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• W2053308116 abstract "Many steroid receptors, including chicken progesterone receptor, have been shown to be activated in the absence of their cognate ligands by modulators of kinases and phosphatases. To investigate the molecular mechanism of ligand-independent activation, chicken progesterone receptor mutants in which either one or all four of the previously identified phosphorylation sites have been changed to nonphosphorylatable alanine were analyzed for their ability to be activated by progesterone, 8-bromoadenosine 3′:5′-cyclic monophosphate, or a dopamine agonist, SKF82958. Our current study shows that the receptor is differently phosphorylated in ligand-dependent and ligand-independent activation. The transcriptional activity of the receptor in response to 8-bromoadenosine 3′:5′-cyclic monophosphate is affected by mutation of either Ser211 or Ser260. In addition, our data demonstrated that none of the four sites is absolutely required for the activation of the receptor by either 8-bromoadenosine 3′:5′-cyclic monophosphate or the dopamine agonist. Treatment with 8-bromoadenosine 3′:5′-cyclic monophosphate did not increase the overall level of receptor phosphorylation or cause phosphorylation of the receptor at alternate sites. These data raise the possibility that ligand-independent activation of the chicken progesterone receptor may be mediated through changes in the phosphorylation of coregulators or other protein factors interacting with the receptors. Many steroid receptors, including chicken progesterone receptor, have been shown to be activated in the absence of their cognate ligands by modulators of kinases and phosphatases. To investigate the molecular mechanism of ligand-independent activation, chicken progesterone receptor mutants in which either one or all four of the previously identified phosphorylation sites have been changed to nonphosphorylatable alanine were analyzed for their ability to be activated by progesterone, 8-bromoadenosine 3′:5′-cyclic monophosphate, or a dopamine agonist, SKF82958. Our current study shows that the receptor is differently phosphorylated in ligand-dependent and ligand-independent activation. The transcriptional activity of the receptor in response to 8-bromoadenosine 3′:5′-cyclic monophosphate is affected by mutation of either Ser211 or Ser260. In addition, our data demonstrated that none of the four sites is absolutely required for the activation of the receptor by either 8-bromoadenosine 3′:5′-cyclic monophosphate or the dopamine agonist. Treatment with 8-bromoadenosine 3′:5′-cyclic monophosphate did not increase the overall level of receptor phosphorylation or cause phosphorylation of the receptor at alternate sites. These data raise the possibility that ligand-independent activation of the chicken progesterone receptor may be mediated through changes in the phosphorylation of coregulators or other protein factors interacting with the receptors. In contrast to peptide hormones whose signals are transduced into the nucleus through membrane receptor-activated signal transduction pathways, steroid hormones act through intracellular receptors which themselves are ligand-regulated transcription factors (1Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2838) Google Scholar, 2Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6276) Google Scholar, 3Green S. Chambon P. Trends Genet. 1988; 4: 309-314Abstract Full Text PDF PubMed Scopus (829) Google Scholar, 4Gronemeyer H. Ann. Rev. Genet. 1991; 25: 89-123Crossref PubMed Scopus (327) Google Scholar, 5O'Malley B.W. Mol. Endocrinol. 1990; 4: 363-369Crossref PubMed Scopus (383) Google Scholar, 6O'Malley B.W. Tsai S.Y. Bagchi M.K. Weigel N.L. Schrader W.T. Tsai M.