From the Department of Molecular and Cellular
Biology, § Protein Chemistry Core Laboratory, and
¶ Department of Immunology, Baylor College of Medicine, Houston,
Texas 77030 and the
Department of Pathology and Program in
Molecular Biology, University of Colorado Health Science Center,
Denver, Colorado 80262
Received for publication, October 26, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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We have previously reported the identification of
seven in vivo phosphorylation sites in the amino-terminal
region of the human progesterone receptor (PR). From our
previous in vivo studies, it was evident that several
phosphopeptides remained unidentified. In particular, we wished to
determine whether human PR contains a phosphorylation site in the hinge
region, as do other steroid receptors including chicken PR, human
androgen receptor, and mouse estrogen receptor. Previously, problematic
trypsin cleavage sites hampered our ability to detect phosphorylation
sites in large incomplete tryptic peptides. Using a combination of mass
spectrometry and in vitro phosphorylation, we have
identified six previously unidentified phosphorylation sites in human
PR. Using nanoelectrospray ionization mass spectrometry, we have
identified two new in vivo phosphorylation sites,
Ser20 and Ser676, in baculovirus-expressed
human PR. Ser676 is analogous to the hinge site identified
in other steroid receptors. Additionally, precursor ion scans
identified another phosphopeptide that contains
Ser130-Pro131, a likely candidate for
phosphorylation. In vitro phosphorylation of PR with Cdk2
has revealed five additional in vitro Cdk2 phosphorylation sites: Ser25, Ser213, Thr430,
Ser554, and Ser676. At least two of these,
Ser213 and Ser676, are authentic in
vivo sites. We confirmed the presence of the Cdk2-phosphorylated
peptide containing Ser213 in PR from in vivo
labeled T47D cells, indicating that this is an in vivo
site. Our combined studies indicate that most, if not all, of the
Ser-Pro motifs in human PR are sites for phosphorylation. Taken
together, these data indicate that the phosphorylation of PR is highly
complex, with at least 14 phosphorylation sites.
Human progesterone receptor
(PR),1 a ligand-activated
transcription factor and member of the steroid receptor superfamily
(1), is expressed as two forms: the full-length PR-B and the shorter form, PR-A, which lacks the first 164 amino acids of PR-B (2, 3). These
proteins differ in their relative ability to activate target genes and
in the unique repressor activity of the PR-A isoform (4-8). Recently,
the physiological roles for the different isoforms have been examined
by the generation of isoform-specific knockout mice. Interestingly,
knockout of the PR-A isoform in mice demonstrated a strong
tissue-specific role for PR-A in uterus not shared by PR-B (9). In
addition, overexpression of either PR-A or PR-B results in aberrant
mammary gland development, and the phenotypes of the two overexpressing
lines differ (10, 11). Moreover, recent studies have suggested that
differential coactivator recruitment may be responsible for the
isoform-specific differences observed in transactivation assays
(8).
In addition to regulation dependent on isoform, PR is a phosphoprotein
(12-17) whose activity can be regulated by phosphorylation. There is
ample evidence that regulation of cell signaling pathways alters the
activity and phosphorylation of PR (18-21) as well as other steroid
receptors (22-30). Some of these changes are due to direct alterations
in receptor phosphorylation, whereas others appear to affect associated
proteins (29, 31-35). Additionally, the role of individual sites has
been examined for PR. Mutation of Ser190 results in
decreased activity of PR (21). Since different kinases target unique
subsets of sites and these phosphorylations may have opposing effects
on receptor activity, it is important to know which sites are
coregulated by each kinase for mutagenesis studies to determine the
impact on receptor function.
We have previously identified seven phosphorylation sites in human PR
isolated from 32P-labeled T47D breast cancer cells. Each of
these sites is located in the amino-terminal (A/B) domain. Three of the
previously identified sites, Ser81, Ser102, and
Ser162, are unique to the B form (15). In vivo
labeling of the endogenous PR in T47D cells showed that the PR is
phosphorylated basally in the absence of hormone at serines 81, 162, 190, and 400. Upon hormone stimulation, the net phosphorylation of
these basal sites is dramatically increased within 5 min of treatment.
In addition, hormone induces the phosphorylation of three new
phosphorylation sites, serines 102, 294, and 345, in a temporally
delayed manner, requiring 2 h for maximal phosphorylation. These
sites are referred to as the hormone-dependent
phosphorylation sites (16). Recombinant human PR expressed in
Sf9 insect cells displays the same phosphorylation pattern as PR
from hormone-treated T47D cells; however, the hormone dependence is
lacking in Sf9 cells (36). Additionally, we have shown that PR
is a substrate for several kinases in vitro. We reported
previously that three in vivo phosphorylation sites, Ser162, Ser190, and Ser400, are
phosphorylated in vitro by the cyclin A-Cdk2 complex (17) and that Ser81 is phosphorylated by casein kinase II
(15).
Although many of the phosphorylation sites have been identified in PR,
several phosphotryptic peptide peaks detected in the HPLC analyses of
in vivo labeled PR have yet to be identified. Most of the
phosphorylation sites in steroid receptors contain Ser/Thr-Pro motifs,
and in the case of the chicken PR all four of these motifs are
phosphorylated (37, 38). Human PR contains 15 of these sequences,
several of which have already been identified as phosphorylation sites
(15-17). We were particularly interested in determining whether the
conserved Ser-Pro in the hinge region between the hormone and DNA
binding domains is phosphorylated as has been described for chicken PR
(37), mouse estrogen receptor (39), and human androgen receptors (26).
Initial analyses by Sheridan et al. had suggested that this
site is not phosphorylated in human PR (13). Arg-Pro and Lys-Pro motifs
are not efficiently cleaved by trypsin (40), and PR has an unusually
large number of these sequences. Analyses of candidate Ser-Pro
phosphorylation sites including Ser676 in the hinge region
reveal that many are located within peptides bounded by these highly
resistant cleavage sites. If left uncleaved, these phosphopeptides will
be very large and difficult to recover by HPLC using a C18
reversed-phase column. Using trypsin modified by reductive alkylation,
which has decreased susceptibility to autolysis, we have increased the
percentage cleavage at these problematic sites, allowing resolution of
new phosphopeptides both in PR phosphorylated in vitro by
Cdk2 and by mass spectrometry of phosphopeptides isolated from PR
expressed in Sf9 cells.
