Identification and Characterization of the Protein-associated Splicing Factor as a Negative Co-regulator of the Progesterone Receptor
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m409187200
ISSN1083-351X
AutoresXuesen Dong, Oksana Shylnova, John Challis, Stephen J. Lye,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoProgesterone is essential in all species for the maintenance of pregnancy, and its withdrawal is required to activate the myometrium and to initiate labor. However, unlike most other species, progesterone levels do not fall at term in humans, raising the paradox as to how labor can occur under the continued influence of progesterone. We hypothesized that an endogenous (myometrial) repressor of the progesterone receptor (PR) could induce a functional withdrawal of progesterone and hence lead to the initiation of labor. We used the human PR as bait in a protein pull-down assay and identified polypyrimidine tract-binding protein-associated splicing factor (PSF) as a PR-interacting protein. PSF functions as a potent inhibitor of PR (but not estrogen receptor) transcriptional activity in mammalian cells. It acts through two novel mechanisms, inducing degradation of the PR through the proteasomal pathway and also interfering with binding of PR to its DNA response element. Importantly, in vivo studies in rats demonstrated a dramatic increase in myometrial PSF expression at term that was temporally associated with reduced levels of the myometrial PR. Accordingly, we propose that PSF acts as a PR corepressor and contributes to the functional withdrawal of progesterone and the initiation of human labor. Progesterone is essential in all species for the maintenance of pregnancy, and its withdrawal is required to activate the myometrium and to initiate labor. However, unlike most other species, progesterone levels do not fall at term in humans, raising the paradox as to how labor can occur under the continued influence of progesterone. We hypothesized that an endogenous (myometrial) repressor of the progesterone receptor (PR) could induce a functional withdrawal of progesterone and hence lead to the initiation of labor. We used the human PR as bait in a protein pull-down assay and identified polypyrimidine tract-binding protein-associated splicing factor (PSF) as a PR-interacting protein. PSF functions as a potent inhibitor of PR (but not estrogen receptor) transcriptional activity in mammalian cells. It acts through two novel mechanisms, inducing degradation of the PR through the proteasomal pathway and also interfering with binding of PR to its DNA response element. Importantly, in vivo studies in rats demonstrated a dramatic increase in myometrial PSF expression at term that was temporally associated with reduced levels of the myometrial PR. Accordingly, we propose that PSF acts as a PR corepressor and contributes to the functional withdrawal of progesterone and the initiation of human labor. Progesterone is an essential regulator of the reproductive events associated with the establishment and maintenance of pregnancy through its ligand-activated progesterone receptor (PR) 1The abbreviations used are: PR, progesterone receptor; PRA, progesterone receptor A; PRB, progesterone receptor B; PRE, progesterone response element; DBD, DNA-binding domain; AF, activation function; CBP, cAMP response element-binding protein-binding protein; GST, glutathione S-transferase; PSF, polypyrimidine tract-binding protein-associated splicing factor; ER, estrogen receptor; SHMs, Syrian hamster myocytes; WCL, whole cell lysate; DMEM, Dulbecco's modified Eagle's medium; MALDI, matrix-assisted laser desorption ionization time-of-flight; RRM, RNA recognition motif. (1.Challis J.R.G. Matthews S.G. Gibb W. Lye S.J. Endocr. Rev. 2000; 5: 514-550Google Scholar). Progesterone actions include the suppression of genes encoding contraction-associated proteins (e.g. oxytocin and prostaglandin receptors and connexin-43) that are required for myometrial activation and the onset of labor. In humans, progesterone levels remain elevated throughout labor, raising a paradox as to how labor can be initiated. Even in species in which progesterone levels fall at term, concentrations are likely sufficiently high to inhibit contraction-associated protein gene expression. This suggests there must be an active mechanism for inducing a functional withdrawal of progesterone at term. We have previously suggested that a blockade of PR signaling in the myometrium could induce a "functional withdrawal" of progesterone that would result in the initiation of labor (2.Challis J.R.G. Lye S.J. Creasy R.K. Resnik R. Maternal-Fetal Medicine Principles and Practice. 