Differential Hormone-Dependent
Phosphorylation of Progesterone
Receptor A and B Forms Revealed
by a Phosphoserine Site-Specific
Monoclonal Antibody
David L. Clemm, Lori Sherman, Viroj Boonyaratanakornkit,
William T. Schrader, Nancy L. Weigel, and Dean P. Edwards
Endocrine Research (D.L.C., W.T.S.)
Ligand Pharmaceuticals, Inc.
San Diego, California 92121
Department of Molecular and Cellular Biology (N.L.W.)
Baylor College of Medicine
Houston, Texas 77030
Department of Pathology and Molecular Biology Program
(L.S., V.B., D.P.E.)
University of Colorado School of Medicine
Denver, Colorado 80262
Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven
sites) in a manner that involves distinct groups of
sites coordinately regulated by hormone and different kinases. Progress on defining a functional
role for PR phosphorylation has been hampered
both by the complexity of phosphorylation and the
lack of simple, nonradioactive methods to detect
the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo.
Toward this end, we have produced monoclonal
antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a
basal site at Ser 190 (MAb P190) and a hormoneinduced site at Ser 294 (MAb P294). Biochemical
experiments showed the differential reactivity of
the P190 and P294 MAbs for phosphorylated and
unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under
different experimental conditions including denatured PR protein by Western blots and PR in its
native conformation in solution or complexed to
specific target DNA. As detected by Western blot of
T47D cells treated with hormone for different
times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This
difference in kinetics suggests that the Ser 294 site
is more labile than basal sites and is acted upon by
distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was
observed on PR-B as compared with the aminoterminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences
are identical on both proteins, suggesting that a
distinct conformation of the N-terminal domain of
PR-A inhibits phosphorylation of this site. That Ser
294 lies within an inhibitory domain that mediates
the unique repressive functions of PR-A raises the
possibility that differential phosphorylation of Ser
294 is involved in the distinct functional properties
of PR-A and PR-B. (Molecular Endocrinology 14:
52–65, 2000)
INTRODUCTION
The human progesterone receptor (PR) is phosphorylated on at least seven serine residues, which are all
located in the amino-terminal (N) domain (1–3). Three
sites (Ser 81, Ser 102, Ser 162) are within the Nterminal segment unique to full-length PR-B, while the
others are located within a region shared by PR-B and
the N-terminally truncated A form of PR (Ser 190, Ser
294, Ser 345, and Ser 400). Identification of phosphorylation sites was made as previously described by
conventional 32P-radiolabeling and phosphopeptide
mapping in conjunction with sequencing of PR phosphopeptides isolated from T47D breast cancer cells
(1–3). Treatment of cells with a progestin agonist stim-
0888-8809/00/$3.00/0
Molecular Endocrinology
Copyright © 2000 by The Endocrine Society
52
Phosphospecific PR Antibodies
ulates phosphorylation of PR through at least two
mechanisms. Four sites are basally phosphorylated
(Ser 81, Ser 162, Ser 190, and Ser 400) and, upon
hormone treatment, exhibit a rapid ' 2-fold increase.
The other sites are hormone inducible (Ser 102, Ser
294, Ser 345) and 1–2 h of treatment are required to
reach maximal phosphorylation. The different kinetics
in response to hormone suggest that these two groups
of phosphorylation sites are targets of different signaling pathways and kinases and serve distinct functional/structural roles. At least three different kinases have
been identified that specifically phosphorylate PR in
vitro on authentic sites including casein kinase II on
Ser 81 (1), cyclin A-cyclin-dependent kinase 2 on Ser
162, Ser 190, and Ser 400 (3), and MAP kinase on Ser
162 and Ser 294 (5). The kinases that modify other
sites is not known and whether these enzymes are the
physiological regulators of PR in vivo also remains
unknown.
The functional role of PR phosphorylation remains
elusive. Agents that either activate or inhibit cellular
serine/threonine kinases and phosphatases in different signaling pathways have been observed to
dramatically potentiate or inhibit the transcriptional
activity of PR in cell transfection experiments (6–8).
Additionally, activation of protein kinase A (PKA)
through cAMP signaling leads to a functional
switching of RU486 from an antagonist to a partial
agonist on the B form of PR, but not through PR-A
(8–10). Whether the influence of these agents on the
transcriptional activity of PR is due to alterations of
receptor phosphorylation directly, or to phosphorylation of other associated proteins involved in PR
transactivation, appears to be dependent on the
signaling pathway involved. We were unable to detect a change in total phosphorylation, or in phosphopeptide maps of PR in response to cAMP (Ref. 6,
and C. A. Beck, N. L.Weigel, and D. P. Edwards,
unpublished). However, we cannot rule out an effect
on phosphorylation of PR directly, since all the
phosphorylation sites have not been identified (1–3).
Based on our phosphopeptide mapping experiments, there are at least two additional phosphorylation sites in human PR that have not yet been
identified by sequencing, bringing the total number
of sites to nine (1–3).
The consequence of phosphorylation on the transcriptional activity of PR remains largely unknown.
Takimoto et al. (11) analyzed a series of serine to
alanine substitution mutations for effects on the
transcriptional activity of PR in cell transfection assays. However, the results are difficult to interpret
since almost all the mutations were introduced into
groups of putative, unproven, serine phosphorylation sites. The exception was an alanine substitution
for Ser 190 that was observed in certain cell and
promoter contexts to exhibit a 50% reduction in
transcriptional activity (11). Thus functional analysis
of most of the PR phosphorylation sites remains
untested by site-directed mutagenesis. Similar
53
studies with other steroid receptors have reported
either partial reductions or enhancements of transcriptional activities by introduction of phosphorylation site mutations ( Refs. 12–18; reviewed in Ref.
19). These limited results with human PR, taken
together with studies of other steroid receptors,
suggest that phosphorylation does not serve as a
regulatory on-off switch for transcriptional activity,
but functions to either amplify or attenuate activity.
Such a modulatory role could be achieved by phosphorylation altering the strength of coactivator association with receptor , as was recently shown for
the estrogen receptor b (20). Alternatively, phosphorylation may be involved in integrating signals
from other pathways, or in regulating cellular trafficking or degradation of receptor protein (18, 21).
A major advancement in the cell signaling field has
resulted from the development of antibodies to specific phosphorylation sites of intracellular signaling
molecules and nuclear transcription factor targets
(22–25). These antibodies have enabled the simple,
rapid, and sensitive detection of specific phosphorylation states of proteins in cells in response to
activators or inhibitors of signaling pathways. Antibodies that recognize specific phosphorylation sites
of nuclear receptors have not been available. Thus,
steroid receptor phosphorylation studies have required cumbersome and laborious 32P-labeling
experiments. Because of the complexity of PR
phosphorylation, meaningful 32P-labeling results
cannot be obtained by simply analyzing net 32P
incorporation. Analysis of individual sites requires
additional phosphopeptide-mapping experiments,
thus severely limiting the number of samples that
can be evaluated simultaneously. Additionally,
phosphopeptide mapping is not quantitative, recovery of individual peptides can vary, and 32P-labeling
conditions may themselves alter some signaling
pathways. What is needed is a rapid and simple
method to detect phosphorylation states of steroid
receptors in whole cells.
Monoclonal antibodies (MAbs) that specifically
recognize human PR phosphorylated on Ser 190 or
Ser 294 were developed in this study. Biochemical
experiments describe the utility of these MAbs for
detection of specific phosphorylation states of PR
within whole cells and for differential recognition of
phosphorylated and unphosphorylated forms of PR
under different experimental conditions. Experiments with the MAb (P294) specific for PR phosphorylated on Ser 294 revealed that this site is preferentially phosphorylated in response to hormone
on PR-B, despite the fact that Ser 294 lies within the
N-terminal domain of identical amino acid sequence
shared by PR-A and PR-B. These results suggest
that differential phosphorylation of Ser 294 may be
involved in the distinct functional activities of PR-A
and PR-B (26–28).
