The Inhibitory Function in Human Progesterone Receptor N Termini Binds SUMO-1 Protein to Regulate Autoinhibition and Transrepression
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m204573200
ISSN1083-351X
AutoresHany Abdel-Hafiz, Glenn S. Takimoto, Lin Tung, Kathryn B. Horwitz,
Tópico(s)NF-κB Signaling Pathways
ResumoAlthough most studies of progesterone receptors (PR) and their two isoforms, PR-A and PR-B, focus on transcriptional stimulation, the receptors exhibit important inhibitory properties.Autoinhibition refers to an inhibitory function located in the PR N terminus, whose deletion increases transcriptional activity at least 6–10-fold. Transrepression refers to the ability of PR-A to suppress the transcriptional activity of PR-B and other nuclear receptors, including estrogen receptors.Self-squelching refers to the observation in transient transfection assays that increasing receptor concentrations paradoxically decrease transcriptional activity. Using a series of N-terminal deletion mutants constructed in both PR isoforms, we have mapped their autoinhibitory and transrepressor activities to a small ubiquitin-like modifier (SUMO-1) protein consensus-binding motif,387IKEE, located in the N terminus upstream of AF1. Self-squelching does not involve this site. SUMO-1 binds PR covalently at 387IKEE, but only if the C-terminal, liganded, hormone-binding domain is also present. A single point K388R mutation within the 387IKEE motif in either PR-A or PR-B leads to a loss of autoinhibitory and transrepressor functions of the liganded, full-length receptors. We conclude that autoinhibition and transrepression involve N-terminal sumoylation combined with intramolecular N/C-terminal communication. Although most studies of progesterone receptors (PR) and their two isoforms, PR-A and PR-B, focus on transcriptional stimulation, the receptors exhibit important inhibitory properties.Autoinhibition refers to an inhibitory function located in the PR N terminus, whose deletion increases transcriptional activity at least 6–10-fold. Transrepression refers to the ability of PR-A to suppress the transcriptional activity of PR-B and other nuclear receptors, including estrogen receptors.Self-squelching refers to the observation in transient transfection assays that increasing receptor concentrations paradoxically decrease transcriptional activity. Using a series of N-terminal deletion mutants constructed in both PR isoforms, we have mapped their autoinhibitory and transrepressor activities to a small ubiquitin-like modifier (SUMO-1) protein consensus-binding motif,387IKEE, located in the N terminus upstream of AF1. Self-squelching does not involve this site. SUMO-1 binds PR covalently at 387IKEE, but only if the C-terminal, liganded, hormone-binding domain is also present. A single point K388R mutation within the 387IKEE motif in either PR-A or PR-B leads to a loss of autoinhibitory and transrepressor functions of the liganded, full-length receptors. We conclude that autoinhibition and transrepression involve N-terminal sumoylation combined with intramolecular N/C-terminal communication. progesterone receptor human progesterone response element hormone-binding domain DNA-binding domain activation function B upstream segment androgen receptor glucocorticoid receptor estrogen receptor inhibitory function ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase synergy control green fluorescent protein estrogen response element Human progesterone receptor A (PR-A)1 and B (PR-B) isoforms are members of the nuclear receptor family of ligand-activated transcription factors (1Evans R. Science. 1988; 240: 889-895Crossref PubMed Scopus (6317) Google Scholar). The two PR are identical except that PR-B contains an additional 164 N-terminal amino acids in the B upstream segment (BUS) (2Horwitz K. Alexander P. Endocrinology. 1983; 113: 2195-2201Crossref PubMed Scopus (223) Google Scholar, 3Krett N. Wei L. 