5th Ed. W. B. Saunders Co., Philadelphia2004: 79-87Google Scholar). A number of mechanisms have been proposed to effect such a functional withdrawal, including changes in the expression of the PR or PR isoforms (3.Mesiano S. Chan E.C. Fitter J.T. Kwek K. Yeo G. Smith R. J. Clin. Endocrinol. Metab. 2002; 6: 2924-2930Crossref Scopus (350) Google Scholar) as well as altered transcriptional activity of the PR as a result of changes in the expression of essential co-regulators (both coactivators and corepressors) (4.Condon J.C. Jeyasuria P. Faust J.M. Wilson J.W. Mendelson C.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9518-9523Crossref PubMed Scopus (243) Google Scholar). The PR is a member of the steroid receptor superfamily of ligand-dependent transcriptional factors. In the human myometrium, the PR is transcribed as full-length PRB and an N-terminally truncated (164 amino acids) PRA isoform (5.Kastner P. Krust A. Turcotte B. Stropp U. Tora L. Gronemeyer H. Chambon P. EMBO J. 1990; 5: 1603-1614Crossref Scopus (1339) Google Scholar). PRA is generally a weaker transcriptional activator than PRB (6.Tora L. Gronemeyer H. Turcotte B. Gaub M.P. Chambon P. Nature. 1988; 333: 185-188Crossref PubMed Scopus (319) Google Scholar, 7.Meyer M.E. Quirin-Stricker C. Lerouge T. Bocquel M.T. Gronemeyer H. J. Biol. Chem. 1992; 267: 10882-10887Abstract Full Text PDF PubMed Google Scholar, 8.Giangrande P.H. McDonnell D.P. Recent Prog. Horm. Res. 1999; 54 (Discussion 313–314): 291-313PubMed Google Scholar) and can also act as a repressor of PRB as well as of other steroid receptors (9.Vegeto E. Shahbaz M.M. Wen D.X. Goldman M.E. O'Malley B.W. McDonnell D.P. Mol. Endocrinol. 1993; 10: 1244-1255Google Scholar, 10.Kraus W.L. Weis K.E. Katzenellenbogen B.S. Mol. Cell. Biol. 1995; 4: 1847-1857Crossref Scopus (175) Google Scholar). Upon ligand binding through the hormone-binding domain, the activated PR undergoes a conformational change enabling it to bind to specific progesterone response elements (PREs) through its DNA-binding domain (DBD). This in turn facilitates recruitment of the general transcriptional machinery, either directly (11.Ing N.H. Beekman J.M. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar) or indirectly via co-regulators (12.Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2063) Google Scholar, 13.Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1086) Google Scholar), which act to positively or negatively modulate the transcription rate of target genes. Two common transcriptional activation domains exist within PRs, a hormone-dependent activation function domain (AF2) in the C-terminal hormone-binding domain and a ligand-independent domain (AF1) in the N-terminal region (14.Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2702) Google Scholar). In addition, PRB possesses a third activation function domain (AF3) within its unique N-terminal region (15.Sartorius C.A. Melville M.Y. Hovland A.R. Tung L. Takimoto G.S. Horwitz K.B. Mol. Endocrinol. 1994; 8: 1347-1360Crossref PubMed Scopus (244) Google Scholar). Interactions between the N-terminal AF domains (AF1 and AF3) and the AF2 domain (either direct or indirect via co-regulators) elicit maximal hormone-dependent activity (16.Tetel M.J. Giangrande P.H. Leonhardt S.A. McDonnell D.P. Edwards D.P. Mol. Endocrinol. 1999; 6: 910-924Crossref Scopus (144) Google Scholar). Our knowledge of nuclear receptor co-regulators has increased markedly over recent years, revealing their multifaceted roles in regulating gene transcription. Besides the autonomous activation domains in steroid receptor coactivators and repression domains in the nuclear receptor corepressor and SMRT, many co-regulators possess acetylase (such as steroid receptor coactivators, p300/CBP, and p300/CBP-associated factor) or deacetylase (such as histone deacetylase-1/2) activities (17.Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Moreover, in the case of E6-AP and RPF-1, nuclear receptor activation by these co-regulators can be enhanced through their ubiquitin ligase activity, which is separable from their coactivation functions (18.Nawaz Z. Lonard D.M. Smith C.L. Lev-Lehman E. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 1999; 19: 1182-1189Crossref PubMed Scopus (354) Google Scholar, 19.McKenna N.J. Xu J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Steroid Biochem. Mol. Biol. 1999; 69: 3-12Crossref PubMed Scopus (365) Google Scholar). In an effort to identify co-regulators within the myometrium that interact with the PR and modulate PR function, we used glutathione S-transferase (GST)-PR fusion proteins to pull-down protein extracts from myometrium smooth muscle cell lysate. One of the associated proteins identified (polypyrimidine tract-binding protein-associated splicing factor (PSF)) was shown to inhibit the transcriptional activity of the PR by mechanisms that involve interference with PR binding to the PRE and degradation of the PR protein through the proteasomal pathway. Furthermore, the finding that the expression of PSF increased dramatically in the rat myometrium at term pregnancy in association with reduced levels of the myometrial PR led us to speculate that this novel PR corepressor might contribute to the functional withdrawal of progesterone and the initiation of labor. Materials—DNA restriction and modification enzymes were obtained from Fermentas (Burlington, Ontario, Canada), Promega (Nepean, Ontario), and Roche (Laval, Quebec). PCR reagents were obtained from Invitrogen (Burlington). Progesterone, 17β-estradiol, and the proteasomal inhibitor MG132 were purchased from Sigma (Oakville, Ontario). Anti-PR primary antibody (C-20 and AB-52), anti-His tag antibody (H-15), and anti-Gal4 DBD antibody (sc-4050) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-PSF antibody (B92) was from Sigma. Protease inhibitor mixture was purchased from Roche. Glutathione-Sepharose 4B affinity matrix was from Amersham Biosciences (Oakville). Plasmid Construction—The PR expression vectors pSG5-PRA and pSG5-PRB were kindly provided by Dr. P. Chambon. With pSG5-PRB used as the template, a series of deletion mutations (PRB amino acids 1–164, 164–456, 456–556, 456–650, 556–650, 556–933, and 650–933) were generated by PCR with a 5′-primer containing an EcoRI site and an ATG start codon and 3′-primer containing a TGA stop codon and a SalI site using high fidelity Platinum Taq DNA polymerase (Invitrogen). PCR fragments were then inserted into expression vectors pM and VP16 (Clontech) and pGEX-5X-2 (Amersham Biosciences). PR-(556–650) were also inserted downstream of the T7 promoter of pcDNA3 at the EcoRI and XhoI site. FLAG-PRB was constructed using pFLAG-CMV2 (Sigma) as the backbone. Full-length PR cDNA were amplified by PCR and cloned in-frame into pFLAG-CMV2 at the EcoRI and BamHI sites. Human PSF cDNA was a kind gift from Dr. J. G. Patton. Full-length PSF and its deletion mutants (amino acids 1–707, 1–662, 1–150, 1–290, 150–290, 290–370, 370–450, 290–707, 370–707, 450–707, and 662–707) were also generated by PCR with a 5′-primer containing an EcoRI site and an ATG start codon and a 3′-primer containing an TGA stop codon and a SalI site. PCR fragments were then inserted into vectors pM and pGEX-5X-2, respectively, at the EcoRI and SalI sites and into pcDNA3.1-His6 at the EcoRI and XhoI sites. The veracity of all PCR-generated fragments was confirmed by DNA sequencing. Expressed proteins were also detected by Western blotting using specific antibodies. Estrogen receptor (ER)-α and ERβ expression vectors were obtained from Dr. Paul Walfish. Construction of the mouse mammary tumor virus-luciferase and 3xERE-luciferase (containing three copies of the estrogen response element) reporter vectors was described previously (20.Dong X. Challis J.R.G. Lye S.J. J. Mol. Endocrinol. 2004; 32: 843-857Crossref PubMed Scopus (28) Google Scholar). Identification of Interacting Proteins by Mass Spectrometry—GST fusion proteins were prepared as described previously (20.Dong X. Challis J.R.G. Lye S.J. J. Mol. Endocrinol. 