Phosphorylation of Human Progesterone Receptor by Cyclin-Dependent Kinase 2 on Three Sites That Are Authentic Basal Phosphorylation Sites In Vivo
Yixian Zhang,
Candace A. Beck,
Angelo Poletti1,
John P. Clement, IV,
Paul Prendergast,
Tai-Tung Yip,
T. William Hutchens,
Dean P. Edwards and
Nancy L. Weigel
Department of Cell Biology (Y.Z., A.P., J.P.C., N.L.W), Baylor
College of Medicine, Houston, Texas 77030,
Department of
Pathology and Molecular Biology Program, (C.A.B., P.P., D.P.E.),
University of Colorado Health Sciences Center, Denver, Colorado
80262,
Department of Food Science and Technology (T-T.Y.,
W.H.), University of California at Davis, Davis, California
95616
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ABSTRACT
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The human progesterone receptor (hPR) in T47D
breast cancer cells is phosphorylated on at least nine different serine
residues. We have previously reported the identification of five sites;
three are hormone inducible (Ser102,
Ser294 and Ser345), and
their phosphorylation correlates with the timing of the change in
receptor mobility on gel electrophoresis in response to hormone
treatment. The other two sites, Ser81 and
Ser162, along with the remaining sites, are
basally phosphorylated and exhibit a general increase in
phosphorylation in response to hormone. With the exception of
Ser81, all of these sites are in Ser-Pro
motifs, suggesting that proline-directed kinases are responsible for
their phosphorylation. We now report that cyclin A-cyclin-dependent
kinase-2 complexes phosphorylate hPR-B in vitro with a high
stoichiometry on three sites that are authentic basal sites in
vivo. One of these is Ser162, which has
been described previously. The other two sites are identified here as
Ser190 and Ser400. The
specificity and stoichiometry of the in vitro
phosphorylation suggest that hPR phosphorylation may be regulated in a
cell cycle-dependent manner in vivo.
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INTRODUCTION
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The progesterone receptor (PR) is a member of the steroid/thyroid
hormone receptor family of ligand-activated transcription factors (1).
All steroid receptors studied to date are phosphorylated on multiple
sites (2, 3, 4, 5, 6, 7, 8, 9, 10) and, in some cases, phosphorylation changes in response
to hormone treatment. The contributions of phosphorylation to steroid
receptor function have been addressed either by analyzing the function
of receptors containing mutated phosphorylation sites or by examining
the modulatory effects of cellular protein kinase/phosphatase
activities on receptor function. Mutagenesis studies have shown that
certain phosphorylation sites are important for maintaining maximum
transcriptional activity of the receptors (6, 7, 10, 11, 12, 13, 14). Cell
signaling studies have shown that the activity of estrogen receptor can
be modulated through phosphorylation of Ser118 by
mitogen activated protein (MAP) kinase pathways (15) and that several
different steroid receptors can be activated by modulation of kinase
activity in the absence of ligand (16, 17, 18, 19, 20, 21, 22, 23). Whether or not human PR
(hPR) exhibits ligand-independent activation is unclear. Modulators of
cellular kinases, however, act synergistically with hormone to further
enhance hPR-mediated transcriptional activity (24). Additionally,
treatment with 8-Br cAMP, an activator of protein kinase A, causes the
antagonist RU486 to act as an agonist for the PR (25, 26).
Human PR is expressed as two forms, a 120-kDa form (hPR-B) and a
shorter 94-kDa form (hPR-A), both of which are derived from the same
gene (27). hPR-B differs from hPR-A only in that it has an additional
164 amino acids (aa) at its amino terminus. We have previously reported
the identification of five phosphorylation sites in hPR (3, 28).
Ser81, Ser102, and Ser162 are
located in the B-specific segment whereas Ser294 and
Ser345 are common to both hPR-A and hPR-B. Three of the
sites, Ser102, Ser294, and Ser345,
are almost exclusively phosphorylated as a result of hormone treatment,
and the time course of these phosphorylations correlates with the
hormone-induced change in mobility of hPR observed by SDS gel
electrophoresis (3). The remaining sites exhibit basal phosphorylation,
and the extent of phosphorylation is rapidly increased in response to
hormone (3). Our previous phosphotryptic peptide mapping studies
indicated that there may be as many as nine phosphorylation sites in
hPR; thus at least four basal sites remain to be identified (3, 28).
Although the identification of the phosphorylation sites in hPR
as well as in many of the other receptors is incomplete, the sites
identified to date predominantly contain Ser-Pro motifs (3, 4, 9, 10).
