Differential Hormone-Dependent Phosphorylation of Progesterone Receptor A and B Forms Revealed by a Phosphoserine Site-Specific Monoclonal Antibody

David L. Clemm, Lori Sherman, Viroj Boonyaratanakornkit, William T. Schrader, Nancy L. Weigel and Dean P. Edwards

Endocrine Research (D.L.C., W.T.S.) Ligand Pharmaceuticals, Inc. San Diego, California 92121
Department of Molecular and Cellular Biology (N.L.W.) Baylor College of Medicine Houston, Texas 77030
Department of Pathology and Molecular Biology Program (L.S., V.B., D.P.E.) University of Colorado School of Medicine Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human progesterone receptor (PR) is phosphorylated on at least seven serine residues, which are all located in the amino-terminal (N) domain (1 2 3 ). Three sites (Ser 81, Ser 102, Ser 162) are within the N-terminal segment unique to full-length PR-B, while the others are located within a region shared by PR-B and the N-terminally truncated A form of PR (Ser 190, Ser 294, Ser 345, and Ser 400). Identification of phosphorylation sites was made as previously described by conventional 32P-radiolabeling and phosphopeptide mapping in conjunction with sequencing of PR phosphopeptides isolated from T47D breast cancer cells (1 2 3 ). Treatment of cells with a progestin agonist stimulates phosphorylation of PR through at least two mechanisms. Four sites are basally phosphorylated (Ser 81, Ser 162, Ser 190, and Ser 400) and, upon hormone treatment, exhibit a rapid {approx} 2-fold increase. The other sites are hormone inducible (Ser 102, Ser 294, Ser 345) and 1–2 h of treatment are required to reach maximal phosphorylation. The different kinetics in response to hormone suggest that these two groups of phosphorylation sites are targets of different signaling pathways and kinases and serve distinct functional/structural roles. At least three different kinases have been identified that specifically phosphorylate PR in vitro on authentic sites including casein kinase II on Ser 81 (1 ), cyclin A-cyclin-dependent kinase 2 on Ser 162, Ser 190, and Ser 400 (3 ), and MAP kinase on Ser 162 and Ser 294 (5 ). The kinases that modify other sites is not known and whether these enzymes are the physiological regulators of PR in vivo also remains unknown.

The functional role of PR phosphorylation remains elusive. Agents that either activate or inhibit cellular serine/threonine kinases and phosphatases in different signaling pathways have been observed to dramatically potentiate or inhibit the transcriptional activity of PR in cell transfection experiments (6 7 8 ). Additionally, activation of protein kinase A (PKA) through cAMP signaling leads to a functional switching of RU486 from an antagonist to a partial agonist on the B form of PR, but not through PR-A (8 9 10 ). Whether the influence of these agents on the transcriptional activity of PR is due to alterations of receptor phosphorylation directly, or to phosphorylation of other associated proteins involved in PR transactivation, appears to be dependent on the signaling pathway involved. We were unable to detect a change in total phosphorylation, or in phosphopeptide maps of PR in response to cAMP (Ref. 6 , and C. A. Beck, N. L.Weigel, and D. P. Edwards, unpublished). However, we cannot rule out an effect on phosphorylation of PR directly, since all the phosphorylation sites have not been identified (1 2 3 ). Based on our phosphopeptide mapping experiments, there are at least two additional phosphorylation sites in human PR that have not yet been identified by sequencing, bringing the total number of sites to nine (1 2 3 ).

The consequence of phosphorylation on the transcriptional activity of PR remains largely unknown. Takimoto et al. (11 ) analyzed a series of serine to alanine substitution mutations for effects on the transcriptional activity of PR in cell transfection assays. However, the results are difficult to interpret since almost all the mutations were introduced into groups of putative, unproven, serine phosphorylation sites. The exception was an alanine substitution for Ser 190 that was observed in certain cell and promoter contexts to exhibit a 50% reduction in transcriptional activity (11 ). Thus functional analysis of most of the PR phosphorylation sites remains untested by site-directed mutagenesis. Similar studies with other steroid receptors have reported either partial reductions or enhancements of transcriptional activities by introduction of phosphorylation site mutations ( Refs. 12 13 14 15 16 17 18 ; reviewed in Ref. 19 ). These limited results with human PR, taken together with studies of other steroid receptors, suggest that phosphorylation does not serve as a regulatory on-off switch for transcriptional activity, but functions to either amplify or attenuate activity. Such a modulatory role could be achieved by phosphorylation altering the strength of coactivator association with receptor , as was recently shown for the estrogen receptor ß (20 ). Alternatively, phosphorylation may be involved in integrating signals from other pathways, or in regulating cellular trafficking or degradation of receptor protein (18 21 ).

A major advancement in the cell signaling field has resulted from the development of antibodies to specific phosphorylation sites of intracellular signaling molecules and nuclear transcription factor targets (22 23 24 25 ). These antibodies have enabled the simple, rapid, and sensitive detection of specific phosphorylation states of proteins in cells in response to activators or inhibitors of signaling pathways. Antibodies that recognize specific phosphorylation sites of nuclear receptors have not been available. Thus, steroid receptor phosphorylation studies have required cumbersome and laborious 32P-labeling experiments. Because of the complexity of PR phosphorylation, meaningful 32P-labeling results cannot be obtained by simply analyzing net 32P incorporation. Analysis of individual sites requires additional phosphopeptide-mapping experiments, thus severely limiting the number of samples that can be evaluated simultaneously. Additionally, phosphopeptide mapping is not quantitative, recovery of individual peptides can vary, and 32P-labeling conditions may themselves alter some signaling pathways. What is needed is a rapid and simple method to detect phosphorylation states of steroid receptors in whole cells.

Monoclonal antibodies (MAbs) that specifically recognize human PR phosphorylated on Ser 190 or Ser 294 were developed in this study. Biochemical experiments describe the utility of these MAbs for detection of specific phosphorylation states of PR within whole cells and for differential recognition of phosphorylated and unphosphorylated forms of PR under different experimental conditions. Experiments with the MAb (P294) specific for PR phosphorylated on Ser 294 revealed that this site is preferentially phosphorylated in response to hormone on PR-B, despite the fact that Ser 294 lies within the N-terminal domain of identical amino acid sequence shared by PR-A and PR-B. These results suggest that differential phosphorylation of Ser 294 may be involved in the distinct functional activities of PR-A and PR-B (26 27 28 ).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Production and Specificity of MAbs to Specific Serine Phosphorylation Sites (Ser 190 and Ser 294) in Human PR
A schematic of human PR structure and the location of previously defined phosphorylated serine residues is shown in Fig. 1AGo. The sites are divided into two groups: basal sites that are phosphorylated in the absence of ligand and increase in response to hormone binding (Ser 81, Ser 162, Ser 190, Ser 400), and the truly hormone-dependent sites (Ser 102, Ser 294, and Ser 345). As a first attempt to produce antibodies that recognize specific phosphorylated forms of PR, we chose one basal (Ser 190) and one hormone-dependent (Ser 294) site. Peptides corresponding to amino acids 184–196 (contains Ser 190) and 288–300 (contains Ser 294) of human PR were synthesized (see Fig. 1AGo), and the serine residues were chemically phosphorylated, conjugated to keyhole limpet hemocyanin (KLH), and the conjugate was used as an antigen to inject mice. Hybridomas produced by standard procedures (29 ) were screened by enzyme-linked immunoabsorption assay (ELISA) for preferential binding to the specific phosphopeptide as compared with the unphosphorylated peptide. This was followed by Western blot screening for reaction with full-length PR from untreated and hormone (synthetic progestin agonist R5020)-treated T47D breast cancer cells. One monoclonal antibody (MAb) was isolated from each cell fusion that recognized the native PR protein in a phosphorylation-dependent manner. These included clone 1154/F12 raised against the Ser 190 phosphopeptide and clone 608/G5 raised against the Ser 294 phosphopeptide. Both MAbs are mouse IgG1s, hereafter termed P190 and P294, respectively.