Here we describe the identification of six candidate phosphorylation
sites identified either in in vitro phosphorylation
experiments using Cdk2 or by mass spectrometry of peptides derived from
PR expressed in Sf9 cells. These sites include
Ser676 in the hinge region, which was identified as both an
in vitro Cdk2 site and by mass spectrometry. Three
additional sites, common to PR-A and PR-B, and two sites unique to the
PR-B form have also been found. Finally, an additional site has been
localized to a region between amino acids 107 and 159 in the portion of
the receptor unique to PR-B. These studies suggest that most, if not all, of the Ser/Thr-Pro motifs in PR are phosphorylated.
Materials--
Phosphate-free Dulbecco's modified Eagle's
medium and Hanks' balanced salt solution were purchased from Life
Technologies, Inc., and fetal bovine serum was from Hyclone (Logan,
UT). Radioinert R5020 was obtained from Amersham Pharmacia Biotech.
[32P]H3PO4 and
[ Baculovirus-expressed His Tag PR Purification--
Infection of
Sf9 insect cells with baculovirus encoding His tag PR-B or PR-A
and treatment with 200 nM R5020 was performed as previously
described (41). Cells from a 500-ml culture were centrifuged to
harvest, and pellets were stored at Purification of HA-Cdk2·Glutathione S-Transferase-cyclin
A Complex--
This purification was performed essentially as
described previously (42). In brief, Sf9 cells were infected
with the baculoviruses encoding HA-Cdk2 and glutathione
S-transferase-cyclin A at a 1:1 ratio for 40 h at
27 °C. Cells were harvested, washed with phosphate-buffered saline
(PBS), and harvested by centrifugation at 2000 rpm. The pellet was then
resuspended in 2 volumes of NETN buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 5 mM NaF, 30 mM
p-nitrophenyl phosphate, 1 µg/ml leupeptin, 1 µg/ml
antipain, 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 10 min. The extract was prepared by sonication and cleared
by centrifugation. Five volumes of kinase buffer (20 mM HEPES, pH 7.5, 15 mM MgCl2, 1 mM
dithiothreitol, 10 mM NaF, 5 µg/ml leupeptin, 1 µg/ml
antipain, 1 mM phenylmethylsulfonyl fluoride) and 1 mM ATP were added to the extract and incubated at 30 °C for 30 min. Glutathione-Sepharose was then added to the lysate for
1 h at 4 °C. The beads were washed with NETN and then with 100 mM Tris-HCl, pH 8, containing 150 mM NaCl. To
elute the active Cdk2-cyclin A complex, 100 mM Tris-HCl, pH
8, containing 150 mM NaCl and 40 mM glutathione
was added to the beads and incubated at 4 °C for 15 min. The
supernatant containing the kinase complex was removed, and aliquots
were frozen at In Vitro Phosphorylation of His Tag PR--
Purified recombinant
His tag PR-A or PR-B (1 µg) was incubated with 5-10 µl of purified
Cdk2-cyclin A and 50 µM [ Preparation and Separation of Phosphopeptides--
Sequencing
grade trypsin modified by reductive alkylation to prevent autolysis
(Promega) was reconstituted in acetic acid as described in the
manufacturer's instructions. Two µg of trypsin were added to each
tube containing a gel slice prepared as described previously (15) in 1 ml of 50 mM ammonium bicarbonate, pH 7.8, and incubated
overnight at 37 °C on a rotating platform. The tryptic digest was
dried in a SpeedVac Concentrator® (Savant, Farmingdale, NY), and the
phosphopeptides were separated on a C18 reversed-phase column using a Beckman System Gold HPLC. A linear 0-45% acetonitrile gradient in 0.1% trifluoroacetic acid was run for 90 min followed by
an increase to 100% acetonitrile by 110 min at a flow rate of 1 ml/min. The phosphopeptide peaks were detected using an online radioactive flow detector as described previously. 2-ml fractions were
collected, counted by Cerenkov counting, split for analysis, and dried
in a SpeedVac.
Manual Edman Degradation--
To determine the cycle or position
of the phosphoamino acid in the peptide, manual Edman degradation was
performed as previously described (43) using the Sequelon-AA Reagent
kit. Briefly, the tryptic phosphopeptides were covalently coupled to
Sequelon disks and subjected to Edman degradation. Each "cycle" of
Edman degradation begins by modifying the first
NH2-terminal amino acid of the peptide, which destabilizes
the first peptide bond. Treatment with trifluoroacetic acid cleaves
this bond and releases the amino acid from the disc. Cerenkov counting
of the supernatant measures the amount of 32P released. The
disc was then subjected to additional cycles of Edman degradation.
Secondary Endoproteinase Digestions--
Cleavage by the
endoproteinases Asp-N, Glu-C, Arg-C, and Lys-C was used to further
characterize the phosphopeptides. For Asp-N digestion, the sample was
resuspended in 50 mM sodium phosphate buffer, pH 8.0, and
incubated with 0.2 µg of Asp-N for 8 h at 37 °C. Glu-C
digestion was performed with 1 µg of Glu-C in 25 mM
ammonium bicarbonate, pH 7.8, for 8 h at 30 °C. The typical Arg-C digest was performed by dissolving the dried peptides in 85 µl
of incubation buffer, adding 10 µl of activation solution (provided
by the manufacturer) and 5 µl (or 0.5 µg) of Arg-C, and incubating
for 12 h at 37 °C. For Lys-C, the sample was redissolved in 100 µl of digestion buffer (25 mM Tris-HCl, pH 8.5, 1 mM EDTA) and incubated with 0.2 µg of Lys-C at 37 °C
for 12 h.