Four of the five sites that we have identified in hPR contain the motif
Ser-Pro. This suggests that proline-directed kinases such as
cyclin-dependent kinases (Cdks) and/or MAP kinases are involved in the
phosphorylation of steroid receptors. A hormone-dependent
phosphorylation site in human estrogen receptor, Ser118,
which is contained in a classic MAP kinase consensus sequence (29, 30),
can be phosphorylated in vitro by MAP kinase (15, 31).
Because the phosphorylated consensus Ser-Pro motifs in hPR that we have
identified do not conform to this sequence, we have examined the
ability of another proline-directed kinase, Cdk2, to phosphorylate hPR.
We report here, that Cdk2 specifically phosphorylates highly purified
hPR in vitro on a subset of three sites. One of the sites
was previously identified as Ser162. The other sites are
Ser190 and Ser400, which we identify here as
two of the additional sites that are basally phosphorylated in
vivo.
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RESULTS
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In Vitro Phosphorylation of hPR
Because the majority of the sites identified in hPR contain
a Ser-Pro motif (3, 28), which is a core consensus sequence for
proline-directed kinases including Cdks and MAP kinases, we
investigated whether hPR is phosphorylated by Cdk2, a Cdk that requires
Ser-Pro as a portion of its recognition sequence (32).
Baculovirus-expressed and highly purified recombinant hPR was used as a
substrate for the in vitro kinase assays (33, 34). Aliquots
of the recombinant hPR were incubated with [
-32P]ATP
and increasing amounts of cyclin A-Cdk2 for 30 min at 37 C, separated
by SDS-gel electrophoresis, and phosphorylated receptor was detected by
autoradiography. Shown in Fig. 1
is the phosphorylation
of both purified A and B forms of hPR at levels of cyclin A-Cdk2 that
give maximal receptor phosphorylation. In addition to the major bands
corresponding to receptor phosphorylation, the phosphorylation of
cyclin A can be seen (see enzyme alone lane). Minor bands in
the receptor preparations appear to be receptor fragments because they
increase with the age of the receptor preparation, and we detect
smaller immunoreactive fragments (data not shown). To determine the
stoichiometry of phosphorylation, the receptor bands were cut from the
gels and counted by Cerenkov counting. Based on the specific activity
of the ATP, the receptor levels determined by silver staining of the
gel, and the amount of 32P incorporated into hPR-B and
hPR-A, about 1.5 mol/mol of phosphate was incorporated into hPR-A and
2.7 mol/mol was incorporated into hPR-B. To determine whether Cdk2
phosphorylates hPR on sites that are phosphorylated in vivo,
we performed HPLC phosphotryptic mapping of in vitro
phosphorylated hPR-A and hPR-B. As shown in Fig. 2
, four
major phosphopeptides were obtained from hPR-B and two phosphopeptides
from hPR-A. The elution time of each peptide corresponds with
phosphotryptic peptides 1, 2, 4, and 6 derived from hPR-B isolated from
R5020-treated T47D cells labeled with [32P] in
vivo (Fig. 3
). Only two of these, peak 6 (retention
time 52 min), and its overdigestion product, peak 4 (retention time 42
min), were identified previously (28). Both contain the hPR-B specific
site, Ser162. Thus, in addition to Ser162, Cdk2
phosphorylates two major basal sites contained in two peptides, HPLC
peptide 1 and peptide 2, common to hPR-A and hPR-B.

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Figure 1. In Vitro Phosphorylation of hPR by
Cyclin A-Cdk2
The results are obtained from two separate experiments. Left
panel, Purified baculovirus hPR-B (0.2 µg) was incubated at
37 C for 30 min in buffer containing 20 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 30 µM
[ -32P]ATP (specific activity, 12,000 dpm/pmol), and
cyclin A-cdk2 (40 nM). Right panel, hPR-A
(0.35 µg) was incubated at 37 C for 30 min in buffer containing 20
mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5
µM [ -32P]ATP (specific activity, 89,000
dpm/pmol), and cyclin A-cdk2 (25 nM). The reactions were
terminated by addition of sample buffer, and samples were subjected to
SDS-PAGE and detected by autoradiography. PR-B in lane 3 of panel A
contained 54000 dpm. PR-A in lane 3 of panel B contained 480,000 dpm.
Lane 1, Cyclin A-Cdk2; lane 2, purified hPR; lane 3, cyclin A-Cdk2 and
purified hPR.