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Figure 1. Schematic of Human PR Structure and Specificity of P190 and P294 MAbs for Phosphorylated Forms of PR

A, Serine phosphorylation sites in human PR and phosphopeptides used to generate P190 and P294 MAbs. B, Whole cell extracts prepared from T47D cells after treatment for 1 h at 37 C with R5020 (100 nM), were analyzed by Western blot with the P190 or P294 MAbs. The MAbs (4 µg/ml of P190 and 10 µg/ml of P294) were preincubated overnight at 4 C before addition to Western blot filters with either no additions (-) or a 200-fold molar excess of the peptides indicated including phosphorylated (p-Ser 190) and unphosphorylated Ser 190 peptide (Ser 190), phosphorylated (p-Ser 294) and unphosphorylated Ser 294 peptide (Ser 294), and phosphorylated Ser 345 peptide (p-Ser 345).

 
Specificity for phosphorylated forms of PR was first tested by comparing the ability of phospho-and dephosphorylated peptides to block MAb binding to PR in a Western blot assay. PR was prepared as a whole cell lysate from hormone-treated (R5020 for 1 h at 37 C) T47D breast cancer cells. T47D cells express both the A (90 kDa) and B (118 kDa) isoforms of PR at similar levels (29 30 ). The P190 and P294 MAbs reacted strongly with proteins of the correct size for PR-A and PR-B (Fig. 1BGo, lanes 1 and 6, respectively). Weak cross-reactions with smaller mol wt protein bands were detected with both MAbs. Whether these are degradation products of PR or epitopes in other proteins recognized by the MAbs is not known. Nonetheless, intact PR-A and B are the major immunoreactive proteins in Western blots of whole-cell extracts. Whereas P190 exhibited comparable reactivity with the A and B forms of PR, P294 reacted preferentially with PR-B (Fig. 1BGo and see Figs. 2Go, 3Go, 4Go, and 6Go). P190 reactivity with both PR proteins was completely blocked by preincubation with excess phosphorylated Ser 190 peptide (Fig. 1BGo, lane 2), but was unaffected by the unphosphorylated Ser 190 peptide or by phosphopeptides containing other phosphorylation sites of PR, including Ser 345 and Ser 294 (Fig. 1BGo, lanes 2–5). P294 reactivity with PR was blocked by an excess of phosphorylated Ser 294 peptide, but not by unphosphorylated Ser 294 peptide or phosphorylated Ser 190 and Ser 345 peptides (Fig. 1BGo, lanes 7–10). Additional phosphopeptides corresponding to other phosphorylation sites in PR and human AR (Ser 81 in PR and Ser 650 in human AR) also had no effect on P190 or P294 immunoreactivity with PR (not shown). Because the majority of phosphorylation sites in steroid receptors are Ser-Pro motifs, including Ser 190 and Ser 294 of human PR, we also tested the P190 and P294 MAbs for cross-reaction with other steroid receptors. By Western blot of Sf9 insect cell extracts, neither P190 nor P294 cross-reacted with baculovirus-expressed human estrogen receptor, glucocorticoid receptor, or androgen receptor. Under the same conditions, both MAbs reacted strongly with baculovirus-expressed human PR-B (data not shown). The peptide competition results, which show specificity for the specific phosphorylated peptide, taken together with the ability of the MAbs to selectively detect full-length PR protein, suggest that P190 and P294 MAbs are specific, respectively, for Ser 190 and Ser 294 phosphorylated forms of PR.



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Figure 2. P190 and P294 Mabs Are Highly Selective for Hormone Activated PR, with P294 Exhibiting a Preference for PR-B

Cytosol and 0.4 M NaCl extractable nuclear fractions were prepared from T47D cells after treatment for 1 h at 37 C with vehicle (ethanol) or R5020 (100 nM). Aliquots of cytosol (C) and nuclear extracts (N) containing equal amounts of total protein (20 µg) were analyzed by Western blot with 1294 (for detection of total PR-A and PR-B), P190 or P294 MAbs.

 


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Figure 3. Two Classes of Progesterone Antagonists, RU486 and ZK98299, Have Distinct Effects on PR Reactivity with P190 and P294 Mabs

A, T47D cells were treated for 1 h at 37 C with vehicle, R5020 (100 nM), RU486 (100 nM), or ZK98299 (200 nM). Whole-cell extracts were prepared from each treatment group and analyzed by Western blot with 1294, P190, or P294 MAbs. B, T47D cells were treated and analyzed as in panel A except that cells were incubated with vehicle, R5020 (5 nM), ZK98299 (200 nM), or R5020 (5 nM) plus ZK98299 (200 nM), and the Western blot assay was done with the P294 MAb only.

 


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Figure 4. Kinetics of Ser 190 and Ser 294 Phosphorylation in Response to Continuous Treatment of T47D Cells with Hormone

T47D cells were treated with vehicle (ethanol) or R5020 (100 nM) for the times indicated. At each time point, cells were harvested, and whole-cell lysates were prepared and analyzed by Western blot with 1294, P190, or P294. MAb binding to PR was detected using ECL Plus (Amersham Pharmacia Biotech) and quantitated on a Molecular Dynamics, Inc. Storm 860 Phosphorimager. A, Autoradiography of the region of Western blots containing PR-A and PR-B. B–D, Phosphorimager analysis of the Western blot results in panel A. Arbitrary Phosphorimager fluorescence units were graphed for separate PR-A and PR-B values with each MAb. For P190 and P294, the Phosphorimager units were normalized to total PR detected by 1294. Values were normalized by setting the Ser 1294 time point to 1.0 and each 1294 time point thereafter as a fraction of 1.0. The P190 and P294 values were then taken as a proportion of the normalized 1294 values. The data are from a single experiment that is representative of five independent experiments.

 


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Figure 6. PR-A and PR-B Expressed from Recombinant Adenovirus Vectors in COS Cells Are Differentially Phosphorylated on Ser 190 and Ser 294 in Response to Hormone

COS cells were transduced for 24 h with PR-A (Ad5dl327CMV-PR-A) or PR-B (Ad5dl327CMV-PR-B) recombinant adenoviruses at an M.O.I. of 50 and were treated with and without R5020 (100 nM) for 1 h before harvest. Whole-cell lysates were prepared and aliquots containing equal amounts of total protein (100 µg) were analyzed by Western blot with 1294, P190, or P294 MAbs.

