(Received for publication, May 8, 1995; and in revised form, October 18, 1995)
From the
Human progesterone receptor (PR) is phosphorylated on multiple
serine residues; three sites (Ser, Ser
, and
Ser
) are inducible by hormone agonist, while at least six
others are basally phosphorylated and exhibit a general increase in
response to hormone. In this study we have used high performance liquid
chromatography phosphopeptide mapping and manual peptide sequencing to
investigate how two different progestin antagonists, RU486 and ZK98299,
affect site-specific phosphorylation of PR isolated from T47D breast
cancer cells. As compared to the progestin agonist R5020, RU486
stimulated a similar increase in overall incorporation of
[
P]phosphate per PR molecule (2.5-2.6-fold
for PR-A and 2.1-fold for PR-B), and at the site-specific level, RU486
stimulated both the basal and inducible sites to the same extent as
R5020. In contrast, ZK98299 produced only a minimal increase in overall
phosphorylation (1.2-fold for PR-A and 1.1-fold for PR-B) which was due
to a reduced stimulation of the basal sites and failure to induce any
of the three hormone-dependent sites. No inappropriate phosphorylation
sites were detected in response to either RU486 or ZK98299. In
cotreatment studies, ZK98299 blocked the increase in overall
phosphorylation of PR induced by R5020, demonstrating that the failure
of this antagonist to stimulate specific phosphorylation sites is not
due to an inefficient interaction with PR in the intact cell. These
results indicate that the biological effects of RU486 are not mediated
by an alteration in the phosphorylation state of PR, whereas failure to
promote phosphorylation of certain sites may contribute to the
antagonist action of ZK98299. Additionally these results support the
concept of two mechanistic classes of anti-progestins that affect PR
differently in vivo.
All members of the steroid/thyroid hormone receptor family that
have been analyzed so far are phosphoproteins, and as a general
property they become hyperphosphorylated as a rapid response to binding
hormone in the
cell(1, 2, 3, 4, 5, 6, 7) .
Sites of phosphorylation have been identified for certain members of
the steroid/thyroid receptor family including mouse glucocorticoid
receptor(8) , chicken (9, 10) and human
progesterone receptor (PR)()(11, 12) ,
human estrogen receptor(13, 14, 15) ,
androgen receptor(16) , and the vitamin D
receptor(17) . The major targets for phosphorylation are serine
residues located in the N-terminal domain that is required for maximal
transcriptional activity. However, sites in the hinge and C-terminal
ligand binding domain have also been identified in certain receptors,
as well as a minor phosphotyrosine site in estrogen
receptor(18, 19) , suggesting that phosphorylation may
serve multiple functional
roles(6, 9, 10, 14, 16, 17, 20) .
Although the functional role for phosphorylation of steroid receptors
remains poorly defined, initial site-directed mutagenesis studies have
provided direct evidence that phosphorylation of certain sites is
important for receptor activity. For example, substitution of a
nonphosphorylatable alanine residue for Ser
of human
estrogen receptor resulted in as much as a 60% reduction in
transcriptional activity(13) , and substitution of alanine for
Ser
of chick PR decreased the sensitivity of the receptor
to progesterone(20) .
Human PR is expressed from a single
gene as two distinct molecular forms, PR-A and PR-B. The B form
contains an additional 164-amino acid N-terminal sequence; otherwise
the two proteins are identical throughout their common N-terminal
domain and DNA and steroid binding domains(21) .
Phosphorylation of human PR is complex. PR-B has at least nine
phosphoserines N-terminal to the DNA binding
region(3, 11, 12) . Three sites identified as
Ser, Ser
, and Ser
(11, 12) are contained in the unique N-terminal
segment of PR-B; the remaining sites are common to PR-A and PR-B. There
appears to be two classes of phosphorylation sites based on how they
are affected by progestin agonists. Three sites found in Ser-Pro
consensus sequences, Ser
, Ser
, and
Ser
, are highly inducible and were detected as major new
sites only after treatment of cells with hormone agonist. The remaining
sites are basally phosphorylated in the absence of hormone and exhibit
a general increase in response to hormone agonist(12) .