MOL ENDO · 2000
54
Vol 14 No. 1
Fig. 1. Schematic of Human PR Structure and Specificity of P190 and P294 MAbs for Phosphorylated Forms of PR
A, Serine phosphorylation sites in human PR and phosphopeptides used to generate P190 and P294 MAbs. B, Whole cell
extracts prepared from T47D cells after treatment for 1 h at 37 C with R5020 (100 nM), were analyzed by Western blot with the
P190 or P294 MAbs. The MAbs (4 mg/ml of P190 and 10 mg/ml of P294) were preincubated overnight at 4 C before addition to
Western blot filters with either no additions (2) or a 200-fold molar excess of the peptides indicated including phosphorylated
(p-Ser 190) and unphosphorylated Ser 190 peptide (Ser 190), phosphorylated (p-Ser 294) and unphosphorylated Ser 294 peptide
(Ser 294), and phosphorylated Ser 345 peptide (p-Ser 345).
RESULTS
Production and Specificity of MAbs to Specific
Serine Phosphorylation Sites (Ser 190 and Ser
294) in Human PR
A schematic of human PR structure and the location of
previously defined phosphorylated serine residues is
shown in Fig. 1A. The sites are divided into two
groups: basal sites that are phosphorylated in the
absence of ligand and increase in response to hormone binding (Ser 81, Ser 162, Ser 190, Ser 400), and
the truly hormone-dependent sites (Ser 102, Ser 294,
and Ser 345). As a first attempt to produce antibodies
that recognize specific phosphorylated forms of PR,
we chose one basal (Ser 190) and one hormone-de-
pendent (Ser 294) site. Peptides corresponding to
amino acids 184–196 (contains Ser 190) and 288–300
(contains Ser 294) of human PR were synthesized (see
Fig. 1A), and the serine residues were chemically
phosphorylated, conjugated to keyhole limpet hemocyanin (KLH), and the conjugate was used as an antigen to inject mice. Hybridomas produced by standard procedures (29) were screened by enzyme-linked
immunoabsorption assay (ELISA) for preferential binding to the specific phosphopeptide as compared with
the unphosphorylated peptide. This was followed by
Western blot screening for reaction with full-length PR
from untreated and hormone (synthetic progestin agonist R5020)-treated T47D breast cancer cells. One
monoclonal antibody (MAb) was isolated from each
cell fusion that recognized the native PR protein in a
Phosphospecific PR Antibodies
55
Fig. 2. P190 and P294 Mabs Are Highly Selective for Hormone Activated PR, with P294 Exhibiting a Preference for PR-B
Cytosol and 0.4 M NaCl extractable nuclear fractions were prepared from T47D cells after treatment for 1 h at 37 C with vehicle
(ethanol) or R5020 (100 nM). Aliquots of cytosol (C) and nuclear extracts (N) containing equal amounts of total protein (20 mg) were
analyzed by Western blot with 1294 (for detection of total PR-A and PR-B), P190 or P294 MAbs.
phosphorylation-dependent manner. These included
clone 1154/F12 raised against the Ser 190 phosphopeptide and clone 608/G5 raised against the Ser
294 phosphopeptide. Both MAbs are mouse IgG1s,
hereafter termed P190 and P294, respectively.
Specificity for phosphorylated forms of PR was first
tested by comparing the ability of phospho-and dephosphorylated peptides to block MAb binding to PR
in a Western blot assay. PR was prepared as a whole
cell lysate from hormone-treated (R5020 for 1 h at 37
C) T47D breast cancer cells. T47D cells express both
the A (90 kDa) and B (118 kDa) isoforms of PR at
similar levels (29, 30). The P190 and P294 MAbs reacted strongly with proteins of the correct size for
PR-A and PR-B (Fig. 1B, lanes 1 and 6, respectively).
Weak cross-reactions with smaller mol wt protein
bands were detected with both MAbs. Whether these
are degradation products of PR or epitopes in other
proteins recognized by the MAbs is not known. Nonetheless, intact PR-A and B are the major immunoreactive proteins in Western blots of whole-cell extracts.
Whereas P190 exhibited comparable reactivity with
the A and B forms of PR, P294 reacted preferentially
with PR-B (Fig. 1B and see Figs. 2, 3, 4, and 6). P190
reactivity with both PR proteins was completely
blocked by preincubation with excess phosphorylated
Ser 190 peptide (Fig. 1B, lane 2), but was unaffected
by the unphosphorylated Ser 190 peptide or by phosphopeptides containing other phosphorylation sites of
PR, including Ser 345 and Ser 294 (Fig. 1B, lanes 2–5).
P294 reactivity with PR was blocked by an excess of
phosphorylated Ser 294 peptide, but not by unphosphorylated Ser 294 peptide or phosphorylated Ser 190
and Ser 345 peptides (Fig. 1B, lanes 7–10). Additional
phosphopeptides corresponding to other phosphorylation sites in PR and human AR (Ser 81 in PR and Ser
650 in human AR) also had no effect on P190 or P294
immunoreactivity with PR (not shown). Because the
majority of phosphorylation sites in steroid receptors
are Ser-Pro motifs, including Ser 190 and Ser 294 of
human PR, we also tested the P190 and P294 MAbs
for cross-reaction with other steroid receptors. By
Western blot of Sf9 insect cell extracts, neither P190
nor P294 cross-reacted with baculovirus-expressed
human estrogen receptor, glucocorticoid receptor, or
androgen receptor. Under the same conditions, both
MAbs reacted strongly with baculovirus-expressed
human PR-B (data not shown). The peptide competition results, which show specificity for the specific
phosphorylated peptide, taken together with the ability
of the MAbs to selectively detect full-length PR protein, suggest that P190 and P294 MAbs are specific,
respectively, for Ser 190 and Ser 294 phosphorylated
forms of PR.
From our earlier 32P-labeling studies, phosphorylation of Ser 190 was shown to be partial in the absence
of hormone, while Ser 294 phosphorylation was highly
dependent on hormone (2). It has long been known
that PR in the absence of hormone distributes between NaCl-extractable nuclear and cytosol fractions
of T47D cells, while hormone treatment stimulates the
major amount of cellular PR to bind tightly to nuclei
(29, 30). We therefore asked whether the reactivity of
P190 and P294 with PR was influenced by hormone
treatment of cells and was generally, or preferentially,
distributed between cytosol and nuclear receptors.
Western blots were performed with cytosol and highsalt extracts of nuclei prepared from T47D cells that
had been treated for 1 h at 37 C with or without R5020.
To determine the total amount of PR (phosphorylated
and nonphosphorylated), another MAb (clone 1294,
mouse IgG1) was used that recognizes a nonphosphorylated epitope in the N-terminal region of human
PR that is common to the A and B isoforms (B. Spaulding, L. Sherman, and D. P. Edwards, unpublished).
Western blots with 1294 show that total PR in the
MOL ENDO · 2000
56
Vol 14 No. 1
Fig. 3. Two Classes of Progesterone Antagonists, RU486 and ZK98299, Have Distinct Effects on PR Reactivity with P190 and
P294 Mabs
A, T47D cells were treated for 1 h at 37 C with vehicle, R5020 (100 nM), RU486 (100 nM), or ZK98299 (200 nM). Whole-cell
extracts were prepared from each treatment group and analyzed by Western blot with 1294, P190, or P294 MAbs. B, T47D cells
were treated and analyzed as in panel A except that cells were incubated with vehicle, R5020 (5 nM), ZK98299 (200 nM), or R5020
(5 nM) plus ZK98299 (200 nM), and the Western blot assay was done with the P294 MAb only.
absence of ligand is distributed comparably between
cytosol and nuclear extracts, while the majority of
receptors were localized in the tight nuclear bound
fraction after hormone treatment (Fig. 2, left panel). In
non-hormone-treated cells, P190 immunoreactive PR
in both cytosol and nuclear fractions was substantially
lower than total PR (compare 1294 and P190, Fig. 2).