 
From our earlier 32P-labeling studies, phosphorylation of Ser 190 was shown to be partial in the absence of hormone, while Ser 294 phosphorylation was highly dependent on hormone (2 ). It has long been known that PR in the absence of hormone distributes between NaCl-extractable nuclear and cytosol fractions of T47D cells, while hormone treatment stimulates the major amount of cellular PR to bind tightly to nuclei (29 30 ). We therefore asked whether the reactivity of P190 and P294 with PR was influenced by hormone treatment of cells and was generally, or preferentially, distributed between cytosol and nuclear receptors. Western blots were performed with cytosol and high-salt extracts of nuclei prepared from T47D cells that had been treated for 1 h at 37 C with or without R5020. To determine the total amount of PR (phosphorylated and nonphosphorylated), another MAb (clone 1294, mouse IgG1) was used that recognizes a nonphosphorylated epitope in the N-terminal region of human PR that is common to the A and B isoforms (B. Spaulding, L. Sherman, and D. P. Edwards, unpublished). Western blots with 1294 show that total PR in the absence of ligand is distributed comparably between cytosol and nuclear extracts, while the majority of receptors were localized in the tight nuclear bound fraction after hormone treatment (Fig. 2Go, left panel). In non-hormone-treated cells, P190 immunoreactive PR in both cytosol and nuclear fractions was substantially lower than total PR (compare 1294 and P190, Fig. 2Go). Hormone treatment increased PR reactivity with P190 exclusively in the nuclear fraction (Fig. 2Go, P190 panel). Interestingly, the reactivity of P190 in lysates of non-hormone-treated cells was preferential for nuclear PR-A, while hormone stimulation of P190 phosphorylation was equally distributed between PR-A and PR-B (Fig. 2Go). Although preferential phosphorylation of Ser190 on PR-A in the absence of hormone was typically observed (see Figs. 2Go, 3Go, 5Go, and 6Go), some variation occurred as shown in the time course experiment of Fig. 4Go, in which Ser 190 phosphorylation on PR-A was only slightly higher than on PR-B. Such variation is likely due to the presence of trace hormone in the culture medium capable of stimulating some phosphorylation of Ser 190 and thus minimizing the differential between PR-A and PR-B. Nonetheless, these results suggest that basal phosphorylation of Ser 190 is preferential for a subpopulation of PR-A tightly associated with nuclear components. Consistent with preferential phosphorylation of unliganded nuclear PR-A, Lim et al. (32 ), using green fluorescent-tagged PR-proteins, reported that unliganded PR-A localizes primarily in the nucleus while PR-B is distributed between nuclear and cytoplasmic compartments.



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Figure 5. Selective Immunoprecipitation of Phosphorylated Forms of PR by P190 and P294

T47D cells were treated for 1 h at 37 C with ethanol vehicle or R5020 (100 nM), and whole-cell extracts containing equal amounts of total protein (100 µg) were immunoprecipitated using protein A Sepharose as an immunoabsorbent. The 1294, P190, and P294 MAbs were prebound to protein A Sepharose through a secondary rabbit antimouse IgG as previously described (43 ). Whole-cell extracts were then incubated in suspension with the MAb-coated protein Sepharose beads for 3 h at 4 C. The beads were washed in buffer (KPFM) containing 0.4 M NaCl (to disrupt PR dimerization), and samples were eluted from the beads with 2% SDS-sample buffer and analyzed by Western blot with 1294 MAb. The heavy and light chains of the MAbs used for immunoprecipitation that react on Western blot are indicated by arrows.

 
The P294 MAb exhibited essentially no reactivity with either cytosol or nuclear PR from non-hormone-treated cells; immunoreactivity was observed only with nuclear PR after hormone treatment of cells. Even though hormone-treated cells contain an equal amount of total nuclear PR-A and PR-B, P294 reactivity was strongly preferential for PR-B, indicating that phosphorylation of this hormone-dependent site is substantially different on the A and B forms of receptor (Fig. 2Go, P294 panel). The influence of R5020 on the reactivity of PR with the P190 and P294 MAbs is consistent with our earlier 32P-labeling studies. Ser 190 was basally phosphorylated and increased in response to hormone binding, while Ser 294 phosphorylation was highly hormone dependent (1 2 3 4 ).

We next examined the influence of two different classes of progesterone antagonists, RU486 and ZK98299, on phosphorylation of Ser 190 and Ser 294 as detected with MAbs. By 32P-labeling and peptide mapping, we previously observed that RU486 stimulated phosphorylation of basal and hormone-dependent PR phosphorylation sites the same as the agonist R5020, whereas ZK98299 inhibited phosphorylation of both types of sites (4 ). Consistent with previous 32P-labeling results, RU486 and R5020 had similar effects on stimulating P190 and P294 MAb interaction with PR. RU486 stimulated binding of both isoforms of PR to P190 MAb and preferentially induced PR-B reactivity with P294 (Fig. 3AGo). In contrast, ZK98299 minimally stimulated P294 and P190 binding to PR (Fig. 3Go, A and B). The low reactivity of the MAbs with PR in the presence of ZK98299 was not due to a loss of PR protein as shown by Western blot with the 1294 MAb that detects total PR (Fig. 3AGo). Because ZK98299 has a lower affinity (10-fold) for PR than R5020 or RU486 (33 34 ), it was important to determine whether the failure to stimulate PR interaction with P190 and P294 was due to an inefficient binding of ZK98299 to PR in cells. When T47D cells were cotreated with R5020 (5 nM) and ZK98299 (200 nM), ZK98299 effectively blocked R5020 stimulation of P294 interaction with PR-B (Fig. 3BGo). These results indicate that ZK98299 competes with R5020 for binding to PR in whole cells, but fails to induce phosphorylation. The influence of different PR ligands (Figs. 2Go and 3Go), taken together with peptide competition (Fig. 1BGo) results, provides strong evidence that the P190 and P294 MAbs specifically recognize, respectively, Ser 190 and Ser 294 phosphorylated forms of PR.

Distinct Kinetics of Ser 190 and Ser 294 Phosphorylation in Response to Hormone and Preferential Phosphorylation of Ser 294 on PR-B
In our earlier 32P-labeling studies we observed that hormone effects on phosphorylation of basal (Ser 190) and hormone-dependent sites (Ser 294) followed different kinetics. Basal sites increased rapidly (10–20 min) in response to hormone treatment while hormone-dependent sites responded more slowly requiring 1–2 h to reach maximal phosphorylation (2 4 ). To examine the kinetics of phosphorylation of Ser 190 (basal site) and Ser 294 (hormone-induced site) in more detail, and further test the utility of the MAbs for PR phosphorylation studies, we performed Western blots with P190 and P294 of T47D extracts taken at different times after R5020 treatment of cells. The 1294 MAb was used in parallel to determine the total amount of PR-A and PR-B (Fig. 4AGo). The relative amount of immunoreactive PR-A and PR-B at each time point was quantitated by phosphorimager analysis, and the results with P190 and P294 were normalized to the total PR levels obtained with the 1294 MAb (Fig. 4Go, B–D). As expected, hormone had minimal effect on total PR between 0.2 h and 1 h of treatment, but resulted in a significant decrease that started at 3 h after hormone treatment and continued for 24 h (Figs. 4Go, A and B). At 24 h of hormone treatment, total PR was reduced by {approx}80%. It should be noted that total PR-A and PR-B were expressed at similar levels ({approx}1:1 ratio) at all time points of R5020 treatment, indicating that the two isoforms of PR down-regulate at the same rate (Fig. 4Go, A and B). This reduction of total PR in response to continuous treatment with progestin agonists has been shown previously to be due to the combined effect of decreased PR mRNA expression and increased turnover of PR protein (30 35 ). Ligand-dependent PR down-regulation represents an 80–90% decrease in the steady-state level of PR that takes 24–48 h to achieve (30 35 ).