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As a result of these isoform-specific functional differences, tissue responses to progesterone are profoundly affected by PR-A:PR-B ratios, which vary considerably among normal target tissues and breast cancers (11Arnett-Mansfield R.L. deFazio A. Wain G. Jaworski R. Byth K. Mote P. Clarke C. Cancer Res. 2001; 61: 4576-4582PubMed Google Scholar). The multiplicity of regulatory effects that impact PR and other nuclear receptors is due in part to an intricate array of coactivators, corepressors, and cointegrators that are recruited to receptor-bound promoters (12Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1176) Google Scholar). Studies of coregulators have focused mainly on transcriptional activation, but transcriptional repression is also critical to understanding gene regulation. Repression can occur over entire chromosomal loci or by global targeting of general transcription factors (13Courey A. Jia S. Genes Dev. 2001; 15: 2786-2796PubMed Google Scholar). However, transcription factor-specific repression also involves mechanisms such as direct interference with DNA binding, blockade of coactivator binding, or nucleosome remodeling by histone deacetylation (14Maldonado E. Hampsey M. Reinberg D. Cell. 1999; 99: 455-458Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Of these, the interaction surfaces presented to coregulators by the receptors, possibly controlled by the DNA-binding site, are clearly important. For example, liganded glucocorticoid receptors (GR) are activators as dimers on palindromic DNA response elements but may be repressors in the monomeric state (15Lefstin J. Yamamoto K. Nature. 1998; 392: 885-888Crossref PubMed Scopus (438) Google Scholar). Even coregulatory proteins, like RIP140, PIAS1, and SRC1, can exhibit either coactivator or corepressor activity, depending on expression levels, the nuclear receptor target, and the promoter (16Ikonen T. 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These post-translational covalent modifications alter protein structure and protein-protein interactions, but in most cases, the mechanisms by which these modifications influence function are unknown. Phosphorylation of nuclear receptors regulates both their ligand-dependent and ligand-independent transcriptional activities (25Power R. Mani S. Codina J. Conneely O. O'Malley B. Science. 1991; 254: 1636-1639Crossref PubMed Scopus (498) Google Scholar). Ubiquitylation, which targets nuclear receptors for degradation, paradoxically increases their transcriptional activity (23Lange C. Shen T. Horwitz K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1032-1037Crossref PubMed Scopus (388) Google Scholar). Sumoylation, about which little is known, involves covalent binding of the SUMO (small ubiquitin-like modifier) protein. The prototype, SUMO-1, is a member of a family of ubiquitin-like proteins that are post-translationally conjugated predominantly to nuclear proteins (18Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (649) Google Scholar). Several important transcription factors, including GR, androgen receptors (AR), p53, c-Jun, and c-Myb are covalently modified by SUMO-1 binding (24Poukka H. Karvonen U. Janne O. Palvimo J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (370) Google Scholar, 26Goodson M. Hong Y. Rogers R. Matunis M. Park-Sarge O. Sarge K. J. Biol. Chem. 2001; 276: 18513-18518Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 27Muller S. Berger M. Lehembre F. Seeler J. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 28Iniguez-Lluhi J. Pearce D. Mol. Cell. Biol. 2000; 20: 6040-6050Crossref PubMed Scopus (178) Google Scholar, 29Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Like ubiquitylation, sumoylation uses a battery of activating (E1), conjugating (E2), and ligating (E3) enzymes for covalent attachment (30Pichler A. Gast A. Seeler J. Dejean A. Melchior F. Cell. 2002; 108: 109-120Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). In contrast to ubiquitylation, polysumoylation does not occur, and sumoylation does not lead to protein degradation, at least directly. Instead, influences on a variety of cellular processes and pathways have been described, including effects on assembly of protein complexes that regulate DNA recombination and repair (31Melchior F. Hengst L. Nat. Cell Biol. 2000; 2: E161-E163Crossref PubMed Scopus (16) Google Scholar, 32Mao Y. Sun M. Desai S. Liu L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar), effects on subcellular localization and targeting of proteins to nuclear pores and nuclear bodies (33Seeler J. Dejean A. Oncogene. 