Hormone treatment increased PR reactivity with P190
exclusively in the nuclear fraction (Fig. 2, P190 panel).
Interestingly, the reactivity of P190 in lysates of nonhormone-treated cells was preferential for nuclear
PR-A, while hormone stimulation of P190 phosphorylation was equally distributed between PR-A and PR-B
(Fig. 2). Although preferential phosphorylation of
Ser190 on PR-A in the absence of hormone was typically observed (see Figs. 2, 3, 5, and 6), some variation occurred as shown in the time course experiment
of Fig. 4, in which Ser 190 phosphorylation on PR-A
was only slightly higher than on PR-B. Such variation
is likely due to the presence of trace hormone in the
culture medium capable of stimulating some phosphorylation of Ser 190 and thus minimizing the differ-
Phosphospecific PR Antibodies
ential between PR-A and PR-B. Nonetheless, these
results suggest that basal phosphorylation of Ser 190
is preferential for a subpopulation of PR-A tightly associated with nuclear components. Consistent with
preferential phosphorylation of unliganded nuclear
PR-A, Lim et al. (32), using green fluorescent-tagged
PR-proteins, reported that unliganded PR-A localizes
primarily in the nucleus while PR-B is distributed between nuclear and cytoplasmic compartments.
The P294 MAb exhibited essentially no reactivity
with either cytosol or nuclear PR from non-hormonetreated cells; immunoreactivity was observed only
with nuclear PR after hormone treatment of cells. Even
though hormone-treated cells contain an equal
amount of total nuclear PR-A and PR-B, P294 reactivity was strongly preferential for PR-B, indicating that
phosphorylation of this hormone-dependent site is
substantially different on the A and B forms of receptor
(Fig. 2, P294 panel). The influence of R5020 on the
reactivity of PR with the P190 and P294 MAbs is
consistent with our earlier 32P-labeling studies. Ser
190 was basally phosphorylated and increased in response to hormone binding, while Ser 294 phosphorylation was highly hormone dependent (1–4).
We next examined the influence of two different
classes of progesterone antagonists, RU486 and
ZK98299, on phosphorylation of Ser 190 and Ser
294 as detected with MAbs. By 32P-labeling and
peptide mapping, we previously observed that
RU486 stimulated phosphorylation of basal and hormone-dependent PR phosphorylation sites the
same as the agonist R5020, whereas ZK98299 inhibited phosphorylation of both types of sites (4).
Consistent with previous 32P-labeling results,
RU486 and R5020 had similar effects on stimulating
P190 and P294 MAb interaction with PR. RU486
stimulated binding of both isoforms of PR to P190
MAb and preferentially induced PR-B reactivity with
P294 (Fig. 3A). In contrast, ZK98299 minimally stimulated P294 and P190 binding to PR (Fig. 3, A and
B). The low reactivity of the MAbs with PR in the
presence of ZK98299 was not due to a loss of PR
protein as shown by Western blot with the 1294 MAb
that detects total PR (Fig. 3A). Because ZK98299
has a lower affinity (10-fold) for PR than R5020 or
RU486 (33, 34), it was important to determine
whether the failure to stimulate PR interaction with
P190 and P294 was due to an inefficient binding of
ZK98299 to PR in cells. When T47D cells were cotreated with R5020 (5 nM) and ZK98299 (200 nM),
ZK98299 effectively blocked R5020 stimulation of
P294 interaction with PR-B (Fig. 3B). These results
indicate that ZK98299 competes with R5020 for
binding to PR in whole cells, but fails to induce
phosphorylation. The influence of different PR ligands (Figs. 2 and 3), taken together with peptide
competition (Fig. 1B) results, provides strong evidence that the P190 and P294 MAbs specifically
recognize, respectively, Ser 190 and Ser 294 phosphorylated forms of PR.
57
Distinct Kinetics of Ser 190 and Ser 294
Phosphorylation in Response to Hormone and
Preferential Phosphorylation of Ser 294 on PR-B
In our earlier 32P-labeling studies we observed that
hormone effects on phosphorylation of basal (Ser 190)
and hormone-dependent sites (Ser 294) followed different kinetics. Basal sites increased rapidly (10–20
min) in response to hormone treatment while hormone-dependent sites responded more slowly requiring 1–2 h to reach maximal phosphorylation (2, 4). To
examine the kinetics of phosphorylation of Ser 190
(basal site) and Ser 294 (hormone-induced site) in
more detail, and further test the utility of the MAbs for
PR phosphorylation studies, we performed Western
blots with P190 and P294 of T47D extracts taken at
different times after R5020 treatment of cells. The
1294 MAb was used in parallel to determine the total
amount of PR-A and PR-B (Fig. 4A). The relative
amount of immunoreactive PR-A and PR-B at each
time point was quantitated by phosphorimager analysis, and the results with P190 and P294 were normalized to the total PR levels obtained with the 1294 MAb
(Fig. 4, B–D). As expected, hormone had minimal effect on total PR between 0.2 h and 1 h of treatment,
but resulted in a significant decrease that started at 3 h
after hormone treatment and continued for 24 h (Figs.
4, A and B). At 24 h of hormone treatment, total PR
was reduced by '80%. It should be noted that total
PR-A and PR-B were expressed at similar levels ('1:1
ratio) at all time points of R5020 treatment, indicating
that the two isoforms of PR down-regulate at the same
rate (Fig. 4, A and B). This reduction of total PR in
response to continuous treatment with progestin agonists has been shown previously to be due to the
combined effect of decreased PR mRNA expression
and increased turnover of PR protein (30, 35). Liganddependent PR down-regulation represents an
80–90% decrease in the steady-state level of PR that
takes 24–48 h to achieve (30, 35).
As detected by Western blot reactivity with the P190
MAb, phosphorylation of Ser 190 increased rapidly on
both PR-A and PR-B (approximately equal with both
isoforms) reaching a maximum between 30 and 60 min
of hormone treatment (Fig. 4A). Between 60 min and
24 h of hormone treatment, P190 immunoreactivity
decreased progressively (Fig. 4A). However, when
normalized to total PR at each time point, P190 reactivity increased only for the first 1 hour of hormone
treatment and remained unchanged relative to total
PR between 60 min and 24 h (Fig. 4C). This result
indicates that the Ser 190 phosphorylation site increased rapidly in response to hormone reaching a
steady state at 30–60 min and thereafter turned over at
the same rate as down-regulation of total PR. As detected by the P294 MAb, phosphorylation of Ser 294
increased more markedly in response to hormone and
at a slower rate than the Ser 190 site, reaching a
maximum between 1 and 3 h of hormone treatment
(Fig. 4, A and D). Additionally, the majority of the
MOL ENDO · 2000
58
Vol 14 No. 1
Fig. 4. Kinetics of Ser 190 and Ser 294 Phosphorylation in Response to Continuous Treatment of T47D Cells with Hormone
T47D cells were treated with vehicle (ethanol) or R5020 (100 nM) for the times indicated. At each time point, cells were
harvested, and whole-cell lysates were prepared and analyzed by Western blot with 1294, P190, or P294. MAb binding to PR was
detected using ECL Plus (Amersham Pharmacia Biotech) and quantitated on a Molecular Dynamics, Inc. Storm 860 Phosphorimager. A, Autoradiography of the region of Western blots containing PR-A and PR-B. B–D, Phosphorimager analysis of the
Western blot results in panel A. Arbitrary Phosphorimager fluorescence units were graphed for separate PR-A and PR-B values
with each MAb. For P190 and P294, the Phosphorimager units were normalized to total PR detected by 1294. Values were
normalized by setting the Ser 1294 time point to 1.0 and each 1294 time point thereafter as a fraction of 1.0. The P190 and P294
values were then taken as a proportion of the normalized 1294 values. The data are from a single experiment that is representative
of five independent experiments.