As detected by Western blot reactivity with the P190 MAb, phosphorylation of Ser 190 increased rapidly on both PR-A and PR-B (approximately equal with both isoforms) reaching a maximum between 30 and 60 min of hormone treatment (Fig. 4AGo). Between 60 min and 24 h of hormone treatment, P190 immunoreactivity decreased progressively (Fig. 4AGo). However, when normalized to total PR at each time point, P190 reactivity increased only for the first 1 hour of hormone treatment and remained unchanged relative to total PR between 60 min and 24 h (Fig. 4CGo). This result indicates that the Ser 190 phosphorylation site increased rapidly in response to hormone reaching a steady state at 30–60 min and thereafter turned over at the same rate as down-regulation of total PR. As detected by the P294 MAb, phosphorylation of Ser 294 increased more markedly in response to hormone and at a slower rate than the Ser 190 site, reaching a maximum between 1 and 3 h of hormone treatment (Fig. 4Go, A and D). Additionally, the majority of the increased phosphorylation of Ser 294 occurred on PR-B. At the peak of hormone stimulation, the ratio of Ser 294 phosphorylation of PR-B to PR-A was more than 5:1 (Fig. 4Go, A and D). When the P294 immunoreactivity was normalized to total PR, the time course experiment shows that hormone stimulated a new steady state level of phosphorylation of Ser 294 that required 1–3 h to achieve. Between 3 and 6 h of hormone treatment, Ser 294 phosphorylation decreased at a faster rate than that of total PR and then turned over at the same rate as total PR between 6 and 24 h (Fig. 4DGo). Thus, the Ser 294 site is more transiently phosphorylated in response to hormone than the Ser 190 site. It should also be noted that R5020 treatment caused a slight upshift in electrophoretic mobility of PR-A and PR-B, which has been shown previously to be associated with phosphorylation of Ser 345 (2 ). As expected, the P190 and P294 MAbs (Fig. 4Go) bind to the upshifted receptors, indicating that most of the upshifted Ser345 phosphorylated receptor is also phosphorylated on Ser190 and Ser294.

Preferential Phosphorylation Of Ser 294 On PR-B in its Native Conformation in Solution and When Complexed to Target DNA
We next sought to determine whether the influence of hormone on phosphorylation of Ser 190 and Ser 294 and the differential phosphorylation of Ser 294 on PR-B detected by Western blot also occurred with PR in its native conformation. PR was immunoprecipitated under nondenaturing conditions with P294 or P190 from lysates of T47D cells that had been treated with and without hormone. The immunoprecipitates were then analyzed by Western blot with 1294 MAb. As a control for total PR, samples were also immunoprecipitated with the 1294 MAb. As expected, 1294 immunoprecipitated approximately the same amount of PR-A and PR-B from hormone and non-hormone-treated cells (Fig. 5Go). However, the P294 MAb preferentially immunoprecipitated PR-B from cells treated acutely (1 h) with hormone, whereas little PR-A from hormone-treated cells or either isoform of PR from non-hormone-treated cells was immunoprecipitated (Fig. 5Go). With the P190 MAb, PR-A was preferentially immunoprecipitated from non-hormone-treated cells, while substantially greater amounts of both PR-A and PR-B were immunoprecipitated from hormone-treated cells (Fig. 5Go). These immunoprecipitation results are consistent with Western blots, indicating that differential reactivity with P190 and P294 reflects the phosphorylation state of PR in its native conformation.

To determine the Ser 190 and Ser 294 phosphorylation state of native PR-A and PR-B complexed to target DNA, we examined the ability of both MAbs to supershift receptor-DNA complexes in electrophoretic gel mobility shift assays (EMSAs). Because T47D cells express both PR-A and PR-B, EMSAs of T47D cell extracts are complicated by the presence of PR-A/PR-B heterodimers (36 ). Therefore, PR-A and PR-B were separately expressed in a PR-negative mammalian cell line (COS) by use of recombinant adenoviral vectors (37 ). We first examined whether hormone influenced the phosphorylation of adenovirus expressed PR-A and PR-B the same or differently than native PR in T47D cells. As detected by Western blot with P294, adenovirus-expressed PR-A was not appreciably phosphorylated on Ser 294 in the presence or absence of hormone, while hormone stimulated a preferential phosphorylation of Ser 294 on PR-B (Fig. 6Go, top panel). As detected by the P190 MAb, phosphorylation of Ser 190 was observed preferentially on adenovirus-expressed PR-A in the absence of hormone and was stimulated by hormone to comparable levels on both PR-A and PR-B (Fig. 6Go, middle panel). The lower panel of Fig. 6Go represents total adenovirus-expressed PR detected by the 1294 MAb. Thus, PR expressed from adenovirus vectors in COS cells was phosphorylated on Ser 190 and Ser 294 in a similar manner as native PR in T47D cells.

Nuclear extracts of COS cells containing hormone-activated adenovirus-expressed PR-A or PR-B were next incubated with a DNA probe containing a consensus progesterone response element (PRE) and analyzed for the formation of PR-DNA complexes by EMSA. Both PR-A and PR-B independently bound to the DNA probe with approximately equal efficiency, producing decreased mobility of a substantial amount of the free DNA (Fig. 7Go). Addition of the 1294 MAb supershifted all of the PR-A and PR-B complexes whereas a control unrelated MAb to estrogen receptor had no effect (Fig. 7Go). The P190 MAb supershifted both PR-A and PR-B complexes, while P294 reduced the mobility of the PR-B-DNA complex only (Fig. 7Go). Thus, PR-B that binds to specific PREs is efficiently phosphorylated on Ser 294 whereas PR-A that binds to PREs is not phosphorylated to a significant extent on Ser 294. We also observed that both the P190 and P294 MAbs (only with PR-B) supershifted the majority but not all of the PR-DNA complexes, indicating that a high percentage of DNA complexes contain phosphorylated PR.



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Figure 7. Differential Phosphorylation of Native PR-A and PR-B DNA Complexes

Adenovirus-expressed PR-A and PR-B from hormone-treated (1 h with 100 nM R5020) COS cells were prepared as whole cell extracts as in Fig. 6Go and incubated in a DNA binding reaction with a 32P-oligonucleotide containing a single consensus PRE. Free and bound 32P-DNA were separated by electrophoresis on nondenatured 5% polyacrylamide gels. The different MAbs (1 µg) indicated were added to the DNA binding reaction including a no-addition control, 1294, P190, P294, and an estrogen receptor MAb (h151).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study monoclonal antibodies were produced that distinguish Ser 190 and Ser 294 phosphorylated forms of human PR from unphosphorylated receptors. Specificity of the MAbs was demonstrated by peptide competition experiments (Fig. 1BGo) and by the observation that treatment of T47D breast cancer cells with hormone agonists and two classes of antagonists, RU486 and ZK89299, influenced MAb reactivity with PR in a manner that correlated with the effects of these ligands on phosphorylation of Ser 190 and Ser 294 observed by previous 32P-labeling experiments (1 2 3 4 ). Moreover, both MAbs recognized specific phosphorylated states whether receptor was in a native conformation or denatured under Western blot conditions. These experiments confirm the utility of the MAbs to selectively recognize specific phosphorylated states of PR under different experimental conditions.