Phosphoamino Acid Analysis--
Phosphoamino acid analysis was
performed as described (44). In brief, phosphopeptide samples
containing 4 µg of each phosphoamino acid standard (phosphoserine,
phosphothreonine, phosphotyrosine) were hydrolyzed in 6 M
HCl and then dried in a SpeedVac. The sample was resuspended in pH 1.9 (2.5% (v/v) formic acid, 7.8% (v/v) acetic acid) buffer and spotted
on a TLC plate, followed by electrophoresis for 24 min at 1500 V in the
first dimension using a HTLE-7000 electrophoresis system (CBS
Scientific). After the plate was dry, the plate was rotated
90o counterclockwise and electrophoresed in the second
dimension in pH 3.5 buffer (5% (v/v) acetic acid, 0.5% (v/v)
pyridine) at 1300 V for 17 min. The negatively charged phosphoamino
acids migrate toward the (+)-terminal during electrophoresis in both
dimensions. The amino acid standards were visualized by spraying the
plate with a 0.5% solution of ninhydrin in acetone. Phosphorylated
amino acids were detected by autoradiography.
In Vivo Labeling of T47D Cells and Extraction--
T47D cells
were plated at a density of 3.5 × 106 cells in
75-cm2 T-flasks in 5% charcoal-stripped fetal calf serum
in Dulbecco's modified Eagle's medium. Prior to labeling, the medium
was replaced with serum-free phosphate-free Dulbecco's modified
Eagle's medium for 1.5 h at 37 °C.
[32P]H3PO4 (0.833 mCi/ml) was
added to the phosphate-free medium, and the cells were incubated
overnight at 37 °C. 2 h prior to harvest, the cells were
treated with hormone at a final concentration of 10 nM
R5020. The medium was aspirated, and the cells were washed once with
calcium and magnesium-free Hanks' balanced salt solution. TEN buffer
(40 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl) was added to the flasks and allowed to incubate
for 5 min. Cells were dislodged by shaking, and the suspension was
pooled and transferred to 50-ml conical tubes. The cells were pelleted
by centrifugation at 3000 rpm. Cell pellets were washed with Hanks'
balanced salt solution and then KPFM (50 mM potassium
phosphate, pH 7.4, 50 mM sodium fluoride, 1 mM
EDTA, 1 mM EGTA, and 12 mM monothioglycerol), with centrifugation between washes. To make whole-cell extract, the
cells were suspended in KPFM containing 0.5 M NaCl plus a mixture of protease and phosphatase inhibitors (18) and homogenized by
50 strokes in a glass-Teflon homogenizer. The homogenates were diluted
1:1 with KPFM buffer to reduce the salt concentration, transferred to
Ti75 tubes, and centrifuged at 40,000 rpm for 30 min. The extract
supernatant was directly added to the prepared antibody-coupled
Sepharose beads.
Immunoprecipitation and Gel Separation of PR--
Protein
A-Sepharose was preswollen in water and washed with PBS. Per six-flask
pool, 200 µl of bead suspension in PBS (3:1 beads/buffer) were
incubated with 25 µg of rabbit anti-mouse for 4 h at 4 °C on
a rocking platform. The beads were then washed twice with PBS. 40 µg
(per six-flask pool) of the PR antibody 1294 (45) was added to the bead
suspension and incubated overnight at 4 °C on a rocking platform.
The antibody-conjugated beads were washed once with PBS and twice with
TEG buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA,
10% glycerol). The cell extract was then added to the prepared beads
and incubated overnight at 4 °C on a rocking platform. The beads
were washed once with TEG, three times with TEG containing 0.3 M NaCl, and again with TEG by suspending in the indicated
buffer followed by centrifugation at 3000 rpm to remove nonspecific
proteins. PR was extracted from the beads by heating at 100 °C in
1× SDS-sample buffer and loaded on a 6.5% SDS-polyacrylamide gel.
Labeled PR was detected by autoradiography.
Protease Digestion of His Tag PR for Mass Spectrometric
Analysis--
Purified His tag PR was dialyzed for 8 h against 50 mM ammonium bicarbonate in a Tube-O-dialyzer (Research
Products International Corp., Mount Prospect, IL). PR (40 µg) was
digested with 1 µg each of trypsin, Asp-N, and Glu-C for 4 h at
37 °C. An additional 1 µg of each enzyme was added after 4 h,
and incubation continued for an additional 4 h. The digest was
then dried in a SpeedVac.
FeNTA Isolation of Phosphopeptides--
FeNTA columns were
prepared and used essentially as described (46) by sequentially washing
a 0.5-ml Ni2+-NTA-Sepharose column with 2 ml of
H2O, 2 ml of 0.1 M EDTA, pH 7.4, 2 ml of
H2O, 2 ml of 0.1 M HOAc, 2 ml of 0.1 M ferric chloride in 0.1 M HOAc and 2 ml of 0.1 M HOAc. Dried digests were dissolved in 0.1 M
HOAc and loaded on the column. The column was sequentially washed with
2 ml of 0.1 M HOAc, 2 ml of H2O, and 2 ml of
0.1% ammonium acetate, pH 8.0. The phosphopeptides were eluted with 2 ml of 0.1% ammonium acetate, pH 9.5. Samples were desalted using C18 resin prior to analysis.
Mass Spectrometry--
Analyses were performed on a PE Sciex
(Foster City, CA) API 3000 tandem quadrupole mass spectrometer equipped
with a Protana (Odense, Denmark) nanoelectrospray source. The samples
were dissolved in an aqueous solution of 50% methanol and 1% formic
acid, and 2-3 µl were deposited in the gold/palladium-coated glass
nanoelectrospray capillaries. The samples were analyzed for
phosphorylated peptide identification using negative ion precursor ion
scanning for m/z 79.1 with nitrogen as the
collision gas and a collision energy of 100 eV. Positive ion full
scanning spectra were then recorded utilizing the first quadrupole as
the mass filter and a cone potential of 70 V. Product ion spectra of
the appropriate positively charged precursor ions were then recorded to
identify the site of phosphorylation with nitrogen as the collision gas
and collision energies of 20-40 eV.