Phosphospecific PR Antibodies
increased phosphorylation of Ser 294 occurred on
PR-B. At the peak of hormone stimulation, the ratio of
Ser 294 phosphorylation of PR-B to PR-A was more
than 5:1 (Fig. 4, A and D). When the P294 immunoreactivity was normalized to total PR, the time course
experiment shows that hormone stimulated a new
steady state level of phosphorylation of Ser 294 that
required 1–3 h to achieve. Between 3 and 6 h of
hormone treatment, Ser 294 phosphorylation decreased at a faster rate than that of total PR and then
turned over at the same rate as total PR between 6 and
24 h (Fig. 4D). Thus, the Ser 294 site is more transiently
phosphorylated in response to hormone than the Ser
190 site. It should also be noted that R5020 treatment
caused a slight upshift in electrophoretic mobility of
PR-A and PR-B, which has been shown previously to
be associated with phosphorylation of Ser 345 (2). As
expected, the P190 and P294 MAbs (Fig. 4) bind to the
upshifted receptors, indicating that most of the upshifted Ser345 phosphorylated receptor is also phosphorylated on Ser190 and Ser294.
Preferential Phosphorylation Of Ser 294 On PR-B
in its Native Conformation in Solution and When
Complexed to Target DNA
We next sought to determine whether the influence of
hormone on phosphorylation of Ser 190 and Ser 294
and the differential phosphorylation of Ser 294 on
PR-B detected by Western blot also occurred with PR
in its native conformation. PR was immunoprecipitated under nondenaturing conditions with P294 or
P190 from lysates of T47D cells that had been treated
with and without hormone. The immunoprecipitates
were then analyzed by Western blot with 1294 MAb.
As a control for total PR, samples were also immunoprecipitated with the 1294 MAb. As expected, 1294
immunoprecipitated approximately the same amount
of PR-A and PR-B from hormone and non-hormonetreated cells (Fig. 5). However, the P294 MAb preferentially immunoprecipitated PR-B from cells treated
acutely (1 h) with hormone, whereas little PR-A from
hormone-treated cells or either isoform of PR from
non-hormone-treated cells was immunoprecipitated
(Fig. 5). With the P190 MAb, PR-A was preferentially
immunoprecipitated from non-hormone-treated cells,
while substantially greater amounts of both PR-A and
PR-B were immunoprecipitated from hormone-treated
cells (Fig. 5). These immunoprecipitation results are
consistent with Western blots, indicating that differential reactivity with P190 and P294 reflects the phosphorylation state of PR in its native conformation.
To determine the Ser 190 and Ser 294 phosphorylation state of native PR-A and PR-B complexed to
target DNA, we examined the ability of both MAbs to
supershift receptor-DNA complexes in electrophoretic
gel mobility shift assays (EMSAs). Because T47D cells
express both PR-A and PR-B, EMSAs of T47D cell
extracts are complicated by the presence of PR-A/
PR-B heterodimers (36). Therefore, PR-A and PR-B
59
Fig. 5. Selective Immunoprecipitation of Phosphorylated
Forms of PR by P190 and P294
T47D cells were treated for 1 h at 37 C with ethanol vehicle
or R5020 (100 nM), and whole-cell extracts containing equal
amounts of total protein (100 mg) were immunoprecipitated
using protein A Sepharose as an immunoabsorbent. The
1294, P190, and P294 MAbs were prebound to protein A
Sepharose through a secondary rabbit antimouse IgG as
previously described (43). Whole-cell extracts were then incubated in suspension with the MAb-coated protein Sepharose beads for 3 h at 4 C. The beads were washed in buffer
(KPFM) containing 0.4 M NaCl (to disrupt PR dimerization),
and samples were eluted from the beads with 2% SDSsample buffer and analyzed by Western blot with 1294 MAb.
The heavy and light chains of the MAbs used for immunoprecipitation that react on Western blot are indicated by
arrows.
were separately expressed in a PR-negative mammalian cell line (COS) by use of recombinant adenoviral
vectors (37). We first examined whether hormone influenced the phosphorylation of adenovirus expressed
PR-A and PR-B the same or differently than native PR
in T47D cells. As detected by Western blot with P294,
adenovirus-expressed PR-A was not appreciably
phosphorylated on Ser 294 in the presence or absence
of hormone, while hormone stimulated a preferential
phosphorylation of Ser 294 on PR-B (Fig. 6, top panel).
As detected by the P190 MAb, phosphorylation of Ser
190 was observed preferentially on adenovirus-expressed PR-A in the absence of hormone and was
stimulated by hormone to comparable levels on both
PR-A and PR-B (Fig. 6, middle panel). The lower panel
of Fig. 6 represents total adenovirus-expressed PR
detected by the 1294 MAb. Thus, PR expressed from
adenovirus vectors in COS cells was phosphorylated
on Ser 190 and Ser 294 in a similar manner as native
PR in T47D cells.
Nuclear extracts of COS cells containing hormoneactivated adenovirus-expressed PR-A or PR-B were
next incubated with a DNA probe containing a consensus progesterone response element (PRE) and analyzed for the formation of PR-DNA complexes by
MOL ENDO · 2000
60
Vol 14 No. 1
Ser 294. We also observed that both the P190 and
P294 MAbs (only with PR-B) supershifted the majority
but not all of the PR-DNA complexes, indicating that a
high percentage of DNA complexes contain phosphorylated PR.
DISCUSSION
Fig. 6. PR-A and PR-B Expressed from Recombinant Adenovirus Vectors in COS Cells Are Differentially Phosphorylated on Ser 190 and Ser 294 in Response to Hormone
COS cells were transduced for 24 h with PR-A
(Ad5dl327CMV-PR-A) or PR-B (Ad5dl327CMV-PR-B) recombinant adenoviruses at an M.O.I. of 50 and were treated with
and without R5020 (100 nM) for 1 h before harvest. Whole-cell
lysates were prepared and aliquots containing equal amounts
of total protein (100 mg) were analyzed by Western blot with
1294, P190, or P294 MAbs.
EMSA. Both PR-A and PR-B independently bound to
the DNA probe with approximately equal efficiency,
producing decreased mobility of a substantial amount
of the free DNA (Fig. 7). Addition of the 1294 MAb
supershifted all of the PR-A and PR-B complexes
whereas a control unrelated MAb to estrogen receptor
had no effect (Fig. 7). The P190 MAb supershifted both
PR-A and PR-B complexes, while P294 reduced the
mobility of the PR-B-DNA complex only (Fig. 7). Thus,
PR-B that binds to specific PREs is efficiently phosphorylated on Ser 294 whereas PR-A that binds to
PREs is not phosphorylated to a significant extent on
In the present study monoclonal antibodies were produced that distinguish Ser 190 and Ser 294 phosphorylated forms of human PR from unphosphorylated receptors. Specificity of the MAbs was demonstrated by
peptide competition experiments (Fig. 1B) and by the
observation that treatment of T47D breast cancer cells
with hormone agonists and two classes of antagonists, RU486 and ZK89299, influenced MAb reactivity
with PR in a manner that correlated with the effects of
these ligands on phosphorylation of Ser 190 and Ser
294 observed by previous 32P-labeling experiments
(1–4). Moreover, both MAbs recognized specific phosphorylated states whether receptor was in a native
conformation or denatured under Western blot conditions. These experiments confirm the utility of the
MAbs to selectively recognize specific phosphorylated
states of PR under different experimental conditions.
To our knowledge this is the first report of phosphorylation state-specific steroid receptor antibodies.