To our knowledge this is the first report of phosphorylation state-specific steroid receptor antibodies. The utility of P190 and P294 for PR phosphorylation studies suggests that it is possible to produce MAbs that recognize other phosphorylation sites in PR, as well as phosphorylation sites of other classes of steroid receptors. Antibodies to tyrosine-phosphorylated forms of signaling molecules have proven to be valuable for study of signal transduction pathways (22 25 ). Although fewer antibodies have been produced that recognize serine/threonine phosphorylation sites, those that interact with the serine phosphorylation states of CREB (cAMP response element binding protein) and c-jun have also been of value for studying the signaling pathways that activate these sequence-specific transcription factors (23 24 ). Phosphorylation state-specific antibodies have most often been produced as polyclonal antibodies in rabbits, which requires epitope purification to separate phosphorylation-specific antibodies from those that detect unphosphorylated protein. The approach used here to produce MAbs to phosphopeptide antigens eliminates the epitope purification step. Phosphorylation-specific antibodies were isolated by screening and subcloning of the hybridomas. Hybridoma cell lines also have the advantage of providing a continuous unlimited supply of antibody.

We anticipate that the P190 and P294 MAbs, and future MAbs produced against other phosphorylation sites within PR, will replace 32P-labeling and peptide mapping for phosphorylation studies. Phosphorylation-specific antibodies for steroid receptors are potentially powerful tools to study cellular signals and pathways that regulate nuclear receptor phosphorylation and function in vivo. The use of Western blots to detect phosphorylation of steroid receptors in whole cells will greatly simplify experiments to analyze the influence of different signaling pathways on specific receptor phosphorylation sites. The ability of the P190 and P294 MAbs to recognize phosphorylated PR in its native conformation also indicates the potential of these MAbs to separate phosphorylated and unphosphorylated forms of PR for biochemical studies. MAb affinity chromatography and elution of the immobilized receptor by phosphopeptide competition under nondenaturing conditions should be a useful method for analyzing functional properties of different phosphorylated forms of receptors in vitro. The partial supershift of DNA complexes obtained with P190 and P294 MAbs, as compared with the complete supershift with the 1294 MAb that recognizes total PR, illustrates that receptors are not uniformly phosphorylated (Fig. 7Go).

To produce biochemical amounts of correctly phosphorylated PR for in vitro functional (DNA-binding) experiments, we chose to use a recombinant adenovirus expression system in mammalian cells. In our previous studies with baculovirus expressed human PR, it was determined by 32P-labeling and phosphopeptide mapping that recombinant PR overexpressed in Sf9 insect cells was correctly phosphorylated on all the same sites as native PR in T47D breast cancer cells. However, hormone regulation was lost such that all sites were constitutively phosphorylated and unaffected by hormone treatment of cells (38 ). Using P190 and P294 MAbs to detect the phosphorylation state of adenovirus-expressed PR in COS cells, Ser 190 and Ser 294 sites were phosphorylated in the same hormone-stimulated (Ser 190) or hormone-dependent (Ser 294) manner as native PR in T47D cells (Fig. 6Go). Although preliminary, these data suggest that use of recombinant adenovirus to express wild-type and mutant PRs in mammalian cells for phosphorylation studies is an improvement over the baculovirus system. The more faithful phosphorylation of adenovirus-expressed PR may be due to the presence of different kinases in insect and mammalian cells, or to PR overexpression in the insect baculovirus system exceeding cell-regulatory systems. Under these conditions, adenovirus-expressed PR levels were equal to or slightly lower than that of native PR in T47D cells (not shown).

Initial experiments with the P190 and P294 MAbs have provided insights into PR phosphorylation and function. First, the time course of P294 immunoreactivity in response to continuous hormone treatment (Fig. 4Go) showed that Ser 294 phosphorylation was down-regulated at a faster rate than total PR and the Ser 190 phosphorylation site. This suggests that the hormone-dependent Ser294 phosphorylation site is more labile than basal sites and is likely to be acted upon by different phosphatases than other sites. Whether accelerated down-regulation signifies that Ser 294 phosphorylation has a role in PR protein stability or turnover is not known. As a precedent for phosphorylation affecting steroid receptor protein half-life, studies by Webster et al. (18 ) showed that specific groups of phosphorylation site mutations stabilized GR protein half-life and abrogated ligand-dependent GR down-regulation. However, our finding that total PR-A and PR-B down-regulate at the same rate (Fig. 5Go, A and B) argues that the phosphorylation of Ser 294 is not the major determinant of ligand-dependent down-regulation of PR in the cell. Second, the phosphospecific MAb experiments revealed a strong preferential phosphorylation of Ser 294 on PR-B. This was unanticipated because Ser 294 and surrounding sequences are identical on PR-A. Our previous 32P-labeling and phosphopeptide mapping experiments detected phosphorylation of Ser 294 on PR-A. Although relatively less of the Ser 294 phosphopeptide was generated from PR-A than PR-B, the dramatic differential phosphorylation of this site was not obvious until the MAb experiments were performed. The inability of peptide mapping to detect this substantial differential phosphorylation of PR-A and PR-B was likely due to the large size of the Ser 294 phosphopeptide and the resultant low yields and poor resolution of the peptide on HPLC columns (2 3 4 ). Thus, the development of the P294 MAb enabled an observation that was missed due to inherent limitations of phosphopeptide mapping.

As a possible explanation for the preferential reactivity of P294 for PR-B, we considered the possibility that Ser 294 might be efficiently phosphorylated on PR-A, but that the P294 MAb has limited access to its epitope in the context of PR-A. However, this is not likely for several reasons. First, we observed the same strong preference of the P294 MAb for PR-B by Western blot, as for PR-B in its native conformation as detected by immunoprecipitation (Fig. 5Go) and EMSA (Fig. 7Go). SDS-denatured PR-A and PR-B proteins on Western blots are not expected to exhibit differential accessibility to the P294 MAb. Second, the peptide- blocking experiment (Fig. 1BGo) suggests that both MAbs efficiently recognize a small phospho-dependent amino acid sequence relatively independent of conformation. Third, purified PR-A can be phosphorylated efficiently on Ser 294 in vitro and reacts strongly with P294 MAb (5 ). Why Ser 294 is preferentially phosphorylated on PR-B in vivo (T47D cells) is not known. This is likely due to the N terminus of PR-A adopting a distinct conformation that either hinders access of cellular kinases to this site or creates a unique active site domain for an interacting protein that blocks phosphorylation of Ser 294. Alternatively, the differential phosphorylation of PR-A and PR-B at Ser294 could be due to the gain of a phosphatase interaction site in PR-A that is not accessible in PR-B. In support of the idea that the N terminus of PR-A and PR-B have different conformations, Bain et al. (39 ), using proteolytic peptide mapping, reported that regions of amino termini common to PR-A and PR-B exhibit distinct ordered structures.