Additionally, progestin agonists such as R5020 cause human PR to
exhibit a slightly slower electrophoretic mobility on SDS-gels which is
associated with phosphorylation of a single site at
Ser
(12) .
Steroid analogs have been developed that function as potent competitive antagonists of progesterone(22) . The mechanism by which these compounds inactivate PR remains unresolved. Because progestin agonists and antagonists induce different conformational changes in the C-terminal domain of human PR, it is generally believed that an altered receptor conformation plays an important role(23, 24, 25) . Additionally, it has been suggested that anti-progestins fall into two mechanistic classes. One class, represented by ZK98299 (Onapristone), has been shown in vitro to fail to induce PR association with target DNA(26, 27) . Another class of compounds, which includes the clinically important antagonist RU486 (Mifapristone), is capable of inducing high affinity association of PR with target DNA(28, 29, 30, 31) . Thus it is generally believed that RU486 interferes with a receptor activation step downstream of DNA binding and that ZK98299 impairs receptor binding to DNA. This hypothesis for two classes of anti-progestins is based largely on in vitro receptor DNA binding studies and has come under challenge by studies that have assessed PR binding to specific target DNA sequences in vivo(32, 33) .
Previous studies have indicated
that progestin agonists affect overall phosphorylation of human PR
differently than antagonists, implying that an altered phosphorylation
state of PR may contribute to the antagonist activity of these
compounds. RU486 was reported to stimulate as much as a 2-3-fold
higher incorporation of [P]phosphate into human
PR as compared to the progestin agonist R5020, and ZK98299 stimulated
60% less total incorporation than R5020(3, 34) .
Additionally, ZK98299 failed to promote the upshift of PR mobility on
SDS-gel electrophoresis that is associated with
phosphorylation(34) . Whether these differences in total levels
of phosphorylation represent general quantitative changes at all
phosphorylation sites and/or qualitative changes, including
phosphorylation of inappropriate sites, has not been investigated. In
this study we have used methods developed previously for identification
of PR phosphorylation sites (11, 12) to directly
analyze the effects of ZK98299 and RU486 on site-specific
phosphorylation of PR in T47D breast cancer cells.
Harvested cell pellets
were lysed in KPFM buffer (50 mM potassium phosphate (pH 7.4),
50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA,
and 12 mM monothioglycerol) containing 0.5 M NaCl and
a mixture of protease inhibitors as described previously(36) .
Cell lysates were centrifuged at 100,000 g for 30 min.
The supernatant, considered as a whole cell extract, was dialyzed
against KPFM or diluted 1:1 with KPFM to reduce the salt concentration
before immunoprecipitation.
Figure 1:
The progestin antagonists RU486 and
ZK98299 exhibit distinct effects on overall phosphorylation of human
PR-A and PR-B. Receptors in T47D cells were uniformly labeled with
[P]orthophosphate or
[
S]methionine and were treated for the last 2 h
of labeling with A, vehicle (ethanol), 500 nM ZK98299 (ZK98) or 80 nM R5020, or B, with vehicle,
RU486 (80 nM) or R5020 (80 nM). Radiolabeled PR from
cell lysates were immunoprecipitated with receptor specific antibody
(AB-52) and subjected to SDS-gel electrophoresis and autoradiography.
Only the region of the gels containing the A (94 kDa) and B (120 kDa)
forms of human PR are shown. Broken lines (PR-A) or brackets (PR-B) indicate the position of upshifted PR and solid lines (PR-A) or brackets the position of
nonupshifted PR.