-J. Recent Prog. Horm. Res. 1991; 47: 1-26PubMed Google Scholar, 7Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2666) Google Scholar). The lipophilic hormones diffuse through the cell membrane, bind the receptors inside the cell, and transform them into active transcription factors. In this way, the signal carried by steroids is directly transduced into long term changes in gene expression without an absolute requirement for a phosphorylation cascade to mediate the effect. Studies in recent years have provided increasing evidence that there is cross-talk between the signaling processes induced by growth factors and steroids, and phosphorylation plays an important role in these processes. First, most, if not all, steroid receptors are phosphoproteins (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar, 9Sheridan P.L. Evans R.M. Horwitz K.B. J. Biol. Chem. 1989; 264: 6520-6528Abstract Full Text PDF PubMed Google Scholar, 10Sullivan W.P. Madden B. McCormick D.J. Toft D.O. J. Biol. Chem. 1988; 263: 14717-14723Abstract Full Text PDF PubMed Google Scholar, 11Housley P.R. Pratt W.B. J. Biol. Chem. 1983; 258: 4630-4635Abstract Full Text PDF PubMed Google Scholar, 12Mendel D.B. Bodwell J.E. Munck A. J. Biol. Chem. 1987; 262: 5641-5648Google Scholar, 13Kemppainen J.A. Lane W.V. Sar M. Wilson E.M. J. Biol. Chem. 1992; 267: 968-974Abstract Full Text PDF PubMed Google Scholar, 14LeGoff P. Montano M.M. Schodin D.J. Katzenellenbogen B.S. J. Biol. Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar). Second, functional analyses of phosphorylation site mutants have demonstrated that phosphorylation regulates the transcriptional activity of many steroid receptors (14LeGoff P. Montano M.M. Schodin D.J. Katzenellenbogen B.S. J. Biol. Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar, 15Bai W. Tullos S. Weigel N.L. Mol. Endocrinol. 1994; 8: 1465-1473Crossref PubMed Google Scholar, 16Bai W. Weigel N.L. J. Biol. Chem. 1996; 271: 12801-12806Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Ali S. Metzger D. Bornert J.M. Chambon P. EMBO J. 1993; 12: 1153-1160Crossref PubMed Scopus (376) Google Scholar, 18Glineur C. Zenke M. Beng H. Ghysdael J. Genes Dev. 1990; 4: 1663-1676Crossref PubMed Scopus (51) Google Scholar, 19Jurutka P.W. Hsieh J.-C. Nakajima S. Haussler C.A. Whitfield G.K. Haussler M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3519-3524Crossref PubMed Scopus (81) Google Scholar). Third, modulators of various signal transduction pathways have been shown to regulate the ligand-stimulated transcriptional activity of the receptors (20Beck C.A. Weigel N.L. Edwards D.P. Mol. Endocrinol. 1992; 6: 607-620Crossref PubMed Scopus (106) Google Scholar, 21Beck C.A. Weigel N.L. Moyer M.L. Nordeen S.K. Edwards D.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4441-4445Crossref PubMed Scopus (193) Google Scholar, 22Culig Z. Hobisch A. Cronauer M.V. Radmayr C. Trapman J. Hittmair A. Bartsch G. Klocker H. Cancer Res. 1994; 54: 5474-5478PubMed Google Scholar, 23Rangarajan P.N. Umesono K. Evans R.M. Mol. Endocrinol. 1992; 6: 1451-1457Crossref PubMed Scopus (143) Google Scholar, 24Power R.F. Lydon J.P. Conneely O.M. O'Malley B.W. Science. 1991; 252: 1546-1548Crossref PubMed Scopus (174) Google Scholar, 25Power R.F. Mani S.K. Codina J. Conneely O.M. O'Malley B.W. Science. 1991; 254: 1636-1639Crossref PubMed Scopus (495) Google Scholar, 26Nordeen S.K. Bona B.J. Moyer M.L. Mol. Endocrinol. 1993; 7: 731-742Crossref PubMed Scopus (103) Google Scholar, 27Jones K.E. Brubaker J.H. Chin W.W. Endocrinology. 1994; 134: 543-548Crossref PubMed Scopus (39) Google Scholar, 28Huggenvik J.I. Collard M.W. Kim Y.-W. Sharma R.P. Mol. Endocrinol. 1993; 7: 543-550PubMed Google Scholar, 29Smith C.L. Conneely O.M. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6120-6124Crossref PubMed Scopus (197) Google Scholar). Most importantly, many steroid receptors have been shown to be activated in the absence of their cognate ligands by modulation of protein kinase or phosphatase activity (22Culig Z. Hobisch A. Cronauer M.V. Radmayr C. Trapman J. Hittmair A. Bartsch G. Klocker H. Cancer Res. 1994; 54: 5474-5478PubMed Google Scholar, 24Power R.F. Lydon J.P. Conneely O.M. O'Malley B.W. Science. 