2004; 32: 843-857Crossref PubMed Scopus (28) Google Scholar). Briefly, GST fusion proteins were produced in Escherichia coli strain BL21(DE3) pLysS cells by incubation with isopropyl β-d-thiogalactopyranoside to a final concentration of 0.2 mm. Bacteria were pelleted and resuspended in NETN buffer (0.5% Nonidet P-40, 1 mm EDTA, 100 mm NaCl, and 20 mm Tris (pH 8.0)) plus protease inhibitor mixture and lysed by mild sonication. Centrifugation-cleared lysates were incubated with 200 μl of a 50% slurry of glutathione-Sepharose 4B affinity matrix. Cytoplasmic and nuclear fractions of Syrian hamster myocytes (SHMs) were prepared using an NE-PER nuclear and cytoplasmic extraction kit (Pierce). Protein extracts (∼1.5 mg) were first precleared by passage through GST-bound glutathione-Sepharose 4B matrix and then incubated with GST fusion proteins bound to Sepharose beads for 2 h at 4°C. The beads were washed three times with NETN buffer and once with NETN buffer containing 100, 150, or 200 mm NaCl. The associated proteins were eluted by the addition of 20 mm glutathione and separated by 10% SDS-PAGE. Gels were stained with Coomassie Blue. Bands were excised, reduced, alkylated, and in-gel digested with trypsin as described (21.Plant P.J. Fawcett J.P. Lin D.C. Holdorf A.D. Binns K. Kulkarni S. Pawson T. Nat. Cell Biol. 2003; 4: 301-308Crossref Scopus (304) Google Scholar). Tryptic peptides were extracted from the gel, desalted using ZipTip desalting columns (Millipore, Bedford, MA), equilibrated in 5% formic acid, washed with equilibration buffer and eluted in a solution of 5% formic acid and 60% methanol. Tandem mass spectrometric analysis was performed using a nanoelectrospray ionization source (Protana A/S) coupled to a high performance hybrid quadrupole time-of-flight API QSTAR™ pulsar mass spectrometer (MDS Sciex, Concord, Ontario). After tryptic ion candidates were identified, product ion spectra were generated by collision-induced dissociation. For product ion scans, the collision energy was determined experimentally. The sequence and mass information of the peptides was used to screen the Natural Resources and Expressed Sequence Tags Databases. We searched the data bases using the Mascot MS/MS search engine (Matrix Science, Ltd., London, UK). Immunoprecipitation and Western Blotting—Co-immunoprecipitation of the transfected PR and His-PSF was performed in 293T cells. Cells were plated in 150-mm diameter dishes and grown to 60% confluency before transfection. A total of 15 μg of plasmid were transfected using ExGen 500 (Fermentas). Eighteen hours after transfection, cells were washed twice with ice-cold phosphate-buffered saline and then lysed in NETN buffer plus protease inhibitor mixture. Protein concentrations of the whole cell lysate (WCL) were determined by the Bradford (46.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) assay, and WCL was diluted to 1 mg/ml in NETN buffer. A 900-μl aliquot of WCL was incubated overnight at 4 °C in suspension with either anti-His tag or anti-PR antibody, followed by the addition of 30 μl of protein A/G Plus-agarose beads (Santa Cruz Biotechnology, Inc.) for another 2 h at4 °C. Resins were washed with NETN buffer, eluted with 1× Laemmli buffer, boiled, and centrifuged. The supernatant was separated by 8% SDS-PAGE, electrophoresed onto polyvinylidene difluoride membrane (Millipore) and visualized by ECL. For immunoprecipitation of the endogenous PR and PSF, T47D cells cultured in 150-mm dishes were lysed in 800 μl of NETN buffer containing 150 mm NaCl plus protease inhibitor mixture. Cell lysates were then incubated overnight with 5 μg of anti-PR or anti-PSF antibody or control mouse IgG at 4 °C, followed by the addition of 30 μl of protein A/G Plus-agarose beads for another 2 h at 4 °C. Resins were washed with NETN buffer containing 250 mm NaCl and eluted with 1× Laemmli buffer. The eluted proteins along with the whole cell extract were Western-blotted using anti-PR or anti-PSF antibody. GST Pull-down Assay—A GST pull-down assay was performed as described previously (20.Dong X. Challis J.R.G. Lye S.J. J. Mol. Endocrinol. 