We have normalized P
incorporation into PR-protein by steady-state labeling of paired T47D
cell cultures with [
S]methionine. The fold
increase in total
P incorporation normalized to PR protein
was calculated by taking the ratio of
P to
S
counts in the PR bands excised from SDS gels. Based on average values
from multiple independent determinations, R5020 stimulated a 2.6- and a
2.1-fold increase in total
P incorporation per receptor
molecule, respectively, for PR-A and PR-B (Table 1). RU486
stimulated similar increases of 2.5- and 2.1-fold, respectively, for
PR-A and PR-B. In contrast, ZK98299 promoted minimal 1.2- and 1.1-fold
increases in PR-A and PR-B (Table 1). It is of interest to point
out that total
P-labeling of PR-B was higher than PR-A ( Fig. 1and Table 1) which is consistent with the fact that
PR-B contains three additional phosphorylation sites in the N-terminal
segment unique to PR-B that are not present in PR-A (11, 12) (see Fig. 3). These results show that
ZK98299 and RU486 have distinct effects on the overall level of
phosphorylation of PR in vivo.
Figure 3:
Tryptic
[P]phosphopeptide mapping of PR-B isolated from
T47D cells treated with R5020. PR was immunoprecipitated from T47D
cells after 2 h treatment with 80 nM R5020 and
[
P]PR-B was excised from gels prepared as
described in Fig. 1and digested with trypsin. Tryptic limit
digests were then resolved by reverse-phase HPLC on a C
column connected to an on-line radioactivity detector. The
results were plotted as a function of retention time versus
P counts. The shaded peaks indicate tryptic
phosphopeptides located in the N-terminal segment unique to PR-B. The
schematic shows the structure of human PR and the relationship of the
tryptic phosphopeptides to the positions of the five phosphorylated
serine residues identified from our previous
studies(11, 12) .
Figure 2:
The progestin antagonist ZK98299 inhibits
hyperphosphorylation of PR induced by R5020. Receptors in T47D cells
were labeled to steady-state with P and treated for the
last 2 h prior to harvest with vehicle, ZK98299 (500 nM), or
R5020 (20 nM), or they were coincubated with ZK98299 (500
nM) and R5020 (20 nM). Radiolabeled PR was
immunoprecipitated from cell lysates, resolved by SDS-gel
electrophoresis, and detected by
autoradiography.
In our earlier studies, phosphopeptide 9
exhibited heterogeneous elution behavior (originally designated as peak
7, 8, and 9) which under the present conditions has been eliminated. As
in our earlier studies, peptide 12 still exhibits a heterogeneous
elution pattern. The reason for the heterogenous behavior of peptide 12
appears to be due to a resistance of Lys-Ser to trypsin when the Ser
residue is phosphorylated, resulting in incomplete digestion products
as described by Allen(40) . Peaks 10 and 11 appear to contain
the same peptide, and peak 4 results from overdigestion of peak 6. All
other HPLC peaks represent a distinct phosphopeptide and a single
phosphorylation site. One other difference between our current and
earlier phosphopeptide maps is the lack of HPLC phosphopeptide 2. In
earlier studies, peptide 2 eluted between peptides 1 and 3. It has
since been found to represent an incomplete digestion product, that
when completely digested to its limit, is too small to be retained by
the C column. Nonetheless, peptide 2 is a distinct
phosphorylation site that is common to both A and B
receptors(11, 12) .
To examine the influence of
progestin antagonists on individual phosphorylation sites, we have
compared tryptic phosphopeptide maps of human PR isolated from cells
treated with vehicle, R5020, RU486, or ZK98299. T47D cells were labeled
with P as in Fig. 1, and PR was immunoprecipitated
from cell lysates and electrophoresed on SDS-gels. PR-A and PR-B bands
were excised, eluted from gel pieces, and digested with trypsin, and
the tryptic phosphopeptides were then separated by HPLC on a C
reverse-phase column.