1991; 252: 1546-1548Crossref PubMed Scopus (174) Google Scholar, 25Power R.F. Mani S.K. Codina J. Conneely O.M. O'Malley B.W. Science. 1991; 254: 1636-1639Crossref PubMed Scopus (495) Google Scholar,30Denner L.A. Weigel N.L. Maxwell B.L. Schrader W.T. O'Malley B.W. Science. 1990; 250: 1740-1743Crossref PubMed Scopus (310) Google Scholar, 31Nazareth L.V. Weigel N.L. J. Biol. Chem. 1996; 271: 19900-19907Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 32Zhang Y. Bai W. Allgood V.E. Weigel N.L. Mol. Endocrinol. 1994; 8: 577-584Crossref PubMed Scopus (67) Google Scholar, 33Ignar-Trowbridge D.M. Nelson K.G. Bidwell M.C. Curtis S.W. Washburn T.F. Machlachlan J.A. Korach K.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4658-4662Crossref PubMed Scopus (448) Google Scholar, 34Aronica S.M. Katzenellenbogen B.S. Endocrinology. 1991; 128: 2045-2052Crossref PubMed Scopus (176) Google Scholar, 35Katzenellenbogen B.S. Norman M.J. Endocrinology. 1990; 126: 891-898Crossref PubMed Scopus (175) Google Scholar). Ligand-independent activation of steroid receptors may have important physiological and clinical implications for the study and treatment of the tumors of hormone-responsive organs. Very little is known about the molecular mechanism of ligand-independent activation for any of the receptors. Since steroid receptors are phosphoproteins, it is possible that alteration of receptor phosphorylation in response to different treatments mediates the ligand-independent activation. Chicken progesterone receptor (cPR) 1The abbreviations used are: cPR, chicken progesterone receptor; EGF, epidermal growth factor; AF, activation function; TAF, transactivation function; HPLC, high performance liquid chromatography; GRE, glucocorticoid response element(s); FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: cPR, chicken progesterone receptor; EGF, epidermal growth factor; AF, activation function; TAF, transactivation function; HPLC, high performance liquid chromatography; GRE, glucocorticoid response element(s); FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis. is one of the steroid receptors that has been shown to be activated in a ligand-independent manner by modulators of kinases and phosphatases such as 8-bromoadenosine 3′:5′-cyclic monophosphate (8-bromo-cAMP), okadaic acid, vanadate, growth factors (e.g. EGF), and neurotransmitters (e.g. dopamine) (25Power R.F. Mani S.K. Codina J. Conneely O.M. O'Malley B.W. Science. 1991; 254: 1636-1639Crossref PubMed Scopus (495) Google Scholar, 30Denner L.A. Weigel N.L. Maxwell B.L. Schrader W.T. O'Malley B.W. Science. 1990; 250: 1740-1743Crossref PubMed Scopus (310) Google Scholar, 32Zhang Y. Bai W. Allgood V.E. Weigel N.L. Mol. Endocrinol. 1994; 8: 577-584Crossref PubMed Scopus (67) Google Scholar). The phosphorylation sites in cPR have been identified (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar, 36Poletti A. Weigel N.L. Mol. Endocrinol. 1993; 7: 241-246PubMed Google Scholar), making it possible to answer the question of whether the phosphorylation of the receptor is involved in or mediates the ligand-independent activation of the receptor. cPR is expressed as two forms, cPRB and cPRA, which lack the amino-terminal 128 amino acids of cPRB. Like the other members of the steroid/thyroid superfamily, cPR is composed of separable domains such as domains for DNA binding, hormone binding, and transcriptional activation (37Gronemeyer H. Turcotte B. Quirin-Stricker C. Bocquel M.T. Meyer M.E. Krozowski Z. Jeltsch J.M. Lerouge T. Garnier J.M. Chambon P. EMBO J. 1987; 6: 3985-3994Crossref PubMed Scopus (164) Google Scholar, 38Carson-Jurica M.A. Tsai M.