Identification of Sites Phosphorylated by Cdk2
HPLC Analysis--
Baculovirus-expressed His tag human PR (A or B)
(1 µg) was phosphorylated by cyclin A-Cdk2 as described under
"Experimental Procedures" and isolated by SDS-PAGE, and the PR was
in-gel digested using modified trypsin. The tryptic digest was loaded
on a C18 reversed-phase HPLC column that yielded the
phosphopeptide patterns seen in the two lower
panels in Fig. 1. Comparing
the phosphopeptide profiles of PR-B and PR-A obtained with the new
digestion procedure (panels III and
IV) to the previous Cdk2 profile obtained with the original
procedure (panel II), we observed several novel
late eluting phosphopeptide peaks (Fig. 1, peaks
A-D) as well as the previously identified peaks 1 (Ser190), 2 (Ser400), and 4/6
(Ser162). These new peaks (A-D) are specific to Cdk2,
since inclusion of the Cdk2 inhibitor roscovitine in the
phosphorylation reaction blocks their induction (data not shown). One
of these peaks, peak C, is clearly found only in PR-B
(arrow). Comparing the new Cdk2 phosphopeptides, peaks A-D
(shown in panel III), with the previously published profile
of phosphopeptides from in vivo labeled PR-B treated with
R5020 (panel I), we observe that the Cdk2 sites
represent only a subset of the total sites found in vivo.
Several of the major phosphopeptide peaks from the in vivo
profile have not yet been identified. Interestingly, although the
elution times vary slightly due to a change in the HPLC apparatus used,
peaks with elution positions comparable with peaks A-D are detected in
the in vivo profile, suggesting that they are previously
unidentified authentic sites. As demonstrated below, even the modified
cleavage procedure is insufficient to produce complete cleavage of
resistant sites; thus, the magnitude of the peaks cannot be used as an
indication of the extent of phosphorylation of these sites.
Phosphopeptide Analysis--
To identify these new
phosphopeptides, we used a combination of manual Edman degradation,
secondary endoproteinase digestion, and phosphoamino acid analysis.
Table I displays the data from the manual
Edman degradation of the phosphopeptides in peaks A-D. Peak A and B
both release in cycle 2, with peak B having an additional smaller
release in cycle 8. Peak C has a weak release in cycle 15, and peak D
releases in cycle 4. Weaker releases are expected in late cycles due to
gradual loss of yield due to incomplete coupling and cleavage as well
as the low efficiency of manual Edman degradation when the reactions
are not performed under an inert atmosphere (40).
To further characterize these phosphopeptides, secondary digests were
performed with the endoproteinases Asp-N and Glu-C followed by
separation on a 40% acrylamide peptide gel. Glu-C digests were also
split for manual Edman degradation and peptide gel analysis. Table
II summarizes the manual Edman
degradation data and secondary digest analysis.
Peptide gel analysis of peak A indicated the presence of a
phosphopeptide that did not cleave with Asp-N but cleaved well with
Glu-C. There was no change in the cycle of release after Glu-C
digestion (cycle 2). Since the possible candidates did not include a
Ser-Pro motif (a Ser/Thr-Pro sequence is required for phosphorylation
by Cdk2), phosphoamino acid analysis was performed. Threonine was the
predominant residue phosphorylated in this peptide pool (Fig.
2A). Of the three threonines
in PR predicted to be in position 2 of a tryptic peptide, only the
Thr430-containing peptide is situated in a Thr-Pro motif
(Fig. 2B). However, the Thr430 tryptic peptide
does not fulfill the secondary digest characteristics. Additionally, it
is a very small peptide and, if bound, should elute early from the HPLC
column. Closer inspection revealed that incomplete tryptic digestion of
the Thr430 peptide would result in a larger phosphopeptide
with the correct digestion patterns. The peptide containing
Thr430 ends with Arg followed by Pro, a sequence very
resistant to cleavage by trypsin (40). To confirm that this peptide is
a larger incomplete tryptic phosphopeptide containing
Thr430, additional digests with Arg-C and Lys-C were
performed. These endoproteinases cleave Arg-Pro and Lys-Pro sequences
more effectively than trypsin. Digestion with Arg-C generates a very
small fragment, consistent with the Thr430 peptide (Fig.
2C). As expected, Lys-C does not cleave the peptide (Fig.
2C). The size of the incomplete tryptic phosphopeptide
containing Thr430 is consistent with its elution time on
the HPLC profile. All of these criteria unambiguously identify
Thr430 as one of the Cdk2 phosphorylation sites.
Peak B contains two phosphopeptides as detected by peptide gel
analysis. Phosphoamino analysis indicates that serine is the predominant modified residue (data not shown). By collecting individual fractions under the peak, the two phosphopeptides could be partially separated. In this case, the fraction containing the phosphopeptide with the cycle 2 release (B2) cleaved with both Asp-N and Glu-C. Of the
three peptide candidates with a cycle 2 release that also contain both
Asp and Glu residues, only two peptides in PR contain a serine in
position 2 followed by a proline (Fig.
3A). Furthermore, since the
first candidate peptide (containing Ser108) is from the
region unique to PR-B, the candidate can be eliminated because the
phosphopeptide was observed in both PR-A and PR-B profiles.
Additionally, the Ser345-containing peptide is not a likely
candidate, since we have previously shown that this phosphopeptide
elutes much earlier than Peak B (see Fig. 1I). Examination
of the remaining candidate peptide containing Ser213
reveals that digestion with Glu-C would greatly change its mobility, whereas the Ser345 peptide would be resistant to cleavage
due to the location of the glutamic acid. Glu-Pro motifs are
inefficiently cleaved, especially at the carboxyl terminus of a peptide
(40). Glu-C digestion of this peptide results in a faster migrating
phosphopeptide (data not shown); thus, the phosphorylated peptide can
only be Ser213. In the case of the phosphopeptide with a
cycle 8 release (B8), digestion of the entire peak B region with Glu-C
prior to manual Edman degradation converts the cycle 8 to a cycle 4 release, while the cycle 2 release remained unchanged as expected (Fig.