Fig. 4shows a comparison of
tryptic phosphopeptide maps of unliganded PR-A with that of PR-A
isolated from T47D cells after 2 h of treatment with ZK98299, RU486, or
R5020. Peptide 9 (Ser) and peptide 12 (Ser
)
were barely detectable in the absence of hormone (Fig. 4A) and become major sites after treatment with
R5020 (Fig. 4B). In contrast, peptides 1, 5, and 10/11
were basally phosphorylated and exhibited a general increase upon
addition of hormone agonist. Treatment with RU486 (Fig. 4B, upper panel) induced phosphorylation
of the two hormone-dependent sites (peptides 9 and 12) and stimulated a
general increase in the basal sites (peptides 1, 5, and 10/11) in a
manner similar to that with R5020 treatment. In contrast, treatment
with 500 nM ZK98299 (Fig. 4A) had little
effect on either of the two hormone-dependent sites or on the basal
sites. Thus, phosphopeptide maps of PR-A after ZK98299 treatment are
more similar to that of the unliganded receptor than hormone
agonist-treated receptor.
Figure 4:
The progestin antagonists RU486 and
ZK98299 have distinct effects on tryptic phosphopeptide mapping of
PR-A. T47D PR labeled to steady-state with P was
immunoprecipitated, gel purified, and digested with trypsin, and the
tryptic [
P]peptides were resolved by HPLC as in Fig. 3. Cells were treated during the last 2 h of labeling with A, vehicle or ZK98299 (500 nM); B, RU486 (80
nM) or R5020 (80 nM). The shaded peaks indicate hormone-dependent
phosphopeptides.
Similar studies were performed with PR-B.
Shown in Fig. 5are tryptic phosphopeptide maps of PR-B isolated
from T47D cells that were treated for 2 h with vehicle, ZK98299, RU486,
or R5020. PR-B contains three additional phosphorylation sites not
present in PR-A, peptide 3 (Ser) and peptide 6
(Ser
), which contain basal sites, and peptide 0
(Ser
) which contains a hormone-inducible site (Fig. 5). Treatment with RU486 increased basal and
hormone-inducible sites of PR-B in a manner that was also
indistinguishable from that observed after R5020 treatment (Fig. 5B). ZK98299 had a minimal effect on
phosphorylation of hormone-inducible and basal sites (Fig. 5A). Thus, similar to results with PR-A, RU486
had the same effect as R5020 on the phosphorylation pattern of PR-B,
whereas the phosphorylation state of PR-B after treatment with ZK98299
more closely resembled that of unliganded PR-B.
Figure 5:
The progestin antagonists RU486 and
ZK98299 have distinct effects on tryptic phosphopeptide mapping of
PR-B. T47D PR was labeled to steady-state with P and
processed as in Fig. 4except that PR-B was gel-purified and
analyzed. Cells were treated with A, vehicle or ZK98299; B, RU486 or R5020 for 2 h. The shaded HPLC peaks
indicate hormone-dependent phosphopeptides.
Figure 6:
Manual Edman degradation to determine
the position of P-labeling in two major tryptic
phosphopeptides. Tryptic phosphopeptides 9 and 12 collected from
reverse-phase HPLC were dried and resuspended in 50% acetonitrile. They
were then covalently coupled to arylamine disks using
ethyl-3-(3-dimethylaminopropyl)carbodiimide and subjected to manual
Edman degradation. The radioactivity in the released amino acid was
determined after each cycle and was detected by Cerenkov counting. For
peptide 9, the majority of
P counts coupled to the disk
(75%) were released in cycle 2, and for peptide 12, the majority of
P counts (82%) were released in cycle
1.
A potentially important question that has not been fully
explored is whether the biological action of anti-progestins is
mediated in part by altering the phosphorylation state of the
progesterone receptor. Previous studies that examined the effects on
net P incorporation into human PR suggested that this
might be the case, since RU486 was reported to stimulate a
substantially greater increase in total phosphorylation than a
progestin agonist (R5020) and ZK98299 effected a much reduced increase
as compared to R5020(3, 34) . Because of the
complexity of human PR phosphorylation(11, 12) , it is
not possible to interpret whether these differences in total
phosphorylation simply represent a general quantitative change in the
degree of phosphorylation of all sites or whether this reflects a more
qualitative change in a few sites. It is also possible that antagonists
may promote the phosphorylation of new inappropriate sites. In contrast
to previous reports with human PR, we did not detect a difference
between the effects of RU486 and R5020 on net
P
incorporation into PR in T47D breast cancer cells. Both ligands
stimulated essentially the same 2.1-fold (PR-B) and 2.5-2.6-fold
(PR-A) increase (Table 1) which differed from the 3-5-fold
increase for R5020 and 5-10-fold increase for RU486 reported in
earlier studies (3, 34) . The reason for this apparent
discrepancy is not known, but is likely due to differences in
methodology. In the present study we have used phosphatase inhibitors
during the isolation of PR to minimize dephosphorylation during in
vitro processing which was not done in earlier studies, and we
have normalized
P incorporation to PR protein by
calculating the ratio of
P to
S counts.