PR-B is generally a stronger transcriptional activator than PR-A. In several cell and promoter contexts, PR-A exhibits minimal activation function and is capable of acting as a trans-dominant repressor of the transcriptional activity of other steroid receptors, including PR-B (26 27 28 ). The mechanism responsible for this repressive function of PR-A is unknown. An inhibitory function was mapped to a discrete region in the first 140 amino acids of the N terminus (amino acids 165–305) of PR-A that was observed to be transferable to other steroid receptors, but not to heterologous proteins (40 41 ). Because Ser 294 lies within this inhibitory domain, it is possible that differential phosphorylation of this site may be involved in the distinct functional properties of the two receptor forms. Phosphorylation at Ser 294 could directly influence receptor association with other coregulatory proteins. Alternatively, differential phosphorylation could be the consequence of a distinct conformation, in which case Ser 294 may be located within an active domain for a novel PR-A interacting protein.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Phosphopeptides
Two peptides were synthesized (Macromolecular Resources at Colorado State University, Fort Collins, CO) corresponding to the following sequences in human progesterone receptor: CGG-(184)VLPRGL[S] PARQLL (196) and CGG-(288) PMAPGR[S]PLATTV(300). Peptides were synthesized to contain extra N-terminal non-PR sequences (indicated in bold lettering) including a Cys for chemical cross-linking and two Gly residues as spacers between the N terminus and the 13 mer PR sequences. The Gly residues were included to reduce steric hindrance after cross-linking to hapten and improve antigenicity. The peptides were produced by standard solid phase peptide synthesis on polystyrene resins. The serine residues indicated by brackets were left unprotected on the resin, and they were chemically phosphorylated after the chain assembly was completed as described by Perich (42 ). A 5-fold molar excess of dibenzyl-N, N-diisopropylphosphoramidite (Novabiochem USA, La Jolla, CA) was added along with 50 molar equivalents of tetrazole in dimethyl formamide (VWR) and reacted for 1 h. The resin was washed and then oxidized by washing several times with an iodine solution to convert the intermediate phosphite ester into the dibenzyl-protected phosphoserine. The peptide was then cleaved from the resin support, and the side chains were deprotected by a 2-h treatment with a cleavage cocktail containing 90% trifluoroacetic acid, 2.5% tiisopropylsilane (TIS, Sigma), 2.5% ethanedithiol (EDT, Sigma), 2.5% water, and 2.5% Anisole (Sigma). The deprotected peptides were then analyzed by Maldi-Tof mass spectrometry to confirm quantitative phosphorylation. The peptides were then chromatographed by reverse phase HPLC to 90% purity. Phosphorylated peptides were coupled to KLH by a sulfhydryl reagent through the N-terminal cysteine.

Other PR Antibodies and PR Ligands
A mouse monoclonal (MAb) IgG1 (clone 1294) antibody was raised against human PR that recognizes an epitope in the N-terminal region (amino acids 165–534) common to PR-A and PR-B (B. Spaulding, L. Sherman, and D. P. Edwards, unpublished). MAb 1294 was used as a control for the total amount of PR present in different cell extracts, independent of the phosphorylation state of PR. The progestin agonist R5020 (Promegestone) was obtained from DuPont-New England Nuclear (Boston, MA), and the progesterone antagonists RU486 (Mifepristone) and ZK98299 (Onapristone) were gifts respectively from Rousell UCLAF (Romainville, France) and Schering AG (Berlin, Germany).

Cell Cultures and Preparation of PR Extracts
PR-rich T47D human breast cancer cells were cultured in MEM medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% FBS (HyClone Laboratories, Inc. Logan Utah) as previously described (29 30 ). At 24 h before treatment with hormone, cells were grown in MEM containing 5% dextran-coated charcoal-treated serum which removes steroids, and the same medium was used for hormone treatments. To prepare cytosol and nuclear extracts, cells were lysed in KPFM buffer containing phosphatase and protease inhibitors as previously described (50 mM potassium phosphate, pH 7.4, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, and 12 mM monothioglycerol) to minimize dephosphorylation and degradation of PR during in vitro processing (1 2 3 4 ). Cytosol and nuclear fractions were prepared by differential centrifugation as previously described. The nuclear pellet was extracted for 1 h at 4 C with lysis buffer containing 0.4 M NaCl and centrifuged at 100,000 x g for 60 min to yield a soluble nuclear extract in the supernatant. To prepare whole-cell extracts, cells were homogenized in lysis buffer containing 0.4 M NaCl and extracted for 1 h at 4 C, and the entire lysate was centrifuged at 100,000 x g for 60 min to yield a soluble supernatant total protein fraction.

Adenovirus Transduction of PR-A and PR-B in Mammalian Cells
Recombinant adenoviral transfer vectors containing human PR-A or human PR-B under the control of the cytomegalovirus (CMV) promoter, were constructed by overlap recombination as described by Schaack et al. (37 ). These are replication-defective viruses lacking the transforming EIA and EIB genes. To construct recombinant viruses, human PR-A or PR-B cDNAs were cloned into the BamHI site of a transfer plasmid (ACCMVC-1) containing the CMV promoter and adenovirus sequences to yield pCMV-PR-A and pCMV-PR-B. Permissive 293 cells were cotransfected using calcium phosphate precipitation with pCMV-PR-A, or pCMV-PR-B and the adenovirus AD5d1327Bst ß-gal (37 ). Homologous recombination of plasmid and viral sequences resulted in generation of infectious viral particles that were plaque purified. Plaques were initially examined by PCR for the presence of either PR-A or PR-B inserts. Positives were picked, grown in 293 cells, and examined for PR-A or PR-B protein expression by Western blot. Purified viruses encoding PR-A or PR-B, named Ad5dl327CMV-PR-A and Ad5dl327CMV-PR-B, respectively, were grown in large scale and concentrated by CsCl gradient centrifugation. The virus concentrations were determined by absorbance at 260 nm where 1 A260 unit represents 1012 viral particles. The particle-plaque forming unit ratio was determined to be close to 100 for both viruses.

To express PR-A or PR-B, monkey kidney COS-1 cells were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FBS (HyClone Laboratories, Inc.) as previously described (42 ). Cells were plated at 1 x 106 cells per 100-mm culture dish and grown for 24–48 h to 70–80% confluency. Ad5dl327CMVPR-A or Ad5dl327CMVPR-B viruses were added to freshly changed medium at multiplicity of infection (M.O.I.) of 50, and cells were incubated for 24 h at 37 C. At 24 h after transduction, medium was changed to DMEM plus 5% FBS, and cells were treated for 1–2 h with 100 nM R5020. Cells were harvested by scraping into ice-cold PBS and homogenized by freeze-thawing in a lysis buffer containing 20 mM HEPES, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 10% glycerol, 1 mM sodium vanadate, 50 mM sodium fluoride, and 0.4 M NaCl.

Western Blotting of PR
Cell extracts or immunoprecipitates containing PR were electrophoresed on 7.5% polyacrylamide SDS-gels, as previously described with modifications (29 30 ). Proteins were transferred to Immobilon-P (Millipore Corp., Bedford, MA) polyvinylidene fluoride transfer membranes at 700 mA for 2 h in a transfer buffer containing 250 mM Tris-base, 1.92 M glycine, 0.01% SDS and 10% methanol. The membranes were blocked for 1 h in 5% milk in PBS-T (PBS, pH 7.4, containing 1% Tween-20). Membranes were then washed three times for 15 min in PBS-T and incubated in the same buffer with primary MAbs overnight at 4 C using the following concentrations for different MAbs: 1294, 1 µg/ml; P190, 4 µg/ml; and P294, 10 µg/ml. Membranes were washed three times for 15 min in PBS-T and incubated for 1 h at room temperature with a 1:5,000 dilution of mouse secondary antibody conjugated to horseradish peroxidase, and detection was by luminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) according to manufacturer’s instructions. Enhanced chemiluminescence (ECL) Plus (Amersham Pharmacia Biotech) was used to quantitate PR Western blots by phosphorimager analysis on a Storm 860 instrument (Molecular Dynamics, Inc., Sunnyvale, CA).