2001; 20: 7243-7249Crossref PubMed Scopus (136) Google Scholar, 34Seeler J. Marchio A. Losson R. Desterro J. Hay R. sChambon P. Dejean A. Mol. Cell. Biol. 2001; 21: 3314-3324Crossref PubMed Scopus (107) Google Scholar), effects on protein stabilization by blocking ubiquitylation (35Desterro J. Rodriguez M. Hay R. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar), and effects on chromatin structure and DNA binding (36Sternsdorf T. Jensen K. Reich B. Will H. J. Biol. Chem. 1999; 274: 12555-12566Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The link between SUMO-1 binding and transcriptional repression is unclear given this multiplicity of cellular effects. However, recently, a novel synergy control (SC) motif was identified in the N-terminal region of GR (28Iniguez-Lluhi J. Pearce D. Mol. Cell. Biol. 2000; 20: 6040-6050Crossref PubMed Scopus (178) Google Scholar) and other transcription factors (27Muller S. Berger M. Lehembre F. Seeler J. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) that acts as a negative regulatory region. The SC motif has no effect when the transcription factor is bound at a single DNA response element, but it blocks synergistic activity resulting from cooperative DNA binding of a transcription factor to multiple DNA response elements. Intriguingly, the SUMO-1 consensus binding motif, ΨKXE (where Ψ is hydrophobic and X is any residue) is identical to the SC motif (28Iniguez-Lluhi J. Pearce D. Mol. Cell. Biol. 2000; 20: 6040-6050Crossref PubMed Scopus (178) Google Scholar). It follows that suppression of transcriptional synergy and repressor activities might work through a common pathway. We (37Hovland A. Powell R. Takimoto G. Tung L. Horwitz K. J. Biol. Chem. 1998; 273: 5455-5460Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and others (38Huse B. Verca S. Matthey P. Rusconi S. Mol. Endocrinol. 1998; 12: 1334-1342Crossref PubMed Scopus (33) Google Scholar, 39Giangrande P. Pollio G. McDonnell D. J. Biol. Chem. 1997; 272: 32889-32900Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) have identified an inhibitory function (IF) in the N-terminal 165–456 amino acids between AF3 and AF1 common to both PR isoforms. Deletion of all or part of thisautoinhibitory region in PR-A increases their transcriptional activity. This autoinhibition is also transferable to related nuclear receptors. When the PR IF domain is fused to the N terminus of human estrogen receptors (ER) (37Hovland A. Powell R. Takimoto G. Tung L. Horwitz K. J. Biol. Chem. 1998; 273: 5455-5460Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) or chicken PR (39Giangrande P. Pollio G. McDonnell D. J. Biol. Chem. 1997; 272: 32889-32900Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), their transcriptional activity is repressed. Similar autoinhibitory regions have been identified in other transcription factors. Examples include c-Jun, c-Fos, and other members of the AP1 protein family (27Muller S. Berger M. Lehembre F. Seeler J. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), ATF-2, a member of the ATF/cAMP regulatory element-binding protein family, and Oct-2a, a lymphoid-specific transcription factor (40Li X. Green M. Genes Dev. 1996; 10: 517-527Crossref PubMed Scopus (106) Google Scholar). Atransrepressor activity, distinct from autoinhibition, also maps to the IF domain of PR-A. This activity is defined by the ability of PR-A to inhibit the transcriptional activity of PR-B, ER, or GR (38Huse B. Verca S. Matthey P. Rusconi S. Mol. Endocrinol. 1998; 12: 1334-1342Crossref PubMed Scopus (33) Google Scholar,39Giangrande P. Pollio G. McDonnell D. J. Biol. Chem. 1997; 272: 32889-32900Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The mechanisms that distinguish autoinhibition from transrepression, both of which originate from the IF domain, are unclear. Recent attempts to precisely map these N-terminal repressor functions have led to ambiguous conclusions, identifying multiple, nonoverlapping regions (38Huse B. Verca S. Matthey P. Rusconi S. Mol. Endocrinol. 