As
compared to the agonist R5020 we did detect a reduced stimulation of
net P incorporation into PR after treatment with ZK98299.
However, the reduction was greater than the 60% reported earlier with
human PR(34) . After normalization to PR protein, we found that
net
P incorporation after ZK98299 treatment was only 10%
(PR-B) to 20% (PR-A) of that obtained after treatment with R5020 (Table 1). Because ZK98299 has a lower affinity for PR than
either progesterone or R5020(32, 39) , we questioned
whether the lack of hyperphosphorylation could be due simply to an
inefficient interaction of ZK98299 with PR in vivo. However,
at concentrations where ZK98299 itself had little effect, it was able
to block hyperphosphorylation of PR induced by R5020 (Fig. 2) or
by RU486 (not shown). These results provide strong evidence that
ZK98299 does in fact bind efficiently to PR in vivo and fails
to induce hyperphosphorylation. This ability of ZK98299 to block
hyperphosphorylation induced by other ligands has not been reported
before.
By phosphotryptic peptide mapping and manual amino acid
sequencing to determine the cycle of P release, we further
showed in this study that R5020 and RU486 phosphorylated all the same
major sites on human PR and to the same relative extent. Additionally,
no alternative sites on the same peptide were phosphorylated after
treatment with RU486, nor were any new inappropriate sites detected.
Thus under the conditions of our assays, we conclude that RU486 and
progestin agonists have indistinguishable effects, both quantitatively
and qualitatively, on the phosphorylation state of human PR. Similar
conclusions were drawn from earlier phosphopeptide mapping studies of
human PR(3) . However, identification of the amino acid
residues phosphorylated after RU486 treatment was not reported, making
it difficult in these earlier studies to rule out whether RU486 was
promoting phosphorylation of alternative sites. Additionally, the HPLC
methodology of the present study was of higher resolution, allowing the
isolation of additional phosphopeptides not detected
earlier(3) .
Phosphopeptide mapping of human PR after
treatment with ZK98299 has not been reported previously; only effects
on net P incorporation have been examined. By
phosphopeptide mapping and manual peptide sequencing we also determined
in this study that ZK98299 failed to induce any of the three hormone
agonist-dependent sites (Ser
, Ser
, and
Ser
). Additionally, the basal phosphorylation sites were
unaffected, and no new inappropriate phosphorylation sites were
detected after ZK98299 treatment ( Fig. 4and Fig. 5).
Therefore, the modest increase in net
P incorporation
stimulated by ZK98299 (1.1-fold for PR-A and 1.2-fold for PR-B; Table 1) would appear to reflect a slight increase in general
phosphorylation of the basal sites. Interestingly, ZK98299 also failed
to produce the same upshift in electrophoretic mobility as progestin
agonist. This is consistent with its failure to phosphorylate
Ser
, since the upshift was shown previously to be
associated with phosphorylation of Ser
(12) .