-J. Conneely O.M. Maxwell B.L. Clark J.H. Dobson A.D.W. Elbrecht A. Toft D.O. Schrader W.T. O'Malley B.W. Mol. Endocrinol. 1987; 1: 791-801Crossref PubMed Scopus (43) Google Scholar, 39Conneely O.M. Sullivan W.P. Toft D.O. Birnbaumer M. Cook R.G. Maxwell B.L. Zarucki-Schulz T. Greene G.L. Schrader W.T. O'Malley B.W. Science. 1986; 233: 767-770Crossref PubMed Scopus (161) Google Scholar). Different from the DNA binding and hormone binding domains which are not further separable, two separable transcriptional activation domains have been identified in chicken progesterone receptor (37Gronemeyer H. Turcotte B. Quirin-Stricker C. Bocquel M.T. Meyer M.E. Krozowski Z. Jeltsch J.M. Lerouge T. Garnier J.M. Chambon P. EMBO J. 1987; 6: 3985-3994Crossref PubMed Scopus (164) Google Scholar, 40Bocquel M.T. Kumar V. Stricker C. Chambon P. Gronemeyer H. Nucleic Acids Res. 1989; 17: 2581-2594Crossref PubMed Scopus (227) Google Scholar). The activation function 2 (AF-2, previously named transactivation function 2 or TAF-2), within the hormone binding domain is known to be regulated by hormone. In contrast to AF-2, the activation function 1 (AF-1, previously named transcriptional activation function 1 or TAF-1) is located in the amino-terminal region (37Gronemeyer H. Turcotte B. Quirin-Stricker C. Bocquel M.T. Meyer M.E. Krozowski Z. Jeltsch J.M. Lerouge T. Garnier J.M. Chambon P. EMBO J. 1987; 6: 3985-3994Crossref PubMed Scopus (164) Google Scholar, 40Bocquel M.T. Kumar V. Stricker C. Chambon P. Gronemeyer H. Nucleic Acids Res. 1989; 17: 2581-2594Crossref PubMed Scopus (227) Google Scholar). The fact that AF-1 is located in the sequence outside the hormone binding domain raises the possibility that the activity of AF-1 might be regulated by means such as phosphorylation rather than by ligand binding. Consistent with this idea, three out of the four phosphorylation sites identified in cPR are located in the A/B region (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar, 36Poletti A. Weigel N.L. Mol. Endocrinol. 1993; 7: 241-246PubMed Google Scholar). Based on the primary sequence of cPRB, the four identified phosphorylation sites are Ser211, Ser260, Ser367, and Ser530 (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar, 36Poletti A. Weigel N.L. Mol. Endocrinol. 1993; 7: 241-246PubMed Google Scholar). All four sites are in Ser-Pro motifs (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar, 36Poletti A. Weigel N.L. Mol. Endocrinol. 1993; 7: 241-246PubMed Google Scholar), and they account for all the Ser-Pro motifs in the receptor. The same phosphorylation pattern was detected in both cPRA and cPRB and is conserved between the endogenous receptor isolated from chicken oviduct and the recombinant receptor expressed in and purified from yeast (41Poletti A. Conneely O.M. McDonnell D.P. Schrader W.T. O'Malley B.W. Weigel N.L. Biochemistry. 1993; 32: 9563-9569Crossref PubMed Scopus (20) Google Scholar). Among the four sites, Ser211 and Ser260 are basally phosphorylated, but their phosphorylation is enhanced in response to hormone stimulation; Ser367 and Ser530 are phosphorylated primarily in response to progesterone treatment (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar,36Poletti A. Weigel N.L. Mol. Endocrinol. 1993; 7: 241-246PubMed Google Scholar). To examine whether any of the known phosphorylation sites in cPR is involved in or mediates the ligand-independent activation of the receptor, the four phosphorylation sites were mutated either individually or simultaneously to nonphosphorylatable alanines, and the responses of these mutant receptors to 8-bromo-cAMP and a dopamine agonist were assayed and compared with that of the wild type receptor. Our data show that receptor phosphorylation is not absolutely required for ligand-independent activation, raising the possibility that the ligand-independent activation of cPR might be mediated through changes in the phosphorylation of receptor-associated