3C). These data conclusively identify the peak B8
phosphopeptide from the candidates (Fig. 3B). Only the
peptide containing Ser554 would cleave to produce a peptide
with a cycle 4 release. Therefore, the two phosphopeptides in peak B
induced by Cdk2 in vitro correspond to the peptides
containing Ser213 and Ser554.
Peak C, observed solely in the PR-B profile, exhibits a weak release on
cycle 15 by manual Edman degradation. Only two of the three candidate
peptides with cycle 15 releases are located in the B-specific region of
PR (Fig. 4A). Of these, only
Ser25 is contained within a Ser/Thr-Pro motif. Examination
of the sequence of the tryptic peptide containing Ser25
shows that redigestion of the tryptic peptide with Glu-C should convert
the cycle 15 release to a cycle 3 release. Indeed, we observe a cycle 3 release after Glu-C digestion (Fig. 4B), whereas the other
B-specific peptide candidate (which does not contain the required
Ser-Pro motif) would produce a release in cycle 4. These results
identify the phosphorylated site in peak C as Ser25.
Peak D elutes very late in the profile, suggesting a large and
hydrophobic peptide. Manual Edman degradation data show that the
phosphoamino acid is in position 4. Of the eight candidates with Ser in
position 4, only one contains a Ser-Pro motif. Secondary digestion with
either Asp-N or Glu-C causes a significant change in mobility of the
peptide observed by peptide gel analysis (Fig. 5B). Of the two peptides with
a serine in position 4 that contain both Asp and Glu residues (Fig.
5A), only the Ser676 site is consistent with
these results. First, Ser676 is the only site located
within a Ser-Pro motif, and second, although the peptide containing
Ser711 contains a glutamic acid, it is adjacent to the
C-terminal arginine, which is known to be resistant to cleavage (40).
Finally, the size and hydrophobicity of the Ser676 tryptic
peptide is consistent with the late elution of this peptide from the
HPLC. By these criteria, we have conclusively identified Ser676 as the Cdk2-induced peptide in peak D. Interestingly, this site is analogous to the "hinge" site
identified in other steroid receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP were obtained from ICN (Irvine, CA). For
affinity purification, nickel-NTA resin was purchased from Qiagen, Inc.
(Valencia, CA), and glutathione-Sepharose 4B and protein A-Sepharose
were obtained from Amersham Pharmacia Biotech. Rabbit anti-mouse
antibody was obtained from Zymed Laboratories Inc.
(South San Francisco, CA). Phenylisothiocyanate and HPLC grade solvents
were purchased from J. T. Baker Inc. Sequencing grade-modified
trypsin was bought from Promega Corp. (Madison, WI). The
endoproteinases Asp-N, Glu-C, Arg-C, and Lys-C were obtained from Roche
Molecular Biochemicals. For manual Edman degradation, triethylamine and
trifluoroacetic acid were obtained from Sigma, and the Sequelon-AA
reagent kit was purchased from Millipore Corp. (Milford, MA). TLC
plates from EM Science (Gibbstown, NJ), ninhydrin from Pierce, and
phosphoamino acids (phosphoserine, phosphotyrosine, and
phosphothreonine) from Sigma were used for the phosphoamino acid analysis.
80 °C until use. Cells were
thawed and homogenized in a glass-Teflon homogenizer in 20 ml of His
tag homogenization buffer (HHB; 20 mM Tris-HCl, pH 8.0, 350 mM NaCl, 15 mM imidazole, 1 mM
B-mercaptoethanol, 10% glycerol) plus 50 mM NaF and a
mixture of protease inhibitors (18). The homogenate was centrifuged at
40,000 rpm for 30 min at 4 °C. Ni2+-NTA resin (2 ml
packed) was washed in HHB twice and resuspended to 40 ml. The
Sf9 extract was then added to the resin and incubated for 1 h at 4 °C in a 50-ml conical tube on a rocking platform. The resin
was centrifuged and washed with HHB twice before transferring the resin
suspension to a 3-ml column. The resin was washed with 30 ml of HHB
followed by 30 ml of HHB containing 0.6 M NaCl. Finally, the column was washed with 10 ml of low salt HHB (NaCl concentration reduced to 50 mM in HHB). His tag PR was eluted in elution
buffer (low salt HHB containing 250 mM imidazole) and
collected as 1-ml fractions. Protein concentrations were determined by
Bradford assay. Estimated purity from silver staining of samples run on SDS gels was greater than 95%.
80 °C.
-32P]ATP
(37,000 cpm/pmol) in Cdk2 buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2) in a final volume of 40 µl for
30 min at 30 °C. The reaction was terminated by the addition of 4×
SDS-Laemmli sample buffer. Samples were separated on a 6.5% SDS-PAGE
gel. The phosphorylated bands corresponding to PR, visualized by
autoradiography, were excised and digested with trypsin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of new phosphopeptide peaks in PR
phosphorylated by Cdk2 in vitro. A,
PR-A and PR-B were phosphorylated in vitro by cyclin A-Cdk2
as described under "Experimental Procedures." PR was separated from
the kinase complex on a 6.5% SDS-polyacrylamide gel, and the
phosphorylated band corresponding to PR was excised and subjected to
trypsin digestion. The phosphopeptides were separated by reversed-phase
HPLC and detected by an online radioactivity detector. The
phosphopeptide profiles are shown here. Panel I
shows the previously published phosphopeptide profile of PR-B from
in vivo labeled T47D cells, which shows all of the in
vivo phosphopeptides identified to date (reproduced and modified
with permission of the American Society for Biochemistry and Molecular
Biology from Fig. 1 of Ref. 15). Panel II shows
the previously identified Cdk2-induced peaks 1, 2, and 4/6 that
correspond to Ser190, Ser400, and
Ser162, respectively (profile reproduced and modified with
permission of the Endocrine Society from Fig. 2 of Zhang, Y., Beck, C. A. Poletti, A., Clement, J. P., IV, Prendergast, P., Yip, T.-T.,
Hutchens, T. W., Edwards, D. P. and Weigel, N. L. (1997)
Phosphorylation of Human Progesterone Receptor by
Cyclin-dependent Kinase 2 on Three Sites That Are Authentic Basal
Phosphorylation Sites in Vivo. Mol Endocrinol. 11, 823-832.
