and forskolin/IBMX-stimulated responses, suggesting that SRC-1
phosphorylation does not specifically modulate
cAMP-dependent activation of ER.
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Fig. 10.
Alanine mutation of SRC-1 phosphorylation
sites decreases its coactivation of ER but is
not specific to cAMP/PKA-dependent signaling.
A, a representative experiment in which HeLa cells were
transiently transfected with 10 ng of expression plasmid for ER
(pCMV5-ER) along with 1 µg of expression plasmid for
either wild type or mutant SRC-1a (SRC1T1179/S1185A or
SRC-17Ala) or the empty vector (pCR3.1) and 1 µg
ERE-E1b-CAT reporter. Cells were subsequently treated with vehicle, 1 nM E2, or 10 µM forskolin + 100 µM IBMX (F/I). B,
combined results from seven experiments. Relative coactivation was
determined by dividing reporter activity in the presence of wild type
SRC-1 by values obtained in the absence of coactivator (pCR3.1) for
each treatment group (vehicle, E2, and F/I) and defining
this value as 100. Values for mutant SRC-1 coactivation are given
relative to wild type SRC-1 coactivation. Results are the averages ± S.E. of seven experiments.
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DISCUSSION |
In this report, we demonstrated that forskolin/IBMX, through
increased intracellular cAMP and activation of PKA, can stimulate ER-dependent transcription, as was shown previously to
be the case for ER. However, there are significant differences in
the ability of ER and ER to be ligand-independently activated by this mechanism. In particular, a TRE upstream from the ERE was necessary for ER-dependent transcription in response to
stimulation with forskolin/IBMX, whereas this sequence contributed to,
but was not required for, activation of full-length ER. Furthermore, functional interactions with the TRE-bound factor(s) could be mediated
by the EF region of ER but not ER, and forskolin/IBMX treatment
increased the phosphorylation of Gal-ER EF but decreased the
phosphorylation of the corresponding ER construct. Overexpression of
the p160 and CBP coactivators enhanced forskolin/IBMX-induced ER and
ER activity, indicating that these coactivators can form functional
complexes with the unliganded receptor. However, in contrast to the
previously reported importance of SRC-1 phosphorylation for
cAMP-mediated activation of chicken PR (34), the contribution of these
phosphorylation sites to SRC-1 coactivation of cAMP-induced ER was
minor and not specific to ER activation by forskolin/IBMX. Taken
together, these data demonstrate that the cAMP signaling pathway can
stimulate ER-dependent transcription by promoting functional interactions among TRE-bound transcription factor(s), coactivators, and either ER or ER bound to an ERE.
There has been considerable interest in understanding the relative
contributions of the ER and ER AF-1 domains in mediating ER-dependent transcription. The A/B domain of ER, which
encompasses the AF-1, is generally more active than that of ER (59).
Interestingly, although a MAP kinase signaling pathway can induce ER
and ER AF-1 activity in the absence of ligand (8-10), our results
demonstrate that cAMP signaling can stimulate the AF-1 activity of
ER, but not ER. Although it has been reported that the cAMP
signaling pathway can stimulate MAP kinase activity, mutation of the
previously identified amino-terminal MAP kinase phosphorylation site in
ER (8, 10) does not inhibit its activation by forskolin/IBMX. Consistent with this result, our work and that of LeGoff et
al. (31) demonstrates cAMP-induced phosphorylation of the ER
carboxyl-terminal domain; however, the location of these
phosphorylation sites in vivo remains to be identified. It
is therefore likely that forskolin/IBMX activation of ER
via the AF-1 domain is caused by cAMP/PKA signaling effects on the
recruitment and/or activity of a coactivator(s) that interacts
selectively with the AF-1 domain of ER. Interestingly, there are
several coactivators, including the p72/p68 RNA-binding DEAD box
proteins (64, 65) and the RNA coactivator,
SRA,2 which have been found
to interact selectively with the AF-1 domain of ER but not that of
ER. Moreover, in addition to p68 being a phosphorylated protein
(66), all three of these coactivators are found in complexes with other
coactivator molecules, such as CBP, SRC-1, TIF2, and AIB1 (64, 65),
which are known to be phosphoproteins (34, 67-69).
The carboxyl-terminal ligand binding domains of ER and ER possess
considerably higher sequence homology than do the A/B domains.
Interestingly, however, there are also differences in the abilities of
the ER and ER EF domains to be activated by cAMP signaling, as
indicated by the ability of Gal-ER EF but not Gal-ER EF to
stimulate 17mer-E1b-CAT activity in response to forskolin/IBMX.
Interestingly, forskolin/IBMX induced opposite changes in overall
phosphorylation for these two constructs. Consistent with a previous
demonstration of cholera toxin and IBMX-induced phosphorylation of an
ER mutant lacking its A/B domains (31), forskolin/IBMX increased the
phosphorylation of the Gal-ER EF chimera. There are four protein
kinase A consensus sites
(XRRXSX or
SKKIXSIX) in the carboxyl terminus of ER:
Ser236, Ser305, Ser338, and
Ser518. However, mutation of the Ser236,
Ser305, and Ser518 sites to alanines does not
block cAMP-induced ER transcriptional activity (35), and the
Ser236 site is not present in the Gal-ER EF construct.
Although a S338A mutation does inhibit cholera toxin/IBMX-induced ER
activity, substitution of a glutamic acid at this position does not
mimic the ligand-independent response, suggesting that mutation of this residue may affect cAMP signaling to the receptor by inducing structural perturbations (35). The only other phosphorylation sites
identified in the ER carboxyl terminus, Thr311 (70) and
Tyr537 (71), are also unlikely to be the targets of the
forskolin/IBMX-induced phosphorylation site because cAMP signaling has
been reported to induce only serine phosphorylation of ER (31). The
location of the phosphorylation site(s) induced by forskolin/IBMX
in vivo, as well as their importance for cAMP activation of
ER transcriptional activity therefore remain to be characterized;
this is however, beyond the scope of this report. It should be noted
that the forskolin/IBMX-induced site need not lie within a protein
kinase A consensus sequence because the cAMP signaling has been shown
to cross-talk with other kinase pathways (34, 60). Apart from an
in vitro demonstration of p38-induced
phosphorylation of the AF2 domain of ER (72), little is known about
phosphorylation of the ER EF domain. Our results therefore provide
the first demonstration that the ER carboxyl-terminal region is
phosphorylated in vivo, as well as novel information
indicating that the basal level of ER EF domain phosphorylation is
reduced by forskolin/IBMX treatment in HeLa cells. Although this may
seem paradoxical, cAMP has been shown to inhibit MAP kinase pathways
through cross-talk with the G protein Rap1 and Raf-1 (60).
The inability of the AF-1-deletion mutants (ER-179C and ER-143C)
and the Gal-ER EF chimera to stimulate the activities of reporters
lacking TREs (ERE-E1b-CAT(mTRE) and 17mer-E1b-CAT(TRE), respectively) in response to forskolin/IBMX also suggests that activation of domains EF of ER and C through F of ER require a
functional interaction between these receptor domains and a TRE-boun
(ER
-dependent Gene Expression
by cAMP Signaling Pathway(s)*
,
TOP
ABSTRACT
INTRODUCTION