The utility of P190 and P294 for PR phosphorylation
studies suggests that it is possible to produce MAbs
that recognize other phosphorylation sites in PR, as
well as phosphorylation sites of other classes of steroid receptors. Antibodies to tyrosine-phosphorylated
forms of signaling molecules have proven to be valuable for study of signal transduction pathways (22, 25).
Although fewer antibodies have been produced that
recognize serine/threonine phosphorylation sites,
those that interact with the serine phosphorylation
states of CREB (cAMP response element binding protein) and c-jun have also been of value for studying the
signaling pathways that activate these sequence-specific transcription factors (23, 24). Phosphorylation
state-specific antibodies have most often been produced as polyclonal antibodies in rabbits, which requires epitope purification to separate phosphorylation-specific antibodies from those that detect
unphosphorylated protein. The approach used here to
produce MAbs to phosphopeptide antigens eliminates
the epitope purification step. Phosphorylation-specific
antibodies were isolated by screening and subcloning
of the hybridomas. Hybridoma cell lines also have the
advantage of providing a continuous unlimited supply
of antibody.
We anticipate that the P190 and P294 MAbs, and
future MAbs produced against other phosphorylation
sites within PR, will replace 32P-labeling and peptide
mapping for phosphorylation studies. Phosphorylation-specific antibodies for steroid receptors are potentially powerful tools to study cellular signals and
Phosphospecific PR Antibodies
61
Fig. 7. Differential Phosphorylation of Native PR-A and PR-B DNA Complexes
Adenovirus-expressed PR-A and PR-B from hormone-treated (1 h with 100 nM R5020) COS cells were prepared as whole cell
extracts as in Fig. 6 and incubated in a DNA binding reaction with a 32P-oligonucleotide containing a single consensus PRE. Free
and bound 32P-DNA were separated by electrophoresis on nondenatured 5% polyacrylamide gels. The different MAbs (1 mg)
indicated were added to the DNA binding reaction including a no-addition control, 1294, P190, P294, and an estrogen receptor
MAb (h151).
pathways that regulate nuclear receptor phosphorylation and function in vivo. The use of Western blots to
detect phosphorylation of steroid receptors in whole
cells will greatly simplify experiments to analyze the
influence of different signaling pathways on specific
receptor phosphorylation sites. The ability of the P190
and P294 MAbs to recognize phosphorylated PR in its
native conformation also indicates the potential of
these MAbs to separate phosphorylated and unphosphorylated forms of PR for biochemical studies. MAb
affinity chromatography and elution of the immobilized
receptor by phosphopeptide competition under nondenaturing conditions should be a useful method for
analyzing functional properties of different phosphorylated forms of receptors in vitro. The partial supershift
of DNA complexes obtained with P190 and P294
MAbs, as compared with the complete supershift with
the 1294 MAb that recognizes total PR, illustrates that
receptors are not uniformly phosphorylated (Fig. 7).
To produce biochemical amounts of correctly phosphorylated PR for in vitro functional (DNA-binding) experiments, we chose to use a recombinant adenovirus
expression system in mammalian cells. In our previous
studies with baculovirus expressed human PR, it was
determined by 32P-labeling and phosphopeptide mapping that recombinant PR overexpressed in Sf9 insect
cells was correctly phosphorylated on all the same
sites as native PR in T47D breast cancer cells. How-
ever, hormone regulation was lost such that all sites
were constitutively phosphorylated and unaffected by
hormone treatment of cells (38). Using P190 and P294
MAbs to detect the phosphorylation state of adenovirus-expressed PR in COS cells, Ser 190 and Ser 294
sites were phosphorylated in the same hormone-stimulated (Ser 190) or hormone-dependent (Ser 294)
manner as native PR in T47D cells (Fig. 6). Although
preliminary, these data suggest that use of recombinant adenovirus to express wild-type and mutant PRs
in mammalian cells for phosphorylation studies is an
improvement over the baculovirus system. The more
faithful phosphorylation of adenovirus-expressed PR
may be due to the presence of different kinases in
insect and mammalian cells, or to PR overexpression
in the insect baculovirus system exceeding cell-regulatory systems. Under these conditions, adenovirusexpressed PR levels were equal to or slightly lower
than that of native PR in T47D cells (not shown).
Initial experiments with the P190 and P294 MAbs
have provided insights into PR phosphorylation and
function. First, the time course of P294 immunoreactivity in response to continuous hormone treatment
(Fig. 4) showed that Ser 294 phosphorylation was
down-regulated at a faster rate than total PR and the
Ser 190 phosphorylation site. This suggests that the
hormone-dependent Ser294 phosphorylation site is
more labile than basal sites and is likely to be acted
MOL ENDO · 2000
62
upon by different phosphatases than other sites.
Whether accelerated down-regulation signifies that
Ser 294 phosphorylation has a role in PR protein stability or turnover is not known. As a precedent for
phosphorylation affecting steroid receptor protein
half-life, studies by Webster et al. (18) showed that
specific groups of phosphorylation site mutations stabilized GR protein half-life and abrogated ligand-dependent GR down-regulation. However, our finding
that total PR-A and PR-B down-regulate at the same
rate (Fig. 5, A and B) argues that the phosphorylation
of Ser 294 is not the major determinant of liganddependent down-regulation of PR in the cell. Second,
the phosphospecific MAb experiments revealed a
strong preferential phosphorylation of Ser 294 on
PR-B. This was unanticipated because Ser 294 and
surrounding sequences are identical on PR-A. Our
previous 32P-labeling and phosphopeptide mapping
experiments detected phosphorylation of Ser 294 on
PR-A. Although relatively less of the Ser 294 phosphopeptide was generated from PR-A than PR-B, the
dramatic differential phosphorylation of this site was
not obvious until the MAb experiments were performed. The inability of peptide mapping to detect this
substantial differential phosphorylation of PR-A and
PR-B was likely due to the large size of the Ser 294
phosphopeptide and the resultant low yields and poor
resolution of the peptide on HPLC columns (2–4).
Thus, the development of the P294 MAb enabled an
observation that was missed due to inherent limitations of phosphopeptide mapping.
As a possible explanation for the preferential reactivity of P294 for PR-B, we considered the possibility that Ser 294 might be efficiently phosphorylated on PR-A, but that the P294 MAb has limited
access to its epitope in the context of PR-A. However, this is not likely for several reasons. First, we
observed the same strong preference of the P294
MAb for PR-B by Western blot, as for PR-B in its
native conformation as detected by immunoprecipitation (Fig. 5) and EMSA (Fig. 7). SDS-denatured
PR-A and PR-B proteins on Western blots are not
expected to exhibit differential accessibility to the
P294 MAb. Second, the peptide- blocking experiment (Fig. 1B) suggests that both MAbs efficiently
recognize a small phospho-dependent amino acid
sequence relatively independent of conformation.
Third, purified PR-A can be phosphorylated efficiently on Ser 294 in vitro and reacts strongly with
P294 MAb (5). Why Ser 294 is preferentially phosphorylated on PR-B in vivo (T47D cells) is not
known. This is likely due to the N terminus of PR-A
adopting a distinct conformation that either hinders
access of cellular kinases to this site or creates a
unique active site domain for an interacting protein
that blocks phosphorylation of Ser 294. Alternatively, the differential phosphorylation of PR-A and
PR-B at Ser294 could be due to the gain of a phosphatase interaction site in PR-A that is not accessible in PR-B. In support of the idea that the N termi-
Vol 14 No. 1
nus of PR-A and PR-B have different conformations,
Bain et al. (39), using proteolytic peptide mapping,
reported that regions of amino termini common to
PR-A and PR-B exhibit distinct ordered structures.