proteins such as coactivators and chaperone proteins that are known to play important roles in receptor function. All cell culture reagents were purchased from Life Technologies, Inc. [3H]Chloramphenicol and carrier-free [32P]H3PO4 were purchased from DuPont NEN. N-Butyryl-coenzyme A and Protein-A Sepharose were purchased from Pharmacia Biotech Inc. The T7-Gen in vitro mutagenesis kit and Sequenase version 2.0 sequencing kit were purchased from United States Biochemical Corp. The oligonucleotides used in the mutagenesis and sequencing were synthesized by GenoSys (The Woodlands, TX). Triethylamine, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, 8-bromo-cAMP, Triton X-100, 8-methoxypsoralen, sequencing grade trifluoroacetic acid, poly-l-lysine and 2,6,10,14-tetra-methylpentadecane were purchased from Sigma. Xylene was purchased from Fisher. Tosylphenylalanyl chloromethyl ketone-treated trypsin was obtained from Worthington. Phenylisothiocyanate and HPLC reagents were obtained from J. T. Baker Inc. The D1 dopamine agonist, SKF82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide) was obtained from Research Biochemicals Inc. (Natick, MA). Monoclonal antibody to the chicken progesterone receptor (PR22) was kindly provided by Dr. David Toft. All other chemicals are of reagent grade. Single site mutations were generated using a conventional, non-polymerase chain reaction mutagenesis strategy as described previously (15Bai W. Tullos S. Weigel N.L. Mol. Endocrinol. 1994; 8: 1465-1473Crossref PubMed Google Scholar). The mutant in which all four phosphorylation sites are changed to alanine was generated by exchanging the restriction fragments between the single site mutants. All mutations were confirmed by direct sequencing using a Sequenase version 2.0 sequencing kit. The subcloning of the mutant and the wild type cPRA into the expression vector, p91023B, has been fully described (15Bai W. Tullos S. Weigel N.L. Mol. Endocrinol. 1994; 8: 1465-1473Crossref PubMed Google Scholar, 38Carson-Jurica M.A. Tsai M.-J. Conneely O.M. Maxwell B.L. Clark J.H. Dobson A.D.W. Elbrecht A. Toft D.O. Schrader W.T. O'Malley B.W. Mol. Endocrinol. 1987; 1: 791-801Crossref PubMed Scopus (43) Google Scholar). GRE2 e1bCAT (provided by Dr. John Cidlowski) is a simple promoter-based reporter composed of two progesterone/glucocorticoid response elements (GREs), the TATA box from the adenovirus e1b gene, and the cDNA sequence for chloramphenicol acetyltransferase (CAT) (42Allgood V.E. Oakley R.H. Cidlowski J.A. J. Biol. Chem. 1993; 268: 20870-20876Abstract Full Text PDF PubMed Google Scholar). cPRA was expressed in cells using a nonrecombinant adenoviral-mediated DNA transfer technique (31Nazareth L.V. Weigel N.L. J. Biol. Chem. 1996; 271: 19900-19907Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 43Allgood V.E. Weigel N.L. Zhang Y. O'Malley B.W. Biochemistry. 1996; 36: 224-232Crossref Scopus (51) Google Scholar) with a few modifications. Replication-deficient adenovirus, dl312, was grown in human 293 embryonic kidney cells. The virus was released from the cells by three successive freeze/thaws of the cell pellet in phosphate-buffered saline. Following centrifugation at 2,500 rpm in a clinical centrifuge, the resulting supernatant was layered on top of a cesium chloride step gradient (1.5, 1.35, and 1.25 g/ml CsCl). Following centrifugation of the CsCl gradient at 150,000 ×g for 1 h at 10 °C, the adenovirus band (between the 1.25 and 1.35 g/ml layers) was removed and brought to a final concentration of 1.35 g/ml CsCl. The adenovirus was centrifuged again at 150,000 × g for 5 h at 10 °C, and the adenovirus band was collected and dialyzed against HEPES-buffered saline (150 mm NaCl, 20 mm HEPES, pH 7.3) for 16 h at 4 °C with 50,000 molecular weight cutoff dialysis tubing. The dialysate was exposed to short wavelength UV light (254 nm) for 3 min. 8-Methoxypsoralen was added to the supernatant at a final concentration of 0.33 mg/ml, and the sample was exposed to long wavelength UV light (366 nm) for 20 min. The preceding treatments inactivated the viral genome without significant loss of infectivity. These treatments were a modification from a previously published method designed to inactivate adenovirus particles (44Cotten M. Saltik M. Kursa M. Wagner E. Maass G. Birnstiel M.L. Virology. 