These in vitro Cdk2 phosphorylation sites,
Ser190, Ser162, and Ser400, have
also been identified in vivo (see panel
I). Panels III and IV show
the new phosphopeptide profiles obtained by Cdk2 phosphorylation of His
tag PR-B (III) and His tag PR-A (IV) after
digestion with specially modified trypsin. Several new peaks were
detected (peaks A-D) after utilizing the new trypsinization method
(compare panels II and III).
Comparison of the new PR-B (panel III) and PR-A
(panel IV) profiles identifies a peak
(C) that is not present in the PR-A profile
(arrow). Additionally, these new peaks A-D
(panel III) elute in similar positions to smaller
peaks from the in vivo profile (panel
I), consistent with peaks A-D containing authentic in
vivo sites. The peak preceding peak 1 in the PR-A profile has not
yet been identified.
Manual Edman degradation
Summary of secondary digests and manual Edman degradation
View larger version (36K):
[in a new window]
Fig. 2.
Identification of Thr430 in peak
A. A, His tag PR-B phosphorylated by Cdk2 in
vitro was digested with trypsin, and the peptides were separated
by reversed-phase HPLC. Fractions corresponding to peak A were
collected and analyzed by phosphoamino acid analysis. Threonine is the
major phosphoamino acid in these fractions. P-Ser,
P-Thr, and P-Tyr, phosphoserine,
phosphothreonine, and phosphotyrosine, respectively. B,
of all of the complete tryptic fragments of PR, only three peptides
contain a Thr residue in position 2. Ser/Thr-Pro motifs are required by
Cdk2 for phosphorylation. C, HPLC fractions of peak A were
split for secondary endoproteinase digestions with Asp-N, Glu-C, Arg-C,
and Lys-C followed by alkaline peptide gel analysis.
View larger version (30K):
[in a new window]
Fig. 3.
Identification of Ser213 and
Ser554 as the phosphorylation sites in peak B. Partial
separation of the two peptides in peak B followed by manual Edman
degradation and secondary endoproteinase digestions indicate that the
peptide with a cycle 2 release contains both Asp and Glu residues.
A, the list of candidate peptides for peak B2 is shown. Glu
residues are underlined. B, comparison of the
sequences of the candidate peptides is shown for the second peptide in
peak B with a cycle 8 release. C, the fraction pool of peak
B (containing both phosphopeptides) was digested with Glu-C followed by
manual Edman degradation. The graph of the release data is presented.
The sequence of the peptide containing Ser554 is shown
prior to and following Glu-C digestion to illustrate how the cycle 8 release is converted to a cycle 4 release. The cycle 2 release of the
Ser213-containing peptide remains unchanged.
View larger version (27K):
[in a new window]
Fig. 4.
Identification of Ser25 as the
phosphorylation site in peak C. A, only three tryptic
peptides contain a phosphorylatable residue in position 15; all of
these are serines. Comparison of the sequences of the candidate
peptides is shown. Glu residues are underlined.
B, HPLC fractions corresponding to peak C were pooled and
digested with Glu-C followed by manual Edman degradation. On the graph
of the release data, the sequence of the peptide containing
Ser25 is shown prior to and following Glu-C digestion to
illustrate how the cycle 15 release was converted to a cycle 3 release.
View larger version (19K):
[in a new window]
Fig. 5.
Identification of Ser676 as the
phosphorylation site in peak D. A, only two tryptic
peptides are candidates for the phosphopeptide in peak D, with the
characteristics of a cycle 4 release and with both Asp and Glu
residues. Both Asp and Glu residues are underlined.
B, secondary endoproteinase digestions of the phosphopeptide
in peak D reveal a drastic alteration in mobility after Asp-N digestion
and a moderate change of mobility after Glu-C digestion observed after
separation on an alkaline peptide gel. This pattern of cleavage is
diagnostic for identification of this phosphopeptide as the
Ser676-containing peptide.
Identification of Additional in Vivo Phosphorylation Sites in PR Expressed in Sf9 Cells
Most of the phosphorylation sites identified in steroid receptors
are in Ser/Thr-Pro motifs. Human PR contains 15 of these motifs, of
which six have been shown previously to be phosphorylated (15-17). An
analysis of the remaining sites reveals that conventional tryptic
digests would produce peptides ranging from about 30 to 70 amino acids
in length, too long for optimal binding and elution from a
C18 column. Our previous radiolabeling studies had
indicated that PR expressed in Sf9 insect cells exhibited the
same phosphopeptide map as PR isolated from T47D cells (36). We
therefore took advantage of the selectivity of mass spectrometers to
look for additional phosphorylation sites in baculovirus-expressed PR
isolated from Sf9 cells. Initial analysis of a tryptic digest of
purified recombinant PR-B in precursor ion mode revealed a number of
potential phosphorylated candidates, but the spectrum in positive ion
mode was too complex to successfully isolate phosphopeptides for
sequence analysis. Fractionation of the digest into 15 fractions by
C18 reversed-phase HPLC revealed a novel, large
phosphopeptide eluting at 50% acetonitrile. Negative ion precursor ion
scan of m/z 79.1 revealed a charge distribution
series corresponding to [M 8H]8
, [M
7H]7
, [M
6H]6
, [M
5H]5
, and [M
4H]4
. The mass
deconvoluted spectrum displayed a peptide of mass 5481.30, corresponding to amino acids 107-159, which contained a single phosphorylation. This peptide contains multiple serines including Ser130, which resides in a Ser-Pro motif (data not shown).
Since none of the serines in this region of PR have been previously
identified as phosphorylation sites, this must represent a novel site.
Phosphopeptides with masses consistent with peptides containing
previously identified sites, Ser162 and Ser345,
were also detected.