PR-B is generally a stronger transcriptional activator
than PR-A. In several cell and promoter contexts,
PR-A exhibits minimal activation function and is capable of acting as a trans-dominant repressor of the
transcriptional activity of other steroid receptors, including PR-B (26–28). The mechanism responsible for
this repressive function of PR-A is unknown. An inhibitory function was mapped to a discrete region in the
first 140 amino acids of the N terminus (amino acids
165–305) of PR-A that was observed to be transferable to other steroid receptors, but not to heterologous proteins (40, 41). Because Ser 294 lies within this
inhibitory domain, it is possible that differential phosphorylation of this site may be involved in the distinct
functional properties of the two receptor forms. Phosphorylation at Ser 294 could directly influence receptor
association with other coregulatory proteins. Alternatively, differential phosphorylation could be the consequence of a distinct conformation, in which case Ser
294 may be located within an active domain for a novel
PR-A interacting protein.
MATERIALS AND METHODS
Phosphopeptides
Two peptides were synthesized (Macromolecular Resources at Colorado State University, Fort Collins, CO) corresponding to the following sequences in human progesterone receptor: CGG-(184)VLPRGL[S] PARQLL (196) and
CGG-(288) PMAPGR[S]PLATTV(300). Peptides were synthesized to contain extra N-terminal non-PR sequences (indicated in bold lettering) including a Cys for chemical cross-linking
and two Gly residues as spacers between the N terminus and
the 13 mer PR sequences. The Gly residues were included to
reduce steric hindrance after cross-linking to hapten and
improve antigenicity. The peptides were produced by standard solid phase peptide synthesis on polystyrene resins.
The serine residues indicated by brackets were left unprotected on the resin, and they were chemically phosphorylated
after the chain assembly was completed as described by
Perich (42). A 5-fold molar excess of dibenzyl-N, N-diisopropylphosphoramidite (Novabiochem USA, La Jolla, CA) was
added along with 50 molar equivalents of tetrazole in dimethyl formamide (VWR) and reacted for 1 h. The resin was
washed and then oxidized by washing several times with an
iodine solution to convert the intermediate phosphite ester
into the dibenzyl-protected phosphoserine. The peptide was
then cleaved from the resin support, and the side chains were
deprotected by a 2-h treatment with a cleavage cocktail
containing 90% trifluoroacetic acid, 2.5% tiisopropylsilane
(TIS, Sigma), 2.5% ethanedithiol (EDT, Sigma), 2.5% water,
and 2.5% Anisole (Sigma). The deprotected peptides were
then analyzed by Maldi-Tof mass spectrometry to confirm
quantitative phosphorylation. The peptides were then chromatographed by reverse phase HPLC to 90% purity. Phosphorylated peptides were coupled to KLH by a sulfhydryl
reagent through the N-terminal cysteine.
Phosphospecific PR Antibodies
Other PR Antibodies and PR Ligands
A mouse monoclonal (MAb) IgG1 (clone 1294) antibody was
raised against human PR that recognizes an epitope in the
N-terminal region (amino acids 165–534) common to PR-A
and PR-B (B. Spaulding, L. Sherman, and D. P. Edwards,
unpublished). MAb 1294 was used as a control for the total
amount of PR present in different cell extracts, independent
of the phosphorylation state of PR. The progestin agonist
R5020 (Promegestone) was obtained from DuPont-New England Nuclear (Boston, MA), and the progesterone antagonists RU486 (Mifepristone) and ZK98299 (Onapristone) were
gifts respectively from Rousell UCLAF (Romainville, France)
and Schering AG (Berlin, Germany).
Cell Cultures and Preparation of PR Extracts
PR-rich T47D human breast cancer cells were cultured in
MEM medium (Life Technologies, Inc., Gaithersburg, MD)
supplemented with 5% FBS (HyClone Laboratories, Inc. Logan Utah) as previously described (29, 30). At 24 h before
treatment with hormone, cells were grown in MEM containing
5% dextran-coated charcoal-treated serum which removes
steroids, and the same medium was used for hormone treatments. To prepare cytosol and nuclear extracts, cells were
lysed in KPFM buffer containing phosphatase and protease
inhibitors as previously described (50 mM potassium phosphate, pH 7.4, 50 mM sodium fluoride, 1 mM EDTA, 1 mM
EGTA, and 12 mM monothioglycerol) to minimize dephosphorylation and degradation of PR during in vitro processing
(1–4). Cytosol and nuclear fractions were prepared by differential centrifugation as previously described. The nuclear
pellet was extracted for 1 h at 4 C with lysis buffer containing
0.4 M NaCl and centrifuged at 100,000 3 g for 60 min to yield
a soluble nuclear extract in the supernatant. To prepare
whole-cell extracts, cells were homogenized in lysis buffer
containing 0.4 M NaCl and extracted for 1 h at 4 C, and the
entire lysate was centrifuged at 100,000 3 g for 60 min to
yield a soluble supernatant total protein fraction.
Adenovirus Transduction of PR-A and PR-B in
Mammalian Cells
Recombinant adenoviral transfer vectors containing human
PR-A or human PR-B under the control of the cytomegalovirus (CMV) promoter, were constructed by overlap recombination as described by Schaack et al. (37). These are replication-defective viruses lacking the transforming EIA and
EIB genes. To construct recombinant viruses, human PR-A or
PR-B cDNAs were cloned into the BamHI site of a transfer
plasmid (ACCMVC-1) containing the CMV promoter and adenovirus sequences to yield pCMV-PR-A and pCMV-PR-B.
Permissive 293 cells were cotransfected using calcium phosphate precipitation with pCMV-PR-A, or pCMV-PR-B and the
adenovirus AD5d1327Bst b-gal (37). Homologous recombination of plasmid and viral sequences resulted in generation of
infectious viral particles that were plaque purified. Plaques
were initially examined by PCR for the presence of either
PR-A or PR-B inserts. Positives were picked, grown in 293
cells, and examined for PR-A or PR-B protein expression by
Western blot. Purified viruses encoding PR-A or PR-B,
named Ad5dl327CMV-PR-A and Ad5dl327CMV-PR-B, respectively, were grown in large scale and concentrated by
CsCl gradient centrifugation. The virus concentrations were
determined by absorbance at 260 nm where 1 A260 unit
represents 1012 viral particles. The particle-plaque forming
unit ratio was determined to be close to 100 for both viruses.
To express PR-A or PR-B, monkey kidney COS-1 cells
were grown in DMEM (Life Technologies, Inc.) supplemented
with 10% FBS (HyClone Laboratories, Inc.) as previously
described (42). Cells were plated at 1 3 106 cells per 100-mm
culture dish and grown for 24–48 h to 70–80% confluency.
63
Ad5dl327CMVPR-A or Ad5dl327CMVPR-B viruses were
added to freshly changed medium at multiplicity of infection
(M.O.I.) of 50, and cells were incubated for 24 h at 37 C. At
24 h after transduction, medium was changed to DMEM plus
5% FBS, and cells were treated for 1–2 h with 100 nM R5020.
Cells were harvested by scraping into ice-cold PBS and
homogenized by freeze-thawing in a lysis buffer containing
20 mM HEPES, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 10%
glycerol, 1 mM sodium vanadate, 50 mM sodium fluoride, and
0.4 M NaCl.
Western Blotting of PR
Cell extracts or immunoprecipitates containing PR were electrophoresed on 7.5% polyacrylamide SDS-gels, as previously described with modifications (29, 30). Proteins were
transferred to Immobilon-P (Millipore Corp., Bedford, MA)
polyvinylidene fluoride transfer membranes at 700 mA for 2 h
in a transfer buffer containing 250 mM Tris-base, 1.92 M
glycine, 0.01% SDS and 10% methanol. The membranes
were blocked for 1 h in 5% milk in PBS-T (PBS, pH 7.4,
containing 1% Tween-20). Membranes were then washed
three times for 15 min in PBS-T and incubated in the same
buffer with primary MAbs overnight at 4 C using the following
concentrations for different MAbs: 1294, 1 mg/ml; P190, 4
mg/ml; and P294, 10 mg/ml. Membranes were washed three
times for 15 min in PBS-T and incubated for 1 h at room
temperature with a 1:5,000 dilution of mouse secondary antibody conjugated to horseradish peroxidase, and detection
was by luminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) according to manufacturer’s instructions.