2004; 32: 843-857Crossref PubMed Scopus (28) Google Scholar). GST and its fusion proteins were first immobilized on glutathione-Sepharose 4B affinity matrix. The matrix was then incubated overnight at 4 °C with rabbit reticulocyte lysate (Promega) containing the PR or His-PSF transcribed and translated in the presence of [35S]methionine. The matrix was washed three times with cold NETN buffer before the addition of 1× Laemmli buffer to elute associated proteins. The eluted proteins were separated by 10% SDS-PAGE. Gels were treated with ENHANCE (PerkinElmer Life Sciences), dried, and analyzed by autoradiography. Cell Culture and Transient Transfection—SHMs and 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) plus 5% fetal calf serum (Sigma) as described (22.Chen Z.Q. Lefebvre D. Bai X.H. Reaume A. Rossant J. Lye S.J. J. Biol. Chem. 1995; 270: 3863-3868Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). For experiments involving steroid exposure, the medium was substituted with phenol red-free DMEM containing 5% charcoal-treated fetal bovine serum (Hyclone Laboratories, Logan, UT). Transfection was performed according to the manufacturer's protocol (Fermentas). Cells were seeded at a density of 5 × 104 to achieve 60–80% confluency the following day. The DNA and transfection reagent were mixed and added to the medium. Cell lysates were collected at least 30 h after transfection. For luciferase assay, cells were collected in 200 μl of lysis buffer (Promega), 10 μl of which were used for the luciferase and β-galactosidase activity assays, respectively. Luciferase activity was determined using the luciferin reagent (Promega) according to the manufacturer's protocol. Transfection efficiency was normalized to β-galactosidase activity. For Western blotting, cell lysates were collected in NETN buffer plus protease inhibitor mixture. About 30 μg of protein extract were separated on SDS gel and electrophoresed, followed by Western blotting with the antibodies of interest. Electrophoretic Mobility Shift Assay—The PR DBD was synthesized in rabbit reticulocyte using the TnT coupled in vitro transcription/translation system (Promega) with vector pcDNA3-PR DBD. Full-length PR protein extract was obtained by transient transfection of 293T cells with the FLAG-PRB vector. Cells were treated with 10–9 m progesterone, and nuclear fractions were extracted using the NE-PER nuclear and cytoplasmic extraction kit. The nuclear fraction containing PRs was confirmed by Western blotting with anti-PR antibody AB-52 and used in a gel shift assay. Double-stranded synthetic oligonucleotide probes containing a 27-bp perfect palindromic consensus PRE were labeled with [32P]dATP and purified by passing through Quick Spin oligonucleotide G-50 columns (Roche). Binding reactions were performed in a total volume of 20 μl in 1× reaction buffer (5% glycerol, 5 mm dithiothreitol, 5 mm EDTA, 250 mm KCl, 100 mm HEPES (pH 7.5), 1 μg of poly(dI-dC), 25 mm MgCl2, 1 mg/ml bovine serum albumin, 1 μg of salmon sperm DNA, and 0.05% Triton X-100), 0.5 ng of labeled probe, and receptor protein. In some cases, bacterially expressed GST or GST-PSF was added as indicated. The binding reaction was allowed to proceed for 20 min at room temperature (the supershift was performed by adding 1.5 μg of anti-PR antibody for an additional 45 min) before the reaction mixtures were loaded onto 5% (60:1) nondenaturing polyacrylamide gel. After 2 h of electrophoresis in 0.5× Tris borate/EDTA at 4 °C, the gels were dried and autoradiographed. Tissue Collection and Northern Blotting—Wistar rats (Charles River Laboratories, St. Constance, Canada) were housed individually under standard environmental conditions (12-h light/12-h dark cycle) and fed Purina rat chow (Ralston Purina, St. Louis, MO) and water ad libitum. Female virgin rats were mated with male Wistar rats. Day 1 of gestation was designated as the day a vaginal plug was observed. The average time of delivery under these conditions was during the morning of day 23. Our criteria for labor were based on delivery of at least one pup. Rats were killed by carbon dioxide inhalation, and myometrial samples were collected on gestational days 6, 12, 15, 17, 19, 21, 22, and 23 or on days 1 and 4 postpartum. Tissue was collected at 10 a.m. on all days with the following exceptions: the labor sample (day 23) was collected once the animals had delivered at least one pup (n = 5). Rat myometrial tissues were placed into ice-cold phosphate-buffered saline, bisected longitudinally, and dissected away from both pups and placentas. The endometrium was carefully removed from the myometrial tissue by mechanical scraping on ice. We have previously shown that this removes the entire luminal epithelium and the majority of the uterine stroma (23.Piersanti M. Lye S.J. Endocrinology. 