1998; 12: 1334-1342Crossref PubMed Scopus (33) Google Scholar, 39Giangrande P. Pollio G. McDonnell D. J. Biol. Chem. 1997; 272: 32889-32900Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). We have now identified a consensus SUMO-1-binding motif,387IKEE within the IF domain of PR that is responsible for and precisely maps both the autoinhibitory and transrepressor activities of PR-A and PR-B. Conservative K388A and K388R single point mutations of the Lys residue within this motif completely abrogate both repressor activities. Loss of repression is directly linked to the loss of SUMO-1 binding at the PR N terminus. Interestingly, SUMO-1-dependent repressor activities arising at the N terminus require the liganded C-terminal HBD, suggesting that the mechanism involves intramolecular communication between the N and C termini. The expression plasmids pSG5 hPR1 and pSG5 hPR2, encoding human PR-B and PR-A, respectively, and HEGO, encoding human ER, cloned into pSG5 were a gift from P. Chambon (Strasbourg, France). Wild type pEGFP-SUMO-1 and mutant pEGFP-SUMO-1G97A were gifts from J. Palvimo and O. Janne (University of Helsinki, Helsinki, Finland). To generate N-terminal deletion mutants of full-length PR-A or NTA (which contain the N terminus, DBD, and NLS of PR-A but lack the hinge and HBD), we used a 5′ sense primer containing an EcoRI site, a Kozak consensus sequence, and the ATG initiation codon. The 3′-antisense primer contained a stop codon and the BglII site. The resulting PCR fragment was cloned into pSG5 digested with EcoRI/BglII. For PR-B deletion mutants, the 5′ primer contained the in frameRsrII site located at the PR-B/PR-A junction, and the 3′-primers were identical to those used for PR-A. The resulting PCR fragment was cloned into pSG5 hPR1 or NTB digested withRsrII/BglII. Other deletions within PR-B and PR-A (PR-BΔ375–397 and PR-AΔ375–397), were created by insertingBamHI sites in PR-A at Pro375 and Ser397 by PCR-mediated mutagenesis and ligating the two sites to produce the deletion. PR-AΔ375–397 was then digested withRsrII/BglII, and the resulting fragment was ligated into RsrII/BglII-digested hPR1 to generate PR-BΔ375–397. To mutate the PR-A ATG initiation codon in PR-B, we used a 5′ primer containing an EcoRI site and a 3′ primer in which the ATG was mutated to GCG (Ala) and linked to anRsrII site. The PCR fragment was cloned into pSG5 hPR1 cut with EcoRI/RsrII. The point mutations, K388A and K388R, were generated by PCR using a 5′ sense primer in which AAG was mutated to AGG or GCG, respectively. The mutated fragments were introduced into pSG5-hPR1 or pSG5-hPR2 usingMluI/HindIII sites. All PCR-based cloning was verified by dideoxy sequencing. HeLa cells were plated at a density of ∼1.2 × 105 cells in 60-mm tissue culture dishes with 3 ml of minimum essential medium supplemented with 7.5% twice charcoal-stripped fetal calf serum. Quadruplicate plates were transfected using calcium phosphate coprecipitation with 2 μg of the reporter plasmids, PRE2-TATA-luciferase (6Tung L. Shen T. Abel M. Powell R. Takimoto G. Sartorius C. Horwitz K. J. Biol. Chem. 2001; 276: 39843-39851Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) or ERE2-TATA-luciferase, variable amounts of PR or ER expression vectors, 2 μg of the β-galactosidase expression plasmid pCH110 (Pharmacia Corp.) to correct for transfection efficiency and Bluescribe (Stratagene, La Jolla, CA) carrier plasmid for a total of 6 μg/plate. After overnight incubation at 37 °C, the cells were washed, the medium was changed to 7.5% minimum essential medium supplemented with 7.5% twice charcoal-stripped fetal calf serum, and the cells were incubated with 10 nm of the synthetic progestin R5020, 10 nm 17β-estradiol, or both for an additional 24 h. The control cells received ethanol only. The cells were harvested, the lysates were normalized to β−galactosidase activity, and the luciferase activity was quantified with a Monolight 3010 luminometer (Analytical Luminescence Lab., Ann Arbor, MI). Whole cell extracts were prepared from HeLa cells transiently transfected with PR expression vectors as described previously (37Hovland A. Powell R. Takimoto G. Tung L. Horwitz K. J. Biol. Chem. 1998; 273: 5455-5460Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The expressed proteins were resolved by electrophoresis on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with our anti-PR monoclonal antibodies, AB-52 and B-30 (41Estes P. Suba E. Lawler-Heavner J. Elashry-Stowers D. Wei L. Toft D. Sullivan W. Horwitz K. Edwards D. Biochemistry. 