Enhanced chemiluminescence (ECL) Plus (Amersham Pharmacia Biotech) was used to quantitate PR Western blots by
phosphorimager analysis on a Storm 860 instrument (Molecular Dynamics, Inc., Sunnyvale, CA).
Immunoprecipitation Assays
PR-specific MAbs were prebound to Protein A Sepharose
(Repligen Corp., Needham, MA) as previously described (29,
43). Resins were incubated with excess (65 mg/100 ml resin)
rabbit antimouse IgG (Cappel, Cochranville, PA), followed by
washing with PBS to remove unbound secondary antibody.
PR-specific MAbs were then bound to the immobilized rabbit
antimouse IgG at concentrations of 10 mg/100 ml of resin. As
a control for nonspecific binding of PR, resins were bound
only with rabbit antimouse IgG and the primary MAb was
omitted. Cell extracts (prepared in KPFM buffer) containing
PR were incubated for 3 h at 4 C on an end-over-end rotator
with MAb-coated protein A Sepharose beads. Resins were
washed four times in KPFM buffer containing 0.4 M NaCl and
extracted with 2% SDS sample buffer, and the extracts were
analyzed by ECL Western blot with the 1294 MAb.
EMSA
A 28-bp double stranded oligonucleotide containing a single
consensus progesterone response element (PRE) was used
as a probe for PR-DNA binding by EMSA as described previously (44). Whole-cell extracts from adenovirus-transduced
COS cells were incubated with 0.3 ng of 32P-labeled PRE
oligonucleotide for 1 h at 4 C in DNA binding buffer containing
10 mM Tris-Base, pH 7.4, 50 mM NaCl, 5 mM dithiothreitol, 2
mM MgCl2, 10% glycerol, and 1 mg of competitor DNA poly
(dA-dT)/poly(dA-dT). DNA binding reactions were electrophoresed on nondenaturing 5% polyacrylamide gels as previously described, and the gels were dried and subjected to
autoradiography (44).
MOL ENDO · 2000
64
Production of MAbs
Male BALB/c mice (8–10 weeks of age, Jackson Laboratories, Inc., Bar Harbor, ME) were injected with phosphorylated
peptide (Ser 190 and Ser 294)-KLH conjugates as an emulsion in MPL/S-TDCM adjuvant (RIBI Immunochemical Research Inc, Hamilton, MT). The primary injections were given
sc with 400 mg of conjugate, or 100 mg of peptide based on
a 1:4 ratio by weight of peptide-KLH. Three booster injections
were given at approximately 3-week intervals with the same
amount of conjugate, alternating between sc and ip routes of
injection. At 3 days before cell fusions, mice were injected ip
with 1 mg of conjugate prepared in PBS.
Sera from immunized mice were tested for antibody titers
by ELISA as previously described (29). ELISA 96-well plates
(Immunolon II, Dynatech Corp., Chantilly, VA) were coated
with 10 mg/ml of free phosphorylated (Ser 190 and Ser 294)
peptide or with free unphosphorylated peptide (Ser 190 and
Ser 294). ELISA plates were blocked with 1% BSA and incubated overnight at 4 C with antisera at different dilutions, and
plates were developed with secondary goat-antimouse IgGhorseradish peroxidase conjugate. Mice that showed strong
ELISA reaction against the specific phosphopeptide and low
reaction with unphosphorylated peptide were subsequently
assayed by Western blot for whether the antisera also contained antibodies reactive with full-length PR in crude extracts of T47D cells. Mice producing antibodies that preferentially reacted with the phosphopeptide and T47D PR
protein by Western blot were taken for cell fusions.
Fusions between mouse spleen cells and the mouse THT
(Fox-NY) myeloma cell line were performed as previously
described (29). Hybridomas were screened by ELISA for antibodies that exhibited preferential reactivity for free phosphopeptides over unphosphorylated peptides. Those showing strong preferential ELISA reaction were further screened
by Western blot for recognition of native PR in extracts of
T47D cells. Since all the serine phosphorylation sites in PR
were observed in previous studies by 32P-labeling studies to
increase in response to hormone treatment, Western blots
were performed with PR taken from cells treated for a short
time (2 h) with and without R5020. For each phosphopeptide
antigen, a MAb was identified that passed the screening
criteria of exhibiting a preferential ELISA reaction with phosphorylated peptide over unphosphorylated and Western blot
reactivity with T47D PR that increased in response to hormone treatment of cells. The hybridomas were subcloned
twice by limiting dilution, and each cell line stably produced
an IgG1 MAb, designated clone 1154 (Ser 190 peptide) and
clone 608 (Ser 294 peptide), respectively. MAbs were purified
by 50% ammonium sulfate precipitation and binding to protein G Sepharose. Antibody was eluted with Tris-glycine
buffer, pH 2.8, followed by neutralization with 1 M Tris-base
and dialysis against PBS.
Acknowledgments
The authors acknowledge the expert technical assistance
of Kurt Christensen, Magda Altmann, and the University of
Colorado Cancer Center Tissue Culture Monoclonal Antibody
Core facility for the production of MAbs. We thank Drs.
Steven K. Nordeen and Jerome Schaack (University of Colorado Health Science Center) for providing recombinant
PR-A and PR-B adenoviruses and Suzzane Wardell for assistance in construction of the viruses.
Received July 30, 1999. Revision received October 11,
1999. Accepted Ooctober 14, 1999.
Address requests for reprints to: Dean P. Edwards, Ph.D.,
University of Colorado Health Sciences Center, Department
Vol 14 No. 1
of Pathology, B-216, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: [email protected].
This work was supported in part by a sponsored research
agreement (D.P.E.) from Ligand Pharmaceuticals, Inc. (San
Diego, CA ), Public Health Service NIH Grant CA-57539
(N.L.W.), and National Cancer Institute University of Colorado
Cancer Center Core Grant P30-CA46934 (D.P.E.).
REFERENCES
1. Zhang Y, Beck CA, Poletti A, Edwards DP Weigel NL
1994 Identification of phosphorylation sites unique to the
B form of human progesterone receptor. J Biol Chem
269:31034–31040
2. Zhang Y, Beck CA, Poletti A, Edwards DP Weigel NL
1995 Identification of a group of Ser-Pro motif hormoneinducible phosphorylation sites in the human progesterone receptor. Mol Endocrinol 9:1029–1040
3. Zhang Y, Beck CA, Poletti A, Clement JPIV, Prendergast
P, Yip T-T, Hutchens TW, Edwards DP, Weigel NL 1997
Phosphorylation of human progesterone receptor by cyclin-dependent kinase 2 on three sites that are authentic
basal phosphorylation sites in vivo. Mol Endocrinol 11:
823–832
4. Beck CA, Zhang Y, Weigel NL, Edwards DP 1996 Two
types of anti-progestins have distinct effects on sitespecific phosphorylation of human progesterone receptor. J Biol Chem 271:1209–1217
5. Keightley M-C, Edwards DP, Weigel NL, Differential regulation of human progesterone receptor A, B phosphorylation by the Erk signaling pathway. J Biol Chem, in
press
6. Beck CA, Weigel NL, Edwards DP 1992 Effects of hormone and cellular modulators of protein phosphorylation
on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Mol Endocrinol
6:607–620
7. Edwards DP, Weigel NL, Nordeen SK, Beck CA 1993
Modulators of cellular protein phosphorylation alter the
trans-activation function of human progesterone receptor and the biological activity of progesterone antagonists. Breast Cancer Res Treat 27:41–56
8. Beck CA, Weigel NL, Moyer ML, Nordeen SK 1993 The
progesterone antagonist RU486 acquires agonist activity
upon stimulation of cAMP signaling pathways. Proc Natl
Acad Sci USA 90:4441–4445
9. Sartorius CA, Tung L, Takimoto GS, Horwitz KB 1993
Antagonist-occupied human progesterone receptors
bound to DNA are functionally switched to transcriptional
agonists by cAMP. J Biol Chem 268:9262–9266
10. Sartorius CA, Groshong SD, Miller LA, Powell RP, Tung
L, Takimoto GS Horwitz KB 1994 New T47D breast cancer cell lines for the independent study of progesterone
B- and A-receptors: Only antiprogestin-occupied B-receptors are switched to transcriptional agonists by
cAMP. Cancer Res 54:3868–3877
11. Takimoto GS, Hovland AR, Tasset DM, Melville MY, Tung
L, Horwitz KB 1996 Role of phosphorylation on DNA
binding and transcriptional functions of human progesterone receptors. J Biol Chem 271:13308–13316