1994; 205: 254-261Crossref PubMed Scopus (76) Google Scholar, 45Cotten M. Wagner E. Zatloukal K. Phillips S. Curiel D.T. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6094-6098Crossref PubMed Scopus (306) Google Scholar). 8-Methoxypsoralen was removed from the virus preparation by G25 gel filtration chromatography. Adenovirus (1.4 × 1011 particles) was mixed with 160 μl of 10 mg/ml poly-l-lysine and 6.5 μl of 80 mm 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a sterile polystyrene tube in a final volume of 4 ml of HEPES-buffered saline and incubated for 4 h on ice with mixing every 30 min. The coupled virus was brought to a final concentration of 1.35 g/ml CsCl. The adenovirus was centrifuged at 150,000 × g for 5 h at 10 °C, and the adenovirus band was collected and dialyzed against HEPES-buffered saline for 16 h at 4 °C. Viral DNA was quantitated by measuring the optical density at 260 nm. Briefly, 25 μl of purified virus was mixed with 465 μl of phosphate-buffered saline and 10 μl of 5% SDS. The mixture was vortexed for 2 min and then centrifuged for 2 min at room temperature in a microcentrifuge. The optical density of the resulting supernatant was measured at 260 nm (1 optical density unit equals approximately 1 × 1012 virus particles/ml). Cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Twenty-four hours before transfection, cells were plated in 6-well plates at a density of 1 × 105 cells per well in the same medium. Before transfection, cells were rinsed with Hank's balanced salt solution, and 2 ml of DMEM was added to each well. DNA and polylysine-coupled viruses were incubated for 30 min at room temperature, and additional polylysine (polylysine/DNA ratio is 1:1.3) was added. The DNA-virus-polylysine complex was incubated at room temperature for another 30 min. During the incubations, the DNA-virus complex was kept in the dark. After the incubations, the complex was added dropwise to the cells. Cell were incubated with DNA-virus complex for 2 h. Then 2 ml of DMEM supplemented with 10% stripped FBS was added to each well. On the next day, progesterone and compounds such as 8-bromo-cAMP at the indicated concentrations were added to the cells, and the cells were incubated for an additional 24 h before being harvested and assayed for CAT activity. For CAT assays, cells were harvested by scraping and subsequently lysed by three cycles of freeze-thawing. The protein concentrations were determined using the Bio-Rad protein assay reagent according to the manufacturer's protocols. Equal amounts of protein (usually 5–10 μg) were then used for a liquid CAT assay that has been described (46Brian S. Sheen J. Gene (Amst.). 1988; 67: 271-277Crossref PubMed Scopus (830) Google Scholar) and used in our previous studies (15Bai W. Tullos S. Weigel N.L. Mol. Endocrinol. 1994; 8: 1465-1473Crossref PubMed Google Scholar, 16Bai W. Weigel N.L. J. Biol. Chem. 1996; 271: 12801-12806Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 32Zhang Y. Bai W. Allgood V.E. Weigel N.L. Mol. Endocrinol. 