1987; 26: 6250-6262Crossref PubMed Scopus (146) Google Scholar), and with an anti-DBD polyclonal antibody, α266 (a gift of D. Toft, Rochester, MN) (42Smith D. Lubahn D. McCormick D. Wilson E. Toft D. Endocrinology. 1988; 122: 2816-2825Crossref PubMed Scopus (38) Google Scholar). The bands were detected by enhanced chemiluminescence (PerkinElmer Life Sciences). For detection of PR-SUMO-1 binding, HeLa cells cotransfected with PR-A, PR-B, or PR mutants and GFP-tagged SUMO-1 or SUMO-1G97A were collected in PBS containing 20 mmN-ethylmaleimide, and the cell extracts were prepared in modified RIPA buffer (50 mmTris-HCl, pH 7.8, 150 mm NaCl, 5 mm EDTA, 15 mm dithiothreitol, protease inhibitor mixture (Roche Molecular Biochemicals), and 20 mmN-ethylmaleimide). The expressed proteins were resolved on SDS-PAGE, and conjugated protein was detected by immunoblotting with AB-52 or an anti-GFP polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Several studies (6Tung L. Shen T. Abel M. Powell R. Takimoto G. Sartorius C. Horwitz K. J. Biol. Chem. 2001; 276: 39843-39851Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 37Hovland A. Powell R. Takimoto G. Tung L. Horwitz K. J. Biol. Chem. 1998; 273: 5455-5460Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 38Huse B. Verca S. Matthey P. Rusconi S. Mol. Endocrinol. 1998; 12: 1334-1342Crossref PubMed Scopus (33) Google Scholar, 39Giangrande P. Pollio G. McDonnell D. J. Biol. Chem. 1997; 272: 32889-32900Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) have identified at least three different inhibitory properties of PRs.Autoinhibition refers to an IF located in the PR N terminus whose deletion increases the activity of PR-A at least 6-fold from a tandem progesterone response element (PRE2)-containing promoter. Transrepression refers to the ability of PR-A to suppress the transcriptional activity of PR-B and other nuclear receptors at low concentrations in which self-squelching is excluded (43Bubulya A. Wise S. Shen X. Burmeister L. Shemshedini L. J. Biol. Chem. 1996; 271: 24583-24589Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 44Shemshedini L., Ji, J. Brou C. Chambon P. Gronemeyer H. J. Biol. Chem. 1992; 267: 1834-1839Abstract Full Text PDF PubMed Google Scholar). For example, the transcriptional activity of ER from a promoter containing an ERE is inhibited by liganded PR-A even in the absence of a PRE. Self-squelching refers to the observation with transiently expressed PRs and other nuclear receptors (6Tung L. Shen T. Abel M. Powell R. Takimoto G. Sartorius C. Horwitz K. J. Biol. Chem. 2001; 276: 39843-39851Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 37Hovland A. Powell R. Takimoto G. Tung L. Horwitz K. J. Biol. Chem. 1998; 273: 5455-5460Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 39Giangrande P. Pollio G. McDonnell D. J. Biol. Chem. 1997; 272: 32889-32900Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) that as the concentration of exogenously transfected receptors increases, there is a paradoxical decrease in transcriptional activity from a cotransfected promoter/reporter. The studies below were designed to address the mechanisms underlying the autoinhibitory and transrepressor properties of PR. We previously mapped IF to the large N-terminal region between residues 165 and 456 in the PR N terminus common to both isoforms (37Hovland A. Powell R. Takimoto G. Tung L. Horwitz K. J. Biol. Chem. 1998; 273: 5455-5460Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). To map IF further, a series of N-terminal deletion mutants were constructed in PR-A and PR-B (Fig. 1A). A concentration (100 ng) of expression vector cDNA for each mutant that yields maximal activity without self-squelching (see Fig. 5) was cotransfected into HeLa cells together with a PRE2-TATA-luciferase reporter, followed by R5020 treatment. The transcriptional activities of the mutants are expressed as percentages of wild type PR-B activity. Stepwise deletions to residue 375 in PR-B (PR-BΔ165–315, PR-BΔ165–345, and PR-BΔ165–375) or PR-A (PR-AΔ290, PR-AΔ315, PR-AΔ345, and PR-AΔ375) had little effect on transcriptional activity compared with the wild type receptors. However, further deletion