Immunoprecipitation Assays
PR-specific MAbs were prebound to Protein A Sepharose (Repligen Corp., Needham, MA) as previously described (29 43 ). Resins were incubated with excess (65 µg/100 µl resin) rabbit antimouse IgG (Cappel, Cochranville, PA), followed by washing with PBS to remove unbound secondary antibody. PR-specific MAbs were then bound to the immobilized rabbit antimouse IgG at concentrations of 10 µg/100 µl of resin. As a control for nonspecific binding of PR, resins were bound only with rabbit antimouse IgG and the primary MAb was omitted. Cell extracts (prepared in KPFM buffer) containing PR were incubated for 3 h at 4 C on an end-over-end rotator with MAb-coated protein A Sepharose beads. Resins were washed four times in KPFM buffer containing 0.4 M NaCl and extracted with 2% SDS sample buffer, and the extracts were analyzed by ECL Western blot with the 1294 MAb.

EMSA
A 28-bp double stranded oligonucleotide containing a single consensus progesterone response element (PRE) was used as a probe for PR-DNA binding by EMSA as described previously (44 ). Whole-cell extracts from adenovirus-transduced COS cells were incubated with 0.3 ng of 32P-labeled PRE oligonucleotide for 1 h at 4 C in DNA binding buffer containing 10 mM Tris-Base, pH 7.4, 50 mM NaCl, 5 mM dithiothreitol, 2 mM MgCl2, 10% glycerol, and 1 µg of competitor DNA poly (dA-dT)/poly(dA-dT). DNA binding reactions were electrophoresed on nondenaturing 5% polyacrylamide gels as previously described, and the gels were dried and subjected to autoradiography (44 ).

Production of MAbs
Male BALB/c mice (8–10 weeks of age, Jackson Laboratories, Inc., Bar Harbor, ME) were injected with phosphorylated peptide (Ser 190 and Ser 294)-KLH conjugates as an emulsion in MPL/S-TDCM adjuvant (RIBI Immunochemical Research Inc, Hamilton, MT). The primary injections were given sc with 400 µg of conjugate, or 100 µg of peptide based on a 1:4 ratio by weight of peptide-KLH. Three booster injections were given at approximately 3-week intervals with the same amount of conjugate, alternating between sc and ip routes of injection. At 3 days before cell fusions, mice were injected ip with 1 mg of conjugate prepared in PBS.

Sera from immunized mice were tested for antibody titers by ELISA as previously described (29 ). ELISA 96-well plates (Immunolon II, Dynatech Corp., Chantilly, VA) were coated with 10 µg/ml of free phosphorylated (Ser 190 and Ser 294) peptide or with free unphosphorylated peptide (Ser 190 and Ser 294). ELISA plates were blocked with 1% BSA and incubated overnight at 4 C with antisera at different dilutions, and plates were developed with secondary goat-antimouse IgG-horseradish peroxidase conjugate. Mice that showed strong ELISA reaction against the specific phosphopeptide and low reaction with unphosphorylated peptide were subsequently assayed by Western blot for whether the antisera also contained antibodies reactive with full-length PR in crude extracts of T47D cells. Mice producing antibodies that preferentially reacted with the phosphopeptide and T47D PR protein by Western blot were taken for cell fusions.

Fusions between mouse spleen cells and the mouse THT (Fox-NY) myeloma cell line were performed as previously described (29 ). Hybridomas were screened by ELISA for antibodies that exhibited preferential reactivity for free phosphopeptides over unphosphorylated peptides. Those showing strong preferential ELISA reaction were further screened by Western blot for recognition of native PR in extracts of T47D cells. Since all the serine phosphorylation sites in PR were observed in previous studies by 32P-labeling studies to increase in response to hormone treatment, Western blots were performed with PR taken from cells treated for a short time (2 h) with and without R5020. For each phosphopeptide antigen, a MAb was identified that passed the screening criteria of exhibiting a preferential ELISA reaction with phosphorylated peptide over unphosphorylated and Western blot reactivity with T47D PR that increased in response to hormone treatment of cells. The hybridomas were subcloned twice by limiting dilution, and each cell line stably produced an IgG1 MAb, designated clone 1154 (Ser 190 peptide) and clone 608 (Ser 294 peptide), respectively. MAbs were purified by 50% ammonium sulfate precipitation and binding to protein G Sepharose. Antibody was eluted with Tris-glycine buffer, pH 2.8, followed by neutralization with 1 M Tris-base and dialysis against PBS.


    ACKNOWLEDGMENTS
 
The authors acknowledge the expert technical assistance of Kurt Christensen, Magda Altmann, and the University of Colorado Cancer Center Tissue Culture Monoclonal Antibody Core facility for the production of MAbs. We thank Drs. Steven K. Nordeen and Jerome Schaack (University of Colorado Health Science Center) for providing recombinant PR-A and PR-B adenoviruses and Suzzane Wardell for assistance in construction of the viruses.


    FOOTNOTES
 
Address requests for reprints to: Dean P. Edwards, Ph.D., University of Colorado Health Sciences Center, Department of Pathology, B-216, 4200 East Ninth Avenue, Denver, Colorado 80262.

This work was supported in part by a sponsored research agreement (D.P.E.) from Ligand Pharmaceuticals, Inc. (San Diego, CA ), Public Health Service NIH Grant CA-57539 (N.L.W.), and National Cancer Institute University of Colorado Cancer Center Core Grant P30-CA46934 (D.P.E.).