1995; 136: 3571-3578Crossref PubMed Google Scholar). The myometrial tissue was flash-frozen in liquid nitrogen. All other tissues from female and male animals (ovary, placenta, heart, liver, lung, small intestine, brain, kidney, skeletal muscle, and testis) were collected at the same time and flash-frozen in liquid nitrogen. All tissues were stored at –70 °C. Total RNA was extracted from the tissues using TRIzol (Invitrogen). Northern blotting and hybridization were carried out as described (24.Shynlova O. Mitchell J.A. Tsampalieros A. Langille B.L. Lye S.J. Biol. Reprod. 2004; 70: 986-992Crossref PubMed Scopus (101) Google Scholar). The probe used to detect PSF mRNA was a PCR-generated 770-bp fragment encompassing sequence 1436–2209 (GenBank™/EBI accession number X70944). The 18 S probe (provided by Dr. David T. Denhardt, Rutgers University) was used as a control probe. A total of five sets of gestational profiles were subjected to one-way analysis of variance, followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) to determine differences between groups, with the level of significance for comparison set at p < 0.05. The expression of the PR protein was determined by Western blotting. Frozen tissue was crushed under liquid nitrogen using a mortar and pestle. The crushed tissue was homogenized for 1 min in radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% (v/v) Triton X-100, 1% (v/v) sodium deoxycholate, and 0.1% (w/v) SDS supplemented with 100 μm sodium orthovanadate and protease inhibitor mixture tablets). Samples were spun at 12,000 × g for 15 min at 4 °C, and the supernatant was transferred to a fresh tube to obtain a crude protein lysate. Protein concentrations were determined using Bio-Rad protein assay buffer. Protein samples (40–50 μg) were resolved by electrophoresis on 8% SDS-polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride membrane in 25 mm Tris-HCl, 250 mm glycine, and 0.1% (w/v) SDS (pH 8.3) at 30 mV for 18 h at 4 °C; blotted with anti-PR antibody C-20; exposed to Eastman Kodak XAR x-ray film; and analyzed by densitometry. The membrane was then stripped and blotted with anti-calponin antibody as a loading control. Four complete sets of gestational profiles were analyzed by Western blotting, and the data were subjected to one-way analysis of variance, followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) to determine differences between groups, with the level of significance for comparison set at p < 0.05. Identification of PSF as a PR-interacting Protein—To identify PR-interacting proteins, GST-PR fusion proteins were bound to glutathione-Sepharose 4B matrix and incubated with either cytosolic or nuclear extracts from SHMs precleared by passage through GST-bound glutathione-Sepharose 4B matrix. Associated proteins were resolved on SDS gel and visualized by Coomassie Blue staining. Two protein bands, present only in nuclear fractions of SHMs, were identified that migrated at the same molecular mass of 100 kDa and bound to PR-(1–164) and PR-(556–933), respectively (Fig. 1A). GST-PR-(556–933) was found to bind p100 at a wider range of NaCl concentrations (100, 150, and 200 mm), whereas GST-PR-(1–164) could bind p100 only at 150 mm NaCl. This suggests that p100 may form a more stable complex with GST-PR-(556–933). These two p100 bands were excised and processed for MALDI mass spectrometry. Four peptide sequences within the proteins were identified. Fig. 1B shows that these two p100 proteins are identical. The sequences matched perfectly within the BLAST Database to a known protein termed PSF, previously identified as an RNA-splicing factor. Two PSF protein isoforms (PSF-A and PSF-F) have been reported (Fig. 1C). These two isoforms are identical through amino acids 1–662, but diverge thereafter, with PSF-F containing 