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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 [{gamma}-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. 1Go 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. 2Go, 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. 3Go). 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 [{gamma}-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 [{gamma}-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. 1Go 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. 3Go 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. 1Go) with the permission of The American Society for Biochemistry and Molecular Biology, Inc.

 
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. 4Go), suggesting the absence of both Asp and Glu in P1. Manual Edman degradation analysis located the phosphorylated residue in the third cycle (Fig. 5Go). 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 1Go). 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. 2Go 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. 4Go). 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 1Go 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 2Go) 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 isn’t 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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Table 1. Potential Tryptic Phosphopeptide Candidates for Peptide I

 

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Table 2. Peptide Sequence Analysis

 
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. 6Go), and the majority of [32P] in peptide 2 was released by manual Edman degradation in cycle 14 (Fig. 7AGo). 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 3Go). 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. 6Go). 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. 6Go, 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 3Go. 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 4Go). Consistent with results in Fig. 6Go and Fig. 7AGo, 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 4Go). To confirm that P2 indeed is the peptide indicated in Table 4Go with phosphorylation at Ser400, the corresponding HPLC fraction from purified, unlabeled baculovirus-expressed hPR was subjected to microsequencing. Table 5Go shows that an amino acid sequence corresponding to the first 10 aa of the predicted peptide shown in Table 4Go 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. 7CGo). 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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Table 3. Potential Tryptic Phosphopeptide Candidates for Peptide 2

 

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Table 4. Result of Protease Digestion of Phosphopeptide 2

 

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Table 5. Peptide 2 Sequence Analysis

 
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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 8Go.



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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.

 
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 3Go) 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 20–25% 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 (5–10 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 1–2 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Earle’s 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 0–45% 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 [{gamma}-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 17–94-J-4202 (to Y.Z.).

1 Current address: Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy. Back

Received for publication December 16, 1997. Accepted for publication March 17, 1997.


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