to residue 397 (PR-BΔ165–397, PR-AΔ397) resulted in 7.5-fold (PR-B) or 6-fold (PR-A) increases in transcriptional activity. These deletions map the autoinhibitory function to residues 375–397. Additional deletions to PR-BΔ456 or PR-AΔ427 lead to some loss of activity because of encroachment into AF1. The location of the autoinhibitory region within IF was confirmed with an internal deletion mutant (Δ375–397) in PR-B and PR-A. This 22-amino acid deletion increased the activity of PR-B 11-fold and that of PR-A 6-fold.Figure 5Δ375–397 and K388R mutants in PR-A and PR-B backgrounds; autoinhibition is lost but self-squelching is retained. HeLa cells were transiently transfected with 10, 100, or 1000 ng of the receptor cDNA expression vectors indicated and 2 μg of PRE2-TATA-luciferase reporter plasmid. The values are expressed as the fold induction over the no hormone control.A, relative dose-dependent activities of wild type (wt) PR-A (open squares), PRAΔ375–397 (closed diamonds), and PRA K388A (open circles).B, relative dose-dependent activities of wild type PR-B (open squares), PRBΔ375–397 (closed diamonds), and PRB K388R (open circles).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Selected N-terminal deletion mutants were then constructed in the background of NTA and NTB, the constitutively active N-terminal fragments of PR-A and PR-B, respectively, which contain the entire N terminus plus the DBD and NLS but lack the HBD (Fig. 1B). Constructs (100 ng) were transfected into HeLa cells and tested for transcriptional activity on PRE2-TATA in the absence of hormone. Analogous to their full-length counterparts, the activity of NTA is ∼10% that of NTB. This residual activity is due to AF1 and is entirely lost in the NTA ΔAF1 construct. Interestingly, in both N-terminal backgrounds (Fig. 1B), deletion of the autoinhibitory region (NTB Δ165−397 and NTA Δ397) identified in Fig. 1A failed to increase transcription. We conclude that the C-terminally liganded HBD is required for N-terminal autoinhibitory activity in both isoforms. Thus, residues 375–397 in the N terminus are necessary but not sufficient for full expression of the autoinhibitory effect. Fig. 2 shows immunoblots using the anti-PR monoclonal antibodies AB-52 (B + A-specific) and B-30 (B-specific) or the anti-DBD polyclonal antibody α266 to document protein expression levels of the wild type and mutant PR-A, PR-B, NTA, and NTB constructs used for the transcription studies of Fig. 1. They demonstrate good expression levels of all of the proteins at their expected molecular masses. The N-terminal autoinhibitory region defined by residues 375–397, contains an 387IKEE consensus SUMO-1-binding motif (Fig. 3A); the only one present in PR. Similar sites are found in GR and AR and in other transcription factors unrelated to PR. This motif binds SUMO-1 covalently. Such binding reportedly blocks synergistic transcriptional activity from promoters that contain multiple response elements for one transcription factor. To determine whether PR are sumoylated, PR-A were transiently transfected into HeLa cells together with GFP-SUMO-1 in the absence or presence of R5020 (Fig. 3B). Denatured cell extracts were prepared in N-ethylmaleimide to prevent GFP-SUMO-1 deconjugation (24Poukka H. Karvonen U. Janne O. Palvimo J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (370) Google Scholar), resolved by SDS-PAGE, and immunoblotted with the anti-PR antibody, AB-52, and the anti-GFP antibody. Lane 1 (Fig. 3B) shows the migration of unliganded PR-A. Despite the presence of GFP-SUMO-1, PR-A migrate at ∼97 kDa, the expected molecular mass of unmodified receptors. However, when R5020 is added (lane 2), a higher molecular mass PR band (∼140 kDa) is formed. That this is due to GFP-SUMO-1 conjugation to PR-A is shown in lane 4 using an anti-GFP antibody. These data demonstrate that PR-A are sumoylated but that this requires ligand binding. To confirm the HBD requirement, NTA and NTB, the constitutively active PR N termini that lack an HBD, were cotransfected with GFP-SUMO-1 (Fig.3C). Lanes 1 and 2 show the migration of NTB (∼97 kDa) and NTA (∼60 kDa) at the positions expected for the unmodified receptors. No higher molecular ma
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