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Figure 2. Phosphopeptide Maps of hPR Phosphorylated in
Vitro
Purified hPR-A and hPR-B phosphorylated by Cdk2 as shown in Fig. 1 were
isolated by SDS-gel electrophoresis, digested with trypsin, separated
by C18 reverse phase HPLC, and detected with an on-line radioactive
detector. Top panel, Phosphotryptic map of hPR-B.
Bottom panel, Phosphotryptic map of hPR-A. The
numbers above the peaks correspond to the elution
positions of peptides phosphorylated in vivo (see Fig. 3
and Ref. 28).
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Figure 3. Phosphotryptic Mapping of hPR from R5020-Treated
T47D Cells
Trypsin-digested immunopurified hPR-B was separated by C18 reverse
phase HPLC and detected with an on-line radioactivity detector. The
major peaks were numbered according to their retention times. Peaks
containing identified phosphorylation sites are labeled with the number
of the serine residue phosphorylated. This figure was modified and
reproduced from Zhang et al. (28) (Fig. 1 ) with the
permission of The American Society for Biochemistry and Molecular
Biology, Inc.
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Identification of the Phosphorylation Site in Peak 1 (P1) as
Ser190
To identify the site in P1, HPLC fractions containing P1 of
receptor labeled in vivo were subjected to secondary
proteinase digestion with the endoproteinases Asp-N and Glu-C and
analyzed by electrophoresis on a 40% alkaline polyacrylamide gel that
resolves small peptides based on their charge/mass ratio. P1 was not
cleaved by either Asp-N or Glu-C because digested and undigested
peptides have the same mobility on the gel (Fig. 4
),
suggesting the absence of both Asp and Glu in P1. Manual Edman
degradation analysis located the phosphorylated residue in the third
cycle (Fig. 5
). Because all phosphorylation sites in hPR
are on Ser residues (35), we have listed the potential tryptic peptides
in hPR-B that contain a Ser in position 3 (Table 1
).
Amino acids are numbered from the first amino acid of hPR-B; hPR-A
begins at Met165. By deduction, the peptide beginning with
Ser188 is the most likely candidate for P1 for several
reasons. First, the peptide beginning with Val160 is hPR-B
specific and therefore is excluded because P1 is common to hPR-A and
hPR-B (Fig. 2
and 28 . The peptides beginning with aa 271, 547,
770, 791, and 900 all contain either a Glu- or Asp-containing sequence
that should be cleaved by secondary digest by Glu-C or Asp-N; P1 is
resistant to these secondary digestions (Fig. 4
). Only the peptide
starting with aa 188 lacks a Glu or Asp. However, we had to consider
the possibility that the peptide beginning with aa 271 may be resistant
to secondary digestion because of the proximity of the Asp and Glu to
the amino terminus of the peptide. Therefore, as a further confirmation
of the identity of P1, we attempted amino acid sequence analysis of the
HPLC-isolated P1 peptide from a tryptic digest of purified unlabeled
baculovirus-produced PR. We showed previously that PR expressed in the
baculovirus system is phosphorylated on all the same major tryptic
peptides as native PR from T47D cells (36). Thus the recombinant PR
gives the same HPLC-eluted P1 phosphopeptide as native T47D PR.
However, the region under HPLC peak 1 contained other contaminating
peptides that prevented identification by this method. As an
alternative approach we determined whether P1 generated from T47D PR
coeluted with a synthetic phosphopeptide corresponding to the peptide
in Table 1
that starts with aa 188. A synthetic peptide was
synthesized, and the sequence of the peptide GLS(P)PAR as well as the
location of the phosphorylation was confirmed by automated sequencing
and mass spectrometry (data not shown). The synthetic phosphopeptide
and a small amount of in vivo labeled P1 were mixed and run
on a C18 reverse phase column. The fraction containing
identified T47D P1 was collected, dried, and subsequently subjected to
electrophoresis on a 40% alkaline gel. The gel was dried and subjected
to autoradiography (data not shown). The band corresponding to P1 was
excised, and the peptide was eluted and subsequently submitted to
automated sequencing for identification of the synthetic peptide. We
found that phosphorylated GLS(P)PAR (Table 2
) coeluted with P1 by HPLC
and 40% gel. Thus we conclude that P1 is phosphorylated on
Ser190.

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Figure 4. Characterization of Endoproteinase-Treated Tryptic
Phosphopeptide 1 by Peptide Gel Electrophoresis
Peptide 1 was treated with Asp-N (cuts on the N-terminal side of Asp)
or Glu-C (cuts on the C-terminal side of Glu provided it isnt within
3 aa of the carboxyl terminus) and analyzed by 40% alkaline
polyacrylamide gel electrophoresis. The gel was dried and peptides were
detected by autoradiography.