Received for publication July 30, 1999. Revision received October 11, 1999. Accepted for publication October 14, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Zhang Y, Beck CA, Poletti A, Edwards DP Weigel NL 1994 Identification of phosphorylation sites unique to the B form of human progesterone receptor. J Biol Chem 269:31034–31040[Abstract/Free Full Text]
  2. Zhang Y, Beck CA, Poletti A, Edwards DP Weigel NL 1995 Identification of a group of Ser-Pro motif hormone-inducible phosphorylation sites in the human progesterone receptor. Mol Endocrinol 9:1029–1040[Abstract]
  3. Zhang Y, Beck CA, Poletti A, Clement JPIV, Prendergast P, Yip T-T, Hutchens TW, Edwards DP, Weigel NL 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[Abstract/Free Full Text]
  4. Beck CA, Zhang Y, Weigel NL, Edwards DP 1996 Two types of anti-progestins have distinct effects on site-specific phosphorylation of human progesterone receptor. J Biol Chem 271:1209–1217[Abstract/Free Full Text]
  5. Keightley M-C, Edwards DP, Weigel NL, Differential regulation of human progesterone receptor A, B phosphorylation by the Erk signaling pathway. J Biol Chem, in press
  6. Beck CA, Weigel NL, Edwards DP 1992 Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Mol Endocrinol 6:607–620[Abstract]
  7. Edwards DP, Weigel NL, Nordeen SK, Beck CA 1993 Modulators of cellular protein phosphorylation alter the trans-activation function of human progesterone receptor and the biological activity of progesterone antagonists. Breast Cancer Res Treat 27:41–56[Medline]
  8. Beck CA, Weigel NL, Moyer ML, Nordeen SK 1993 The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways. Proc Natl Acad Sci USA 90:4441–4445[Abstract]
  9. Sartorius CA, Tung L, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone receptors bound to DNA are functionally switched to transcriptional agonists by cAMP. J Biol Chem 268:9262–9266[Abstract/Free Full Text]
  10. Sartorius CA, Groshong SD, Miller LA, Powell RP, Tung L, Takimoto GS Horwitz KB 1994 New T47D breast cancer cell lines for the independent study of progesterone B- and A-receptors: Only antiprogestin-occupied B-receptors are switched to transcriptional agonists by cAMP. Cancer Res 54:3868–3877[Abstract]
  11. Takimoto GS, Hovland AR, Tasset DM, Melville MY, Tung L, Horwitz KB 1996 Role of phosphorylation on DNA binding and transcriptional functions of human progesterone receptors. J Biol Chem 271:13308–13316[Abstract/Free Full Text]
  12. Ali S, Metzger D, Bornert J-M, Chambon Pierre 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160[Abstract]
  13. Goff PL, Montano MM, Schodin DJ, Katzenellenbogen 1994 Phosphorylation of the human estrogen receptor. J Biol Chem 269:4458–4466[Abstract/Free Full Text]
  14. Bai W, Weigel NL 1996 Phosphorylation of ser211 in the chicken progesterone receptor modulates its transcriptional activity. J Biol Chem 271:12801–12806[Abstract/Free Full Text]
  15. Bai W, Tullos S, Weigel NL 1994 Phosphorylation of ser530 facilitates hormone-dependent transcriptional activation of the chicken progesterone receptor. Mol Endocrinol 8:1465–1473[Abstract]
  16. Mason SA, Housley PR 1993 Site-directed mutagenesis of the phosphorylation sites in the mouse glucocorticoid receptor. J Biol Chem 268:21501–21504[Abstract/Free Full Text]
  17. Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ 1997 Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol 17:3947–3954[Abstract]
  18. Webster JC, Jewell CM, Bodwell JE, Munck A, Sar M, Cidlowski JA 1997 Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein. J Biol Chem 272:9287–9293[Abstract/Free Full Text]
  19. Weigel NL 1996 Steroid hormone receptors and their regulation by phosphorylation Biochem J 319:657–667[Medline]
  20. Tremblay A, Tremblay GB, Labrie F, Giguère V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[Medline]
  21. DeFranco DB, Qi M, Borror KC, Garabedian MJ, Brautigan DL 1991 Protein phosphatase types 1 and/or 2A regulate nucleocytoplasmic shuttling of glucocorticoid receptors. Mol Endocrinol 5:1215–1228[Abstract]
  22. Kawakatsu H, Sakai T, Takagaki Y, Shinoda Y, Saito M, Owada MK, Yano J 1996 A new monoclonal antibody which selectively recognizes the active form of src tyrosine kinase. J Biol Chem 271:5680–5685[Abstract/Free Full Text]
  23. Ginty DD, Kornhauser JM, Thompson MA, Bading H, Mayo KE, Takahashi JS, Greenberg ME 1993 Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238[Medline]
  24. Eilers A, Whitfield J, Babij C, Rubin LL, Ham J 1998 Role of the jun kinase pathway in the regulation of c-jun expression and apoptosis in sympathetic neurons. J Neuroscience 18:1713–1724[Abstract/Free Full Text]
  25. Grammer TC, Blenis J 1997 Evidence for MEK-independent pathways regulating the prolonged activation of the ERK-MAP kinases. Oncogene 14:1635–1642[CrossRef][Medline]
  26. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  27. Tung L, Kamel Mohamed M, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate transcription without binding to progesterone response elements and are dominantly inhibited by A-receptors. Mol Endocrinol 7:1256–1265[Abstract]
  28. Wen DX, Xu YF, Mais DE, Goldman ME, McDonnell DP 1994 The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. Mol Cell Biol 14:8356–8364[Abstract]
  29. Weigel NL, Beck CA, Estes PA, Prendergast P, Altmann M, Christensen K, Edwards DP 1992 Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody. Mol Endocrinol 6:1585–1597[Abstract]
  30. El-ashry D, Oñate SA, Nordeen SK, Edwards DP 1989 Human progesterone receptor complexed with the antagonist RU486 binds to hormone response elements in a structurally altered form. Mol Endocrinol 3:1545–1558[Abstract]
  31. Deleted in proof
  32. Lim CS, Baumann CT, Htun H, Xian W, Irie M, Smith CL, Hager GL 1999 Differential localization and activity of the A- and B-form of the human progesterone receptor using green fluorescent protein chimeras. Mol Endocrinol 13:366–375[Abstract/Free Full Text]
  33. Dekabre JA, Guiochon-Mantel A, Milgram E 1993 in vivo evidence against the existence of antiprogestins disrupting receptor binding to DNA. Proc Natl Acad Sci USA 90:4421–4425[Abstract]
  34. Gass EK, Leonhardt SA, Nordeen SK, Edwards DP 1998 The antagonists RU486 and ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid in vitro and in vivo, but have distinct effects on receptor conformation. Endocrinology 139:1905–1919[Abstract/Free Full Text]
  35. Read LD, Snider CE, Miller JS, Greene GL, Katzenellenbogen BS 1988 Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines. Mol Endocrinol 2:263–271[Abstract]
  36. DeMarzo AM, Oñate SA, Nordeen SK, Edwards DP 1992 Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor. Biochem 31:10491–10501[Medline]
  37. Schaack J, Langer S, Guo X 1995 Efficient selection of recombinant adenoviruses by vectors that express ß-galactosidase. J Virol 69:3920–3923[Abstract]
  38. Beck CA, Zhang Y, Altmann M, Weigel NL, Edwards DP 1996 Stoichiometry and site-specific phosphorylation of human progesterone receptor in native target cells and in the baculovirus expression system. J Biol Chem 271:19546–19555[Abstract/Free Full Text]
  39. Bain DL, Franden MA, Takimoto GS, Horwitz KB, Structural analysis of the two human progesterone receptor N-termini explain their isoform-specific functional differences. Program of the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 187 (Abstract)
  40. Giangrande PH, Pollio G, McDonnell DP 1997 Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor. J Biol Chem 272:32889–32900[Abstract/Free Full Text]
  41. Hovland AR, Powell RL, Takimoto GS, Tung L, Horwitz KB 1998 An N-terminal inhibitory function, IF, suppresses transcription by the A-isoform but not the B-isoform of human progesterone receptors. J Biol Chem 273:5455–5460[Abstract/Free Full Text]
  42. Perich JW 1991 Synthesis of O-phosphoserine and O-phosphothreonine containing peptides. Methods Enzymol 201:234–244[Medline]
  43. Tetel MJ, Jung S, Carbajo P, Ladtkow T, Skafar DF, Edwards DP 1997 Hinge and amino-terminal sequences contribute to solution dimerization of human progesterone receptor. Mol Endocrinol 11:1114–1128[Abstract/Free Full Text]
  44. Boonyaratanakornkit V, Melvin V, Prendergast P, Altmann M, Ronfani L, Bianchi ME, Taraseviviene L, Nordeen SK, Allegretto EA, Edwards DP 1998 High-mobility group chromatin proteins 1 and 2 functionally interact with steroid hormone receptors to enhance their DNA binding in vitro and transcriptional activity in mammalian cells. Mol Cell Biol 18:4471–4487[Abstract/Free Full Text]