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
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ABSTRACT |
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INTRODUCTION |
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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 7 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 9 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or in phosphopeptide maps of PR in response to cAMP (Ref. 6 , and C. A. Beck, N. L.Weigel, and D. P. Edwards, unpublished).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 2 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 2 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 studies with other steroid receptors have reported either partial reductions or enhancements of transcriptional activities by introduction of phosphorylation site mutations ( Refs. 12 13 14 15 16 17 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 ß (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 23 24 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 27 28 ).
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RESULTS |
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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.
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 (1020 min) in response to hormone treatment
while hormone-dependent sites responded more slowly requiring 12 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
, BD). 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 ).
Ligand-dependent PR down-regulation represents an 8090% decrease in
the steady-state level of PR that takes 2448 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 3060 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 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 13 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-hormone-treated 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 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 hormone-activated
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 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 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.
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DISCUSSION |
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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 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. However, 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, adenovirus-expressed 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 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
ligand-dependent 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 3 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 terminus 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 27 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 165305) 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.
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MATERIALS AND METHODS |
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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 165534) 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 2 3 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 x 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 x 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 ß-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 x 106 cells per 100-mm culture dish and grown for 2448 h to 7080% confluency. 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 12 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 µg/ml; P190,
4 µg/ml; and P294, 10 µg/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 manufacturers
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 µg/100 µl 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 µg/100
µl 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 µg
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 ).
Production of MAbs
Male BALB/c mice (810 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 µg of conjugate, or 100 µg 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 µg/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 IgG-horseradish 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.).
Received for publication July 30, 1999. Revision received October 11, 1999. Accepted for publication October 14, 1999.
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REFERENCES |
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