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Figure 5. Identification of the Position of the Phosphoamino
Acid by Manual Edman Degradation
Peptide 1 was covalently coupled to arylamine membrane discs using
carbodiimide and subjected to manual Edman degradation. The
radioactivity in the released amino acid was determined after each
cycle using a scintillation counter. The background counts (24 ±
4, n >10) were not subtracted from the counts. The cycle containing
the released 32P is the cycle containing the phosphoamino
acid.
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Identification of Ser400 as the
Phosphorylation Site in Peptide 2 (P2)
In contrast to other phosphopeptides generated from hPR, the yield
of P2 has been variable from experiment to experiment. In fact, in some
studies P2 has been barely detectable (28, 36). Upon secondary
digestion of P2 with Glu-C or Asp-N, cleavage was obtained with Glu-C
but not Asp-N (Fig. 6
), and the majority of
[32P] in peptide 2 was released by manual Edman
degradation in cycle 14 (Fig. 7A
). Only two predicted
tryptic peptides of hPR have a serine in position 14 and of these only
the one beginning with aa 434 has Glu residues and should be sensitive
to further cleavage by Glu-C and not by
Asp-N (Table 3
). The peptide beginning
with aa 434 has Asp residues and should be sensitive to cleavage by
Asp-N. However, the peptide beginning with aa 434 does not contain the
anticipated Ser-Pro motif according to the in vitro Cdk2
phosphorylation results, which made us suspicious that this may not be
P2. To further analyze peptide 2, we performed manual Edman degradation
of the Glu-C secondary digested phosphopeptide (see Fig. 6
). The
majority of [32P] was released in cycle 5 of the
Glu-C-treated peptide, not in cycle 11 as predicted for the peptide
that begins with aa 434. To determine whether peptide 2 was a result of
incomplete digestion with trypsin, we redigested it with trypsin. The
redigestion with trypsin, shown in Fig. 6
, released a smaller
phosphopeptide from the original, indicating that P2 is not a limit
digest. Thus we conclude that P2 is an incomplete tryptic digest that
is not the initial predicted peptide indicated in Table 3
. Further
analysis of hPR sequence for possible partial tryptic digests revealed
that P2 could be a peptide beginning with Ile387 that would
contain Ser400 in position 14 (Table 4
). Consistent with
results in Fig. 6
and Fig. 7A
, cleavage of this peptide with Glu-C
places the Ser in position 5. The peptide beginning with
Ile387 contains a trypsin site that, when cleaved, results
in a very small phosphopeptide of 3 aa with the Ser in position 1
(Table 4
). To confirm that P2 indeed is the peptide
indicated in Table 4
with phosphorylation at Ser400, the
corresponding HPLC fraction from purified, unlabeled
baculovirus-expressed hPR was subjected to microsequencing. Table 5
shows that an amino acid sequence corresponding to the
first 10 aa of the predicted peptide shown in Table 4
was detected.
Since complete tryptic digestion of P2 should yield a very small
hydrophilic peptide S(P)PR that would not be expected to bind to a
C18 column, we next analyzed the flow-through fraction of a
typical phosphotryptic peptide digest. The flow-through contains a
mixture of free [32P] phosphate and peptides that are not
retained by the column. Manual Edman degradation of the flow-through of
the HPLC after coupling to an arylamine filter revealed that it
contains a peptide with [32P] in cycle 1 as predicted for
this peptide (Fig. 7C
). Thus we conclude that HPLC P2, which has a
retention time of 32 min on HPLC, is the result of incomplete tryptic
digestion producing a peptide of 16 aa starting at Ile387
with the phosphoserine, Ser400, in the 14th position. The
complete digest results in a 3-aa peptide that is not retained and has
Ser400 as the phosphorylated amino acid in the first
position.

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Figure 6. Characterization of Endoproteinase-Treated Tryptic
Phosphopeptide 2
Peptide 2 was treated with Asp-N, Glu-C, or trypsin. Treated and
untreated peptide 2 were separated by 40% alkaline polyacrylamide gel
electrophoresis. The gel was dried and peptides were detected by
autoradiography.