12. Ali S, Metzger D, Bornert J-M, Chambon Pierre 1993
Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor
A/B region. EMBO J 12:1153–1160
13. Goff PL, Montano MM, Schodin DJ, Katzenellenbogen
1994 Phosphorylation of the human estrogen receptor.
J Biol Chem 269:4458–4466
14. Bai W, Weigel NL 1996 Phosphorylation of ser211 in the
chicken progesterone receptor modulates its transcriptional activity. J Biol Chem 271:12801–12806
Phosphospecific PR Antibodies
15. Bai W, Tullos S, Weigel NL 1994 Phosphorylation of
ser530 facilitates hormone-dependent transcriptional activation of the chicken progesterone receptor. Mol Endocrinol 8:1465–1473
16. Mason SA, Housley PR 1993 Site-directed mutagenesis
of the phosphorylation sites in the mouse glucocorticoid
receptor. J Biol Chem 268:21501–21504
17. Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ
1997 Mitogen-activated and cyclin-dependent protein
kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol
Cell Biol 17:3947–3954
18. Webster JC, Jewell CM, Bodwell JE, Munck A, Sar M,
Cidlowski JA 1997 Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the
receptor protein. J Biol Chem 272:9287–9293
19. Weigel NL 1996 Steroid hormone receptors and their
regulation by phosphorylation Biochem J 319:657–667
20. Tremblay A, Tremblay GB, Labrie F, Giguère V 1999
Ligand-independent recruitment of SRC-1 to estrogen
receptor b through phosphorylation of activation function
AF-1. Mol Cell 3:513–519
21. DeFranco DB, Qi M, Borror KC, Garabedian MJ, Brautigan DL 1991 Protein phosphatase types 1 and/or 2A
regulate nucleocytoplasmic shuttling of glucocorticoid
receptors. Mol Endocrinol 5:1215–1228
22. Kawakatsu H, Sakai T, Takagaki Y, Shinoda Y, Saito M,
Owada MK, Yano J 1996 A new monoclonal antibody
which selectively recognizes the active form of src tyrosine kinase. J Biol Chem 271:5680–5685
23. Ginty DD, Kornhauser JM, Thompson MA, Bading H,
Mayo KE, Takahashi JS, Greenberg ME 1993 Regulation
of CREB phosphorylation in the suprachiasmatic nucleus
by light and a circadian clock. Science 260:238
24. Eilers A, Whitfield J, Babij C, Rubin LL, Ham J 1998 Role
of the jun kinase pathway in the regulation of c-jun expression and apoptosis in sympathetic neurons. J Neuroscience 18:1713–1724
25. Grammer TC, Blenis J 1997 Evidence for MEK-independent pathways regulating the prolonged activation of the
ERK-MAP kinases. Oncogene 14:1635–1642
26. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley
BW, McDonnell DP 1993 Human progesterone receptor
A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol
7:1244–1255
27. Tung L, Kamel Mohamed M, Hoeffler JP, Takimoto GS,
Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate transcription without binding
to progesterone response elements and are dominantly
inhibited by A-receptors. Mol Endocrinol 7:1256–1265
28. Wen DX, Xu YF, Mais DE, Goldman ME, McDonnell DP
1994 The A and B isoforms of the human progesterone
receptor operate through distinct signaling pathways
within target cells. Mol Cell Biol 14:8356–8364
29. Weigel NL, Beck CA, Estes PA, Prendergast P, Altmann
M, Christensen K, Edwards DP 1992 Ligands induce
conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody. Mol Endocrinol
6:1585–1597
30. El-ashry D, Oñate SA, Nordeen SK, Edwards DP 1989
Human progesterone receptor complexed with the an-
65
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
tagonist RU486 binds to hormone response elements in
a structurally altered form. Mol Endocrinol 3:1545–1558
Deleted in proof
Lim CS, Baumann CT, Htun H, Xian W, Irie M, Smith CL,
Hager GL 1999 Differential localization and activity of the
A- and B-form of the human progesterone receptor using
green fluorescent protein chimeras. Mol Endocrinol 13:
366–375
Dekabre JA, Guiochon-Mantel A, Milgram E 1993 in vivo
evidence against the existence of antiprogestins disrupting receptor binding to DNA. Proc Natl Acad Sci USA
90:4421–4425
Gass EK, Leonhardt SA, Nordeen SK, Edwards DP 1998
The antagonists RU486 and ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid in vitro
and in vivo, but have distinct effects on receptor conformation. Endocrinology 139:1905–1919
Read LD, Snider CE, Miller JS, Greene GL, Katzenellenbogen BS 1988 Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein
in human breast cancer cell lines. Mol Endocrinol
2:263–271
DeMarzo AM, Oñate SA, Nordeen SK, Edwards DP 1992
Effects of the steroid antagonist RU486 on dimerization
of the human progesterone receptor. Biochem 31:
10491–10501
Schaack J, Langer S, Guo X 1995 Efficient selection of
recombinant adenoviruses by vectors that express b-galactosidase. J Virol 69:3920–3923
Beck CA, Zhang Y, Altmann M, Weigel NL, Edwards DP
1996 Stoichiometry and site-specific phosphorylation of
human progesterone receptor in native target cells and in
the baculovirus expression system. J Biol Chem 271:
19546–19555
Bain DL, Franden MA, Takimoto GS, Horwitz KB, Structural analysis of the two human progesterone receptor
N-termini explain their isoform-specific functional differences. Program of the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 187 (Abstract)
Giangrande PH, Pollio G, McDonnell DP 1997 Mapping
and characterization of the functional domains responsible for the differential activity of the A and B isoforms of
the human progesterone receptor. J Biol Chem 272:
32889–32900
Hovland AR, Powell RL, Takimoto GS, Tung L, Horwitz
KB 1998 An N-terminal inhibitory function, IF, suppresses transcription by the A-isoform but not the Bisoform of human progesterone receptors. J Biol Chem
273:5455–5460
Perich JW 1991 Synthesis of O-phosphoserine and Ophosphothreonine containing peptides. Methods Enzymol 201:234–244
Tetel MJ, Jung S, Carbajo P, Ladtkow T, Skafar DF,
Edwards DP 1997 Hinge and amino-terminal sequences
contribute to solution dimerization of human progesterone receptor. Mol Endocrinol 11:1114–1128
Boonyaratanakornkit V, Melvin V, Prendergast P, Altmann M, Ronfani L, Bianchi ME, Taraseviviene L, Nordeen SK, Allegretto EA, Edwards DP 1998 High-mobility
group chromatin proteins 1 and 2 functionally interact
with steroid hormone receptors to enhance their DNA
binding in vitro and transcriptional activity in mammalian
cells. Mol Cell Biol 18:4471–4487