In subsequent analyses, ferric ion affinity resin was used to
preferentially absorb the phosphopeptides from digests. Purified recombinant PR-B was digested with trypsin, Asp-N, and Glu-C, and the
phosphopeptides were purified on the ferric ion affinity resin as
described under "Experimental Procedures." Fig.
6A shows the full scan of the
m/z 400-1100 range for the ion affinity column eluate, and Fig. 6B shows the corresponding precursor
m/z 79 scan. Collisionally activated
decomposition products of the doubly charged peak at
m/z 598 yielded a sequence for amino acids
11-22 produced by cleavage with trypsin and Glu-C containing a
phosphoserine at amino acid 20, the only serine in the peptide (Fig.
7A and Table
III). Analysis of the products of
the phosphopeptide at m/z 863 reveals that it
corresponds to 672-679 produced by a double trypsin and Asp-N cut,
which contains phosphoserine at position 676 (Fig. 7B).
Previously identified phosphopeptides detected in this analysis
included the doubly charged peptide at m/z 492 identified as the phosphotryptic peptide containing Ser162,
the doubly charged peptide at m/z 571 identified
as the phosphotryptic peptide containing Ser102, and the
phosphopeptide at m/z 680 as the phosphopeptide
containing Ser190 (data not shown).
|
|
|
Analysis of T47D Receptor for Newly Identified Sites
To determine whether any of the newly detected sites
phosphorylated by Cdk2 in vitro are in vivo
phosphorylation sites, we performed in vivo labeling of
endogenous PR from T47D cells in the absence and presence of hormone.
PR was purified by immunoprecipitation, separated by SDS-PAGE, and
processed as previously indicated for the in vitro samples.
HPLC peaks with identical retention times from in vivo and
in vitro samples were analyzed simultaneously by alkaline
peptide gel electrophoresis. Most of the analyses were inconclusive,
presumably due to the poor efficiency of cleavage and extraction from
gel slices and the background from additional peptides. However,
comparison of the HPLC fractions from in vivo labeled PR
with the same retention time as the fractions containing Ser213 phosphorylated by Cdk2 reveal that they contain a
phosphopeptide with the same mobility on alkaline gels as the
phospho-Ser213 peptide (Fig.
8). To determine whether phosphorylation
of this site is strictly hormone-dependent or whether the
site is basally phosphorylated, its phosphorylation was compared in the
presence and absence of hormone with that of the Ser162
phosphopeptide, which has previously been shown to be a basal phosphorylation site and whose phosphorylation is increased upon treatment with hormone. Fig. 8 shows that the relative change in
intensity of Ser213 phosphorylation as a result of hormone
treatment is similar to that of Ser162. These data suggest
that Ser213 is phosphorylated in vivo in T47D
cells and has the characteristics of a basal phosphorylation site.
|
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DISCUSSION |
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The presence of unidentified peptides in our phosphopeptide maps (17) and the failure to identify Ser676 (the conserved Ser-Pro that is homologous to the Ser530 hinge site in chicken PR (37)) as a phosphorylation site led us to develop new means to identify phosphorylation sites in PR. An analysis of PR sequence revealed that the Ser-Pro sequences that had not been identified as phosphorylation sites are likely to reside in large peptides produced by incomplete digestion at resistant trypsin cleavage sites. Using trypsin modified to prevent autolysis as well as other proteases, we have identified Ser676 as a phosphorylation site in PR by mass spectrometry and have shown that it is a substrate for Cdk2. Using these techniques, we have also identified five other candidate phosphorylation sites. We previously identified three phosphorylation sites, Ser162, Ser190, and Ser400, which were phosphorylated by Cdk2 in vitro (17). Using the specially modified trypsin that cleaves Lys-Pro and Arg-Pro sequences at a low but detectable efficiency, we have detected four small, late eluting peaks in the HPLC profile and have identified five additional Cdk2 sites in these peaks. Our studies here underscore the difficulty in detecting phosphorylation sites in large peptides produced by incomplete digestion. Comparison of the trypsin we used in our previous studies with the new modified trypsin showed that there was a significant difference in the phosphopeptide peaks detected by HPLC. Although the new peaks appear to be minor compared with those previously identified, Arg/Lys-Pro motifs will still be poorly cleaved relative to other trypsin cleavage sites (40). Evidence that we are only achieving partial cleavage is shown in Fig. 2. Redigestion of the purified peptide with excess Arg-C cleaves the resistant bond. Therefore, peak size cannot be considered representative of the extent of phosphorylation for peptides bounded by these difficult cleavage sites. This problem probably contributes to our failure to unambiguously identify most of these new peptides in digests of T47D receptor. The increased difficulty in releasing large peptides from gel slices coupled with a higher background as a well as phosphorylation by other kinases in vivo greatly increases the complexity of the analyses. For example, although some of the new peptides elute in positions similar to peaks 10 and 11, our previous characterization of peptides derived from PR from T47D cells suggests that there is at least one additional phosphopeptide in this region that has not been identified.
Employing the techniques of mass spectrometry to analyze peptides derived from PR expressed in Sf9 cells and in vivo labeling of T47D breast cancer cells, we have identified two new phosphorylation sites (Ser20 and Ser676) and two novel phosphopeptides (a peptide containing Ser130 and a peptide that comigrates with the Cdk2-phosphorylated Ser213-containing peptide). The mass spectrometric analyses have allowed us to identify additional phosphorylation sites in PR as well as to confirm previously identified sites. However, mass spectrometric methods also have limitations, requiring purified protein in larger quantities than the analyses following peptides only by radiolabel. For our studies, we used His tag PR amplified by baculovirus infection of Sf9 cells and purified by affinity column to achieve the amount and quality needed for analysis. Although we could detect phosphopeptides in a negative ion precursor ion scan, we found that some form of fractionation of peptides was required to enrich for phosphopeptides for sequencing by tandem mass spectrometry. FeNTA chromatography allowed us to preferentially obtain the phosphopeptides. Although this approach was useful in obtaining phosphopeptides of moderate length, we did not detect the very large phosphopeptides arising from incomplete digestion with trypsin in the FeNTA column eluate.