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Figure 7. Identification of the Cycle Containing
Phosphoserine by Manual Edman Degradation
Peptide 2, Glu-C-digested peptide 2, and HPLC column drop-through were
each covalently coupled to arylamine membrane discs using carbodiimide
and subjected to manual Edman degradation. The radioactivity in the
released amino acid was determined after each cycle using a
scintillation counter. The background counts (24 ± 4, n >10)
were not subtracted from the total counts. The cycle containing the
released 32P is the cycle containing the phosphoamino acid.
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Analyses of the phosphopeptides isolated from in vitro
phosphorylated hPR exhibited elution times on HPLC, mobility in
alkaline gel electrophoresis, protease sensitivity, and positions of
the phosphoamino acids that were indistinguishable from those of
in vivo phosphorylated peptides 1, 2, and 6. Hence we
conclude that cyclin A-Cdk2 phosphorylates Ser162,
Ser190, and Ser400 in hPR.
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DISCUSSION
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We had previously identified five phosphorylation sites in hPR-B
(3, 28). Of these, four contain a Ser-Pro motif (3, 28), and the fifth
is phosphorylated by casein kinase II. In an attempt to identify a
kinase that phosphorylates the other sites, we examined the ability of
cyclin A-Cdk2 to phosphorylate hPR-B and hPR-A. In this study, we have
shown that highly purified cyclin A-Cdk2 phosphorylates hPR with high
stoichiometry on three sites that are basally phosphorylated in
vivo. As expected for cyclin A-Cdk2 substrates (37), the PR
phosphorylation was inhibited by addition of the Cdk2 inhibitor, p21
(data not shown). One of the sites phosphorylated by Cdk2,
Ser162, was identified previously as a basal hPR-B specific
site (28), and the other two Cdk2 sites corresponded to phosphopeptides
that we had detected previously in in vivo labeled receptor,
but whose sequence had not been determined. The other two were
identified in the present study as Ser190 and
Ser400. We predicted, based on peptide mapping, that the
hPR-B contains at least nine different phosphorylated serine residues.
With the additional two sites identified here, we have thus far
identified seven of the sites. A scheme of the seven sites is shown in
Fig. 8
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Figure 8. Summary of Human PR Phosphorylation Sites
Ser102, Ser294, and Ser345 are
hormone-inducible sites whose phosphorylation correlates with the
timing of the change in mobility on gel electrophoresis in response to
hormone treatment (3). Ser81, Ser162,
Ser190, and Ser400 are basally phosphorylated
and exhibit an increase in phosphorylation in response to hormone
treatment.
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In these and in previous studies, we have used a combination of tryptic
digestion, HPLC reverse phase chromatography, secondary protease
digestion, manual Edman degradation, and recombinant hPR produced in a
baculovirus overexpression system to identify the phosphorylation sites
in hPR. Although identification of some sites has been straightforward,
the sites identified in this study highlight some of the difficulties
in identifying sites in proteins that are expressed at low levels so
that direct protein sequencing of highly purified peptides is
impractical. The identification of peptides then relies on the
proteases cleaving as predicted or on sequences obtained from
carrier recombinant protein. In the case of the
baculovirus-expressed hPR-B, we have shown that the same
phosphopeptides are present as in authentic T47D receptor although the
extent of phosphorylation of the individual sites is lower (36). P1 is
a small peptide whose mobility on the C18 reverse phase
column is greatly altered by phosphorylation. Consequently, the use of
a recombinant carrier protein, which is only partially phosphorylated
on this site (36), was insufficient to produce an adequate mass of
peptide to be distinguished from other comigrating peptides when the
partially purified fraction was subjected to automated Edman
degradation. In this case, synthesis and analysis of the predicted
phosphopeptide was sufficient to confirm the identification of
Ser190 in P1.
A limitation of the phosphate release and double digest approach to
identifying phosphorylation sites is the necessary assumption that the
proteases are cleaving with the appropriate specificity. In the case of
P2, neither of the predicted tryptic peptides containing a serine in
position 14 (Table 3
) met the criteria for our analyses: a Ser-Pro site
and a peptide whose cleavage with Glu-C resulted in a phosphoserine in
position 5. In this case, we were able to confirm, by re-treating the
peptide with trypsin, that there was a protease-resistant peptide bond.
Lys-S(P) and Arg-S(P) bonds are known to cleave poorly (38) so that
this is not surprising in retrospect. Fortunately, the recombinant
protein produced the same partial digestion product, and thus the
predicted peptide that contains Ser400 could be verified by
direct sequencing.