Phosphopeptide mapping studies (without identification of the
phosphorylated residues) with rabbit PR expressed in COS cells yielded
somewhat different results with respect to the effects of progestin
agonist and antagonists. On a similar note, both RU486 and R5020
stimulated the same fold increase in net P incorporation
with rabbit PR(41) . However, rabbit PR generated seven
phosphopeptides, all of which were basally phosphorylated, and both
ligands stimulated a general increase in phosphorylation of all
phosphopeptides in a proportionately similar manner. Thus, induction of
major new phosphorylation sites by progestin agonist or RU486, as
occurs with human PR in T47D cells, was not detected with rabbit PR (41) . Additionally, ZK98299 effects on site-specific
phosphorylation of rabbit PR differed from our results with human PR in
that it produced a general decrease in phosphorylation of all basal
sites. Rabbit and human PR phosphorylation may simply respond
differently to ligands, or alternatively, these results may be due to
differences in methods. Rabbit PR was overexpressed in COS cells by
transient transfection, whereas human PR were analyzed in their natural
cellular environment as an endogenous gene product. When human PR was
expressed from baculovirus vectors in insect cells, we found that
phosphorylation occurred on correct sites, but hormone-dependent
phosphorylation was not detected. (
)Thus it is possible that
ligand effects on receptor phosphorylation can change when receptors
are expressed in heterologous cells.
Based largely on in vitro binding studies, it has been suggested that progestin antagonists can be categorized by two mechanistic classes, those such as RU486 that promote efficient binding of PR to DNA and others such as ZK98299 that fail to do so(26, 27) . However, two studies that have analyzed PR binding to specific target DNA in vivo do not support this hypothesis. Cotransfection studies that were able to detect competition for binding of PR to progesterone response elements (PREs) in vivo have suggested that both types of progestin antagonists stimulate PR-DNA binding in the intact cell(32) . In contrast in vivo footprinting studies have suggested that neither RU486 nor ZK98299 are capable of promoting the binding of PR to PREs in the intact cell(33) . Biological studies have suggested that RU486 and ZK98299 do in fact affect PR differently in the intact cell. For example, under conditions where ZK98299 behaves as a complete antagonist, RU486 can exhibit weak partial agonist activity(29, 42) . Additionally, we and others have shown that activators of cAMP signaling pathways can promote functional switching of RU486 from that of a potent progestin antagonist to a relatively strong agonist. Under the same conditions ZK98299 continues to behave as a pure antagonist(43, 44) . The present study showing that RU486 and ZK98299 have different effects on phosphorylation of PR in the intact cell further supports the concept of two mechanistic classes of anti-progestins. We conclude from our analysis of specific phosphorylation sites that RU486 promotes phosphorylation of human PR on all the same serine residues as progestin agonists and that the antagonist activity of this compound cannot therefore be mediated by altering the phosphorylation state of the receptor. In contrast, the dramatic effect of ZK98299 on blocking hyperphosphorylation of PR suggests that the antagonist activity of ZK98299 may be mediated in part by preventing phosphorylation of sites important for receptor activation. Thus we propose that RU486 and ZK98299 inhibit PR activation by different mechanisms; RU486 antagonism does not involve alteration in receptor phosphorylation, whereas blocking of certain phosphorylation sites may be involved in the action of ZK98299.
At the present time, the functional role of phosphorylation of human PR is unknown. Thus far, only a few studies have addressed the functional role of phosphorylation of any steroid hormone receptor directly by mutating authentic phosphorylation sites and determining the effect that lack of a specific phosphorylation has on receptor activity in vivo. Initial studies with estrogen receptor (13) and chick PR (20) have shown that substitution of a single serine phosphorylation site, among several sites, had a substantial effect on receptor activity in vivo. In general, phosphorylation of eukaryotic transcription factors is an important means for modulating activity, and like steroid receptors, other transcription factors are frequently phosphorylated on multiple sites(45) . In some cases specific sites can serve different functional roles and thus provide a means to receive and integrate signals from multiple signal transduction pathways. We are presently conducting site-directed mutagenesis studies to determine the effect of each specific phosphorylation site on the transcriptional activity of human PR in cotransfection assays. Because of the complexity of human PR phosphorylation we suspect that phosphorylation will also modulate multiple receptor functions and that it will be the combination of all phosphorylation sites that determines the overall effect on activity of receptor. The three hormone agonist-induced phosphorylation sites may be particularly important for activation of human PR. Results from this study raise the possibility that blocking of these sites by ZK98299 may contribute in part to the antagonist action of this compound.