1994; 8: 577-584Crossref PubMed Scopus (67) Google Scholar). The samples were heated at 60 °C for 8 min before the addition of the substrates, and the reactions were terminated and extracted before the substrates become a limiting factor to ensure the accurate determination of the CAT activity. Duplicate samples were assayed for each data point. CV-1 monkey kidney cells were plated on 150-mm dishes (2 × 106 cells/dish) and cultured at 37 °C with 5% CO2 in DMEM with 10% FBS that had been stripped of steroid hormones by treatment with dextran-coated charcoal. After 24 h in culture, cells were subjected to adenovirus infection (virus to cell ratio, 750:1) with 0.2 μg of cPRA expression vector/plate and cultured for an additional 24 h. To remove endogenous phosphate pools from the cells, the culture medium was removed, and phosphate-free DMEM was added to the cells for 1 h at 37 °C. Subsequently, this medium was removed, and cells were cultured in phosphate-free DMEM with 1% dialyzed, stripped FBS. 6 mCi of [32P]H3PO4(2 mCi/ml) was added to each plate of CV-1 cells, and cells were cultured for 1 h at 37 °C. Treatment was as follows. For 10-h treatments, either 8-bromo-cAMP (final concentration 2 mm) or progesterone (final concentration 10−8m) was added to cell cultures 1 h after [32P]H3PO4 addition, and cells were cultured for 10 h prior to harvest. For 30-min treatments, 8-bromo-cAMP (final concentration 2 mm) was added to cell cultures 10.5 h after [32P]H3PO4 addition, and cells were cultured for 30 min prior to harvest. cPR protein was purified and subjected to trypsin digestion and HPLC analysis as described previously (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Briefly, cells were scraped from the plates in phosphate-buffered saline and centrifuged at 500 × g for 10 min at 4 °C. Cell pellets were resuspended in homogenization buffer (50 mmpotassium phosphate, pH 7.4, 10 mm sodium molybdate, 50 mm sodium fluoride, 2 mm EDTA, 2 mmEGTA, 0.4 m sodium chloride, 5 mmα-monothioglycerol) (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar) with 1% Triton X-100 and vortexed for 30 s. Following centrifugation at 100,000 × g for 30 min at 4 °C, 3 volumes of homogenization buffer without Triton X-100 was added to the supernatant to reduce the detergent concentration. The receptor was then purified by passing the entire supernatant over a 1-ml PR22-Protein-A-Sepharose immunoaffinity column as described previously (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar). Purified receptor was electrophoresed on a 6.5% SDS-PAGE gel, and the wet gel was exposed to X-AR film (Eastman Kodak) for 2–4 h at 4 °C. The phosphorylated receptor band was cut out of the gel, and the gel slice was digested with trypsin to prepare the tryptic cPRA peptides (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar). HPLC analysis of tryptic peptides was performed as described previously (8Denner L.A. Schrader W.T. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1990; 265: 16548-16555Abstract Full Text PDF PubMed Google Scholar). Briefly, phosphopeptides were loaded onto a C-18 reverse phase HPLC column in HPLC-grade H20 containing 0.1% trifluoroacetic acid. Phosphopeptides were separated by elution from the C-18 column using a 0–45% gradient of acetonitrile containing 0.1% trifluoroacetic acid over a period of 90 min. Purified cPRA was analyzed by SDS-PAGE and transferred to a nitrocellulose membrane. The receptor was detected using the monoclonal antibody, PR22, followed by chemiluminescent detection using enhanced chemiluminescence reagent (Amersham Corp.) as described (47Weigel N.L. Carter T.H. Schrader W.T. O'Malley B.W. Mol. Endocrinol. 1992; 6: 8-14PubMed Google Scholar). To determine the molecular mechanism of the ligand-independent activation of cPR by 8-bromo-cAMP, we individually mutated each of the four known phosphorylation sites to alanine, and the transcriptional activity of each of the four single-site cPRA mutants was analyzed and compared with the wild type receptor after treatment with either 2 mm8-bromo-cAMP or 10 nm progesterone. As shown in Fig.1, the transcriptional activity of the Ala211 and Ala260 mutants in response to either progesterone or 8-bromo-cAMP is significantly lower than that of the wild type. As reported previously (15Bai W. Tullos S. Weigel N.L. Mol. Endocrinol. 