Another difficulty in detecting and identifying phosphopeptides is the
tendency to assume that all of the peptides will be detected by a
single method such as reverse phase HPLC. Large hydrophobic peptides
may simply not elute as a discrete peak, and very small peptides may
not be retained. Complete tryptic digestion of P2 produces a peptide
that does not bind to the C18 column and coelutes with free
phosphate. Once we were aware of this potential peptide, we were able
to detect the peptide by alkaline gel electrophoresis of the
flow-through fraction and to show by manual Edman degradation that a
peptide with S(P) in the first position was in this fraction.
The roles of the phosphorylation sites in hPR function have yet to be
determined. Takimoto et al. (39) have mutated singly or in
combination many of the serines that are potential phosphorylation
sites in hPR-B and have measured the activity of the resulting mutants.
Although many of the mutants exhibited no change in activity, mutation
of Ser190, which we have now demonstrated is an authentic
site, resulted in a 2025% decrease in transcriptional activity,
indicating that this site is necessary for full activity of the
receptor (39).
Six of the seven identified sites in hPR contain a Ser-Pro motif, and
most of the other sites identified in other steroid receptors,
including PRs in other species (4, 40), estrogen receptors (6, 7, 41),
glucocorticoid receptors (9), and androgen receptors (10), also contain
this sequence. For the most part, the Ser-Pro directed kinases that
phosphorylate steroid receptors have not yet been identified. The
estrogen receptor contains a site that conforms to the consensus
sequence for a MAP kinase (15, 42), and several investigators have
shown that this site is phosphorylated by MAP kinase (15, 31). Arnold
et al. (31) have reported that the human estrogen receptor
is not a substrate for Cdc2. To our knowledge, this is the first report
that a steroid receptor can be phosphorylated by a cell cycle-dependent
kinase. Although there have been no other reports of cell
cycle-dependent kinases phosphorylating steroid receptors, Hsu and
DeFranco (43) have reported cell cycle-dependent changes in
glucocorticoid receptor activity and phosphorylation. Hu et
al. (44) have also detected cell cycle-dependent changes in
glucocorticoid receptor phosphorylation.
Although hPR-B contains a minimum of six sites with Ser-Pro as a
portion of the phosphorylation site, only three of these are substrates
phosphorylatable by Cdk2 under in vitro conditions. The
three sites that are phosphorylated by Cdk2 in vitro are
basal phosphorylation sites that are partially phosphorylated in
vivo in the absence of hormone and whose phosphorylation increases
rapidly (510 min) in response to hormone treatment. In contrast, the
three Ser-Pro sites that are not targeted by Cdk2 are phosphorylated
slowly in vivo in response to hormone treatment requiring
12 h for maximal phosphorylation (3). The receptor used in these
experiments had been treated with hormone in vivo, and we
have detected no additional Cdk2-dependent phosphorylation when
additional ligand is added to the receptor. This suggests that the
other Ser-Pro sites are phosphorylated by one or more additional
kinases and that subsets of phosphorylation sites in hPR-B are
phosphorylated by different kinases, allowing regulation by multiple
signal transduction pathways.
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MATERIALS AND METHODS
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Materials
MEM was purchased from Irvine Scientific (Santa Ana, CA).
Phosphate-free MEM was obtained from GIBCO BRL (Grand Island, NY).
AB-52 is a mouse monoclonal antibody that recognizes both hPR-A and
hPR-B (45). R5020 (promegestone) and carrier-free
[32P]H3PO4 were purchased from
Dupont/New England Nuclear Products (Boston, MA). Protein-A Sepharose
was purchased from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ).
Tosylphenylalanyl chloromethyl ketone-treated trypsin was purchased
from Worthington Biochemical Corp. (Freehold, NJ). Sequencing grade
endoproteinases Asp-N and Glu-C were purchased from Boehringer Mannheim
(Indianapolis, IN). Phenylisothiocyanate and sequencing grade
trifluoroacetic acid (TFA) and HPLC reagents were purchased from J. T.
Baker Chemical Corp. (Phillipsburg, NJ). Triethylamine, and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) were purchased
from Sigma (St. Louis, MO). Sequelon-AA membranes and Mylar sheets were
obtained from Millipore Corp. (Milford, MA). Highly purified
baculovirus- expressed glutathione-S-transferase cyclin
A-Cdk2, provided by J. Wade Harper (Department of Biochemistry, Baylor
College of Medicine), was purified and characterized as described
previously (37, 46).