669 amino acids and PSF-A containing 707 amino acids (25.Patton J.G. Porro E.B. Galceran J. Tempst P. Nadal-Ginard B. Genes Dev. 1993; 7: 393-406Crossref PubMed Scopus (310) Google Scholar). PSF contains two RNA recognition motifs (RRMs I and II, within amino acids 290–450) and an unusual N-terminal region rich in proline and glutamine residues and appears to migrate anomalously as an ∼100-kDa protein on SDS gel. Our MALDI mass spectrometric analysis did not detect any sequences specific to PSF-F, but the peptide FGQGGAGPVGGQGP did specifically match PSF-A. Confirmation of PSF Interaction with the PR in Vivo—We preformed immunoprecipitation to confirm the interaction between the PR and PSF in vivo. His-PSF was constructed by insertion of the PSF open reading frame into the C terminus of the His6 tag. 293T cells were transiently transfected with the PR and/or His-PSF expression plasmid as indicated (Fig. 2A). WCLs were first Western-blotted with anti-PR and anti-His-PSF antibodies to ensure that these two proteins were appropriately expressed in the cells (Fig. 2A, lower panel). We then performed immunoprecipitation with either anti-PR or anti-His-PSF antibody, followed by Western blotting with the same antibody to ensure that target proteins could be precipitated by the protein A/G Plus-agarose beads (Fig. 2A, middle panel). Finally, whole cell extracts expressing PRB and/or His-tagged PSF were incubated with anti-PR antibody and then incubated with protein A/G Plus-agarose beads. The associated proteins were washed and analyzed for the presence of His-PSF. Overexpressed His-PSF was specifically co-immunoprecipitated only in the presence of the PR (Fig. 2A, upper panel). Similarly, when anti-His tag antibody was used to immunoprecipitate WCL, we observed that the association of the PR could be detected only in the presence of His-PSF. Endogenous PSF was also co-immunoprecipitated with the endogenous PR from the T47D cell extract (Fig. 2B). No immunoprecipitation of the PR or PSF was observed when anti-PSF or anti-PR antibody was replaced with control mouse IgG. These in vivo data confirm the interaction between the PR and PSF found in the GST pull-down experiment. Further evidence to support an in vivo interaction between PSF and the PR was obtained using the mammalian two-hybrid system (Fig. 2C). PSF was fused to the C terminus of the Gal4 DBD in the pM vector, and PRB or PRA was fused down-stream of the Gal4 activation domain in the VP16 vector. When cotransfected with G5-luciferase, pM-PSF resulted in a 70% reduction in luciferase activity compared with the empty pM vector. However, cotransfection of both activation domain-tagged PRs (VP16-PRA and VP16-PRB) with pM-PSF induced a dramatic increase in luciferase activity as a result of the interaction between PSF and PRs. This interaction was ligand-independent because the addition of progesterone did not cause a significant difference in luciferase activity. These data provide further evidence that the PR and PSF interact in vivo. Mapping Interaction Sites within the PR and PSF—To determine whether the interaction between PSF and the PR is direct and, if so, to define the physical location of the interaction sites, an in vitro GST pull-down assay was performed. PSF was first 35S-labeled using the coupled in vitro transcription/translation system, and its binding to a series of GST-PR fusion proteins was assessed (Fig. 3A). PSF bound strongly to GST-PR fusion proteins containing the DBD and, to a lesser extent, to the AF3 domain of PRB, but did not bind to other segments of the PR. Additional experiments were also carried out to assess the direct binding of the full-length PR to GST-PSF fusion proteins containing the proline/glutamine-rich domain, RRM I, RRM II, and the C terminus, respectively. Specific binding of 35S-labeled PRB was detected only with GST-PSF fusion proteins containing RRM II (Fig. 3B). These differences in PRB binding to GST-PSF fusion proteins were not due to different inputs of GST fusion proteins because electrophoresis of the same mass of GST-PSF fusion proteins produced similar densities of protein bands (Fig. 3C). These data indicate that the interaction betwe
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