Previous studies from our laboratory have shown that in vivo phosphorylation patterns of PR from hormone-treated T47D and baculovirus-infected Sf9 cells are indistinguishable, and no aberrant phosphorylation was observed in Sf9 cells (36). Therefore, we expect that the new phosphorylation sites identified by mass spectrometry are also present in receptor expressed in T47D cells. The poor yield of these peptides suggests that it will be difficult to detect the sites in T47D PR either by radiolabeling or by mass spectrometry of purified protein. Phosphorylation site-specific antibodies have been produced to the Ser190 and Ser294 sites, and we have utilized them to study hormone-dependent phosphorylation of PR in T47D cells (45). Therefore, in the case of many of these new phosphorylation sites, generation of phosphorylation site-specific antibodies will be useful in confirming their phosphorylation in vivo and studying their regulation by hormone and cell signaling pathways.
Including the three sites previously identified, our laboratory has
identified a total of eight sites phosphorylated by Cdk2 in
vitro; five of these have been confirmed in vivo (162, 190, 213, 400, and 676). Notably, only subsets of the 15 Ser-Pro motifs in PR are substrates for Cdk2 phosphorylation, illustrating the site-specific discrimination by Cdk2. In fact, we observed that Cdk2
phosphorylates Ser25, but not Ser20, despite
the fact that both phosphorylation sites are located within the same
tryptic peptide (Table I and Fig. 4). Seven of the sites phosphorylated
by Cdk2 in vitro, Ser25, Ser162,
Ser190, Ser213, Ser400,
Thr430, and Ser554, are situated within the
phosphorylation-rich A/B domain (Fig. 9).
Interestingly, both Thr430 and Ser554 border
the ligand-independent activation domain AF-1. Perhaps most
significantly, Ser554 is located within only 13 amino acids
of the DNA binding domain. The proximity of these sites to the
functional domains suggests their potential ability to regulate or be
regulated by activation or DNA binding.
|
Only one Cdk2 site, Ser676, was identified outside of the A/B domain, located in the hinge region between the DNA and ligand binding domains (Fig. 9). Ser676 is analogous to the in vivo phosphorylation sites Ser530 identified in chicken PR (37) and Ser650 in human androgen receptor (26). Ser676 was not detected as a phosphorylation site in a CNBr digest of radiolabeled PR (13). However, this peptide would have been rather small (about 6500 kDa) for detection by SDS-polyacrylamide gel electrophoresis followed by nitrocellulose transfer and would have contained only one site, whereas the other peptides detected contain multiple (up to five) sites. In androgen receptor and chicken PR, mutation of the serine to alanine resulted in decreased transactivation (26); however, for chicken PR, the effect was only observed at subsaturating hormone conditions (47). Interestingly, this site in human PR has been mutated, and the effect of an alanine substitution was examined. Decreases in activity range from 20 to 50% depending upon the cell and promoter context, suggesting that this is an important site in human PR (21). We have confirmed by mass spectrometry that Ser676 is an in vivo phosphorylation site. Due to its proximity to the LBD, it is tempting to speculate that agonist- and antagonist-induced conformational changes could dramatically alter the phosphorylation of Ser676.
The role of Cdk2 phosphorylation in receptor function has been examined for several steroid receptors. In the case of the human estrogen receptor, overexpression of cyclin A, a regulatory partner of Cdk2, increases estrogen receptor-mediated transactivation (28). Furthermore, two serine residues, Ser104 and Ser106, have been shown by in vitro phosphorylation and site-directed mutagenesis studies to be involved in this pathway (30). Rat glucocorticoid receptor has also been shown to be a substrate for Cdk2. In vitro, Cdk2 complexes modify two serines, Ser224 and Ser232 (27). Analysis of glucocorticoid receptor transactivation in yeast reveals that hormone-induced transactivation is greatly diminished by perturbation of the Cdk pathway (27). Since Cdk2 and its cyclin partners are key players in cell cycle progression, it is interesting to note that the phosphorylation of mouse glucocorticoid receptor is cell cycle-dependent (48). All of this evidence points to a role for Cdk2-cyclin complexes in regulating steroid receptor function by direct phosphorylation. In the case of PR, the large number of Cdk2 phosphorylation sites strongly suggests regulation of PR via the Cdk pathway.
In summary, six new phosphorylation sites have been identified in PR
(Fig. 9). Five of these are phosphorylated in vitro by Cdk2.
One Cdk2-induced site, Ser676, as well as two B-specific
phosphorylation sites, Ser20 and a peptide containing the
putative site Ser130, were identified as in vivo
phosphorylation sites by mass spectrometric methods. The phosphopeptide
containing the newly identified Cdk2 site Ser213 exhibited
characteristics of a basal phosphorylation site in vivo.
Identification of these additional phosphorylation sites in PR argues
that we have only begun to understand the complex nature of PR phosphorylation.
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ACKNOWLEDGEMENTS |
---|
We thank William E. Bingman III for expert technical assistance with His tag PR purification and preparation of phosphopeptides for mass spectrometry, Dr. Brian G. Rowan for the purified Cdk2-cyclin A, and Kurt Christensen and the University of Colorado Cancer Center Tissue Culture Core Facility for expression of baculovirus PR in Sf9 insect cells.
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FOOTNOTES |
---|
* This work was supported in part by Public Health Service Grant R01 CA57539 (to N. L. W.) and the University of Colorado Cancer Center Core Grant P30 CA46934 (to D. P. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-6234; Fax: 713-790-1275; E-mail: nweigel@bcm.tmc.edu.
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M009805200
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ABBREVIATIONS |
---|
The abbreviations used are: PR, progesterone receptor; Cdk2, cyclin-dependent kinase-2; HPLC, high performance liquid chromatography; NTA, nitrilotriacetic acid; FeNTA, ferric ion nitrilotriacetic acid resin; PBS, phosphate-buffered saline.
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