Cell Culture, PR Labeling, and Receptor Preparation
T47D human breast cancer cells were maintained and grown in
75-cm2 T flasks with frequent changes of medium as
previously described (24). Cells were incubated for 24 h in MEM
containing 5% dextran charcoal-treated FCS. Steady state labeling with
[32P]orthophosphate was carried out by preincubation of
cells in phosphate-free serum-free medium for 1 h at 37 C followed
by incubation in phosphate-free MEM containing
[32P]orthophosphate (0.83 mCi/ml) for 6 h at 37 C.
Cells were incubated with hormone for the final 2 h before
harvest.
Cells were harvested in 1 mM EDTA in Earles balanced salt
solution and homogenized at 4 C in a Teflon-glass Potter-Elvehjem
homogenizer (Fisher, Pittsburgh, PA) 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
proteinase inhibitors as described previously (24). The whole-cell
extract was prepared by centrifuging the homogenates at 100,000 x
g for 30 min, followed by dialysis of the supernatant in
KPFM to remove the salt before immunoprecipitation.
Immunoprecipitation and Gel Purification of hPR
Absorption of AB-52 to Protein A-Sepharose was performed as
previously described (24). Dialyzed whole-cell extracts containing hPR
were incubated with the antibody-bound Protein A-Sepharose on an
end-over-end rotator for 4 h at 4 C, and the resin was then washed
three times with KPFM containing 0.3 M NaCl to remove
nonspecifically bound protein. Receptors were eluted with 2% SDS
sample buffer and electrophoresed on a 7.0% discontinuous SDS
polyacrylamide gel. 32P-Labeled receptors were located by
autoradiography of the gels, and bands containing either hPR-A or hPR-B
were excised.
HPLC Analysis of Tryptic Peptides
The individual gel slices containing hPR were washed with 50%
methanol for 1 h followed by H2O for 30 min and 50
mM ammonium bicarbonate for 5 min in 1.5-ml microfuge
tubes. Twenty micrograms of trypsin were then added to each tube. After
the tubes were incubated for 4 h at 37 C, another 20 µg of
trypsin were added, and this was repeated three times. The digested
peptides were dried in a Speedvac (Savant Instruments, Hicksville, NY),
dissolved in 150 µl 50% formic acid, loaded on a Vydac (Hesperia,
CA) C18 reverse phase column in 0.1% TFA in water, run at
a flow rate of 1 ml/min, and eluted with a linear gradient from 045%
acetonitrile over 90 min. The labeled peptides were detected with an
on-line model IC Flo-One Beta-radioactivity flow detector (Radiomatic
Instruments, Inc., Tampa, FL) and collected as 2-ml fractions.
Phosphorylation Site Identification
HPLC fractions corresponding to each labeled peptide were dried
and analyzed by electrophoresis on a 40% polyacrylamide gel. Labeled
peptides were detected by autoradiography of the dried gel, excised,
and eluted with H2O as previously described (28).
The position of the phosphoamino acid was determined by manual Edman
degradation as described by Sullivan and Wong (47). In brief, the
peptide to be analyzed was dissolved in 30 µl 50% acetonitrile and
spotted on an arylamine-Sequelon disc, which was placed on a Mylar
sheet on top of a heating block set at 50 C. After 5 min, the aqueous
solvent was evaporated and the disc was removed from the heating block.
Five microliters of EDAC solution (50 mM in Mes, pH 5.0)
were added to the disc to covalently link the peptide. After 30 min at
room temperature, the disc was washed five times with water and five
times with TFA to remove unbound peptide. The disc was then washed
three times with methanol and subjected to Edman degradation: The disc
was treated at 50 C with 0.5 ml coupling reagent
(methanol-water-triethylamine-phenylisothiocyanate; 7:1:1:1, vol/vol)
for 10 min. After five washes with 1 ml methanol, the disc was treated
at 50 C for 6 min with 0.5 ml TFA to cleave the amino-terminal amino
acid. The TFA solution was placed in a scintillation vial, and the disc
was washed with 1 ml TFA and 42.5% phosphoric acid (9:1, vol/vol). The
wash was combined with the TFA solution, and the released
32P was determined by Cerenkov counting. The next cycle was
begun after the disc was washed five times with 1 ml methanol.
To further characterize the phosphotryptic peptides, they were digested
with secondary endoproteinases Glu-C and Asp-N. Glu-C digestion was
performed in 200 µl 25 mM ammonium bicarbonate, pH 7.8,
for 8 h at 37 C. Asp-N digestion was performed in 200 µl 50
mM sodium phosphate buffer, pH 8, containing 0.2 µg Asp-N
and incubated at 37 C for 4 h. Glu-C cuts on the C-terminal side
of Glu, except for Glu-Pro bonds (38). Glu-X bonds within three
residues of the end of a peptide are also cleaved poorly (38). Asp-N
cuts on the N-terminal side of Asp residues (48).