1994; 8: 1465-1473Crossref PubMed Google Scholar), the activity of Ala530 is not statistically different from that of the wild type at this hormone concentration. The mutation of Ser367to alanine appears not to affect the transcriptional activity of the receptor. From Fig. 1, it is obvious that the transcriptional activity of Ala211 and Ala260 in response to 8-bromo-cAMP is affected by the mutations to a magnitude similar to the activity in response to progesterone, suggesting that the receptor phosphorylation at these two sites can regulate the ligand-independent activation. The fact that all four mutants can still be activated by 8-bromo-cAMP suggests that phosphorylation at any of the individual sites is not absolutely required for the ligand-independent activation. However, this experiment did not rule out the possibility that phosphorylation of cPR at the remaining sites compensates for the loss at one position in mediating the receptor activation. To investigate the possibility that phosphorylations at the four known sites compensate for one another during the ligand-independent activation of the receptor, all four sites were mutated simultaneously to alanines, and the activity of this quadruple mutant (AAAA) was compared with that of the wild type after stimulation with either 8-bromo-cAMP or 10 nm progesterone. As shown in" @default.
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• W2053308116 date "1997-04-01" @default.
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• W2053308116 title "Differential Phosphorylation of Chicken Progesterone Receptor in Hormone-dependent and Ligand-independent Activation" @default.
• W2053308116 cites W139202857 @default.
• W2053308116 cites W1482969059 @default.
• W2053308116 cites W1484666375 @default.
• W2053308116 cites W1520299947 @default.
• W2053308116 cites W1539653470 @default.
• W2053308116 cites W1540224528 @default.
• W2053308116 cites W1567716082 @default.
• W2053308116 cites W1602722523 @default.
• W2053308116 cites W1843244908 @default.
• W2053308116 cites W1988679331 @default.
• W2053308116 cites W1997514802 @default.
• W2053308116 cites W2003347015 @default.
• W2053308116 cites W2003580491 @default.
• W2053308116 cites W2006602286 @default.
• W2053308116 cites W2008494281 @default.
• W2053308116 cites W2008822380 @default.
• W2053308116 cites W2009558707 @default.
• W2053308116 cites W2012945894 @default.
• W2053308116 cites W2014155618 @default.
• W2053308116 cites W2016930558 @default.
• W2053308116 cites W2021828774 @default.
• W2053308116 cites W2024291973 @default.
• W2053308116 cites W2024803067 @default.
• W2053308116 cites W2050712584 @default.
• W2053308116 cites W2058354524 @default.
• W2053308116 cites W2060903877 @default.
• W2053308116 cites W2066855267 @default.
• W2053308116 cites W2068025089 @default.
• W2053308116 cites W2068113812 @default.
• W2053308116 cites W2071741297 @default.
• W2053308116 cites W2072787342 @default.
• W2053308116 cites W2072832822 @default.
• W2053308116 cites W2077644752 @default.
• W2053308116 cites W2079128738 @default.
• W2053308116 cites W2085124463 @default.
• W2053308116 cites W2091472325 @default.
• W2053308116 cites W2092950196 @default.
• W2053308116 cites W2097246067 @default.
• W2053308116 cites W2099558657 @default.
• W2053308116 cites W2116889329 @default.
• W2053308116 cites W2128840922 @default.
• W2053308116 cites W2129289858 @default.
• W2053308116 cites W2131474904 @default.
• W2053308116 cites W2135564507 @default.
• W2053308116 cites W2141109722 @default.
• W2053308116 cites W2143241514 @default.
• W2053308116 cites W2147427274 @default.
• W2053308116 cites W2160402926 @default.
• W2053308116 cites W2343796556 @default.
• W2053308116 cites W4246404158 @default.
• W2053308116 cites W49585494 @default.
• W2053308116 cites W8163034 @default.
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