Endoproteinase-treated phosphopeptides were analyzed by electrophoresis
on 40% alkaline gel and/or by manual Edman degradation.
Baculovirus Expression of hPR and Affinity Purification
Full-length A or B forms of hPR were expressed from recombinant
baculovirus vectors in Sf9 insect cells as previously described (33).
Expressed hPR-A and hPR-B, bound to R5020 (100 nM), were
purified from whole-cell extracts of infected Sf9 cells (300 x
106 cells) by monoclonal antibody affinity chromatography
with AB-52 cross-linked to protein G-Sepharose as previously described
(34). Receptors bound to the AB-52 resins were eluted using alkaline
conditions (pH 11.0) immediately followed by neutralization to pH 7.4.
This method results in purification of hPR to more than 95% as judged
by silver-stained SDS-PAGE and Western blot. Additionally, purified hPR
retains the majority of bound R5020 and is biologically active with
respect to its DNA-binding ability. We have previously shown (36) that
baculovirus-expressed PR is correctly phosphorylated, although the
extent of phosphorylation is much less than the receptor from
hormone-treated T47D cells (
30%). We have found that isolation in
the absence of phosphatase inhibitors results in almost complete loss
of phosphorylation. Hence, the substrate used in these studies is
essentially dephosphorylated.
Preparation and Isolation of Peptides for Sequencing
Purified baculovirus-expressed hPR-B was digested with trypsin
(5% wt/wt). Tryptic peptides were separated by reverse phase HPLC as
described earlier (28). Fractions with retention times corresponding to
the 32P-labeled tryptic phosphopeptides from T47D cells
were collected, dried, and sequenced using an automated sequencer
(4).
Synthesis of Peptide GLSPAR
Peptide GLSPAR was synthesized on an Applied Biosystems model
430A automated peptide synthesizer using Fmoc/NMP
(9-fluorenylmethyloxycarbonyl/N-methylpyrrolidone) chemistry
(Fast MOC, Applied Biosystems, Foster City, CA). The serine was
incorporated without side-chain protection to allow phosphorylation.
The serine was phosphorylated as described by Otvos et al.
(49) after removal of the Fmoc amino terminus-protecting group. The
phosphopeptide was deprotected and cleaved from the resin with 35% TFA
and scavengers including thioanisole, 1,2-ethanedithiol, and phenol.
Both phosphorylated and nonphosphorylated peptide were purified by a
combination of reverse phase and ion exchange HPLC. The amino acid
sequence was confirmed by sequential Edman degradation with an Applied
Biosystems model 473A automated peptide sequence analyzer.
In Vitro Phosphorylation of hPR with Cyclin A-Cdk2
Complexes
Purified baculovirus-expressed hPR-A and hPR-B were incubated in
a buffer containing 20 mM Tris-HCl, pH 7.5, 10
mM MgCl2, and 50 µM
[
-32P]ATP. Purified cyclin A-Cdk2 (46) was added to
initiate the reaction (final volume 40 µl) and incubated for 30 min
at 37 C. The reaction was terminated by the addition of Laemmli sample
buffer and subjected to SDS-gel electrophoresis.
32P-Labeled PR was detected by autoradiography. The band
containing hPR-A or hPR-B was excised and subjected to subsequent
analyses including tryptic digestion, HPLC, and peptide gel
separations, protease digestion, and manual 32P
release.
 |
ACKNOWLEDGMENTS
|
---|
We thank Richard Cook for peptide sequencing; Chee Ming Li for
assisting T. William Hutchens and Tai-Tung Yip in the synthesis,
purification, and mass spectrometric analysis of the peptides; J. Wade
Harper for providing cyclin A-Cdk2 complexes; and Kurt Christensen for
technical assistance with baculovirus expression and purification of
PR.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Nancy L. Weigel, Ph.D., Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
This work was supported in part by United States Public Health Service
Grants CA-57539 (to N.L.W.) and CA-46938 (to D.P.E.), University of
Colorado Cancer Center Tissue Culture Laboratory, National Service
Research Fellowship Award HD-0743 (to C.A.B.), and United States Army
Breast Cancer Fellowship AMD 1794-J-4202 (to Y.Z.).
1 Current address: Istituto di Endocrinologia, via Balzaretti 9, 20133
Milano, Italy. 
Received for publication December 16, 1997.
Accepted for publication March 17, 1997.
 |
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