Estradiol-induced Phosphorylation of Serine 118 in the Estrogen Receptor Is Independent of p42/p44 Mitogen-activated Protein Kinase
1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês
10.1074/jbc.273.21.13317
ISSN1083-351X
AutoresPeteranne B. Joel, Abdulmaged M. Traish, Deborah A. Lannigan,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoPhosphorylation of Ser118 of human estrogen receptor α (ER) enhances ER-mediated transcription and is induced by hormone binding and by activation of the mitogen-activated protein kinase (MAPK) pathway. We discovered that phosphorylation of Ser118 reduces the electrophoretic mobility of the ER. Using this mobility shift as an assay, we determined the in vivo stoichiometry and kinetics of Ser118 phosphorylation in response to estradiol, ICI 182,780, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA). In human breast cancer MCF-7 cells, estradiol induced a steady state phosphorylation of Ser118 within 20 min with a stoichiometry of 0.67 mol of phosphate/mol of ER. Estradiol did not activate p42/p44 MAPK, and basal p42/p44 MAPK activity was not sufficient to account for phosphorylation of Ser118 in response to estradiol. In contrast, both EGF and PMA induced a rapid, transient phosphorylation of Ser118 with a stoichiometry of ~0.25, and the onset of Ser118 phosphorylation correlated with the onset of p42/p44 MAPK activation by these agents. Either the EGF- or PMA-induced Ser118 phosphorylation could be inhibited without influencing estradiol-induced Ser118phosphorylation. The data suggest that a kinase other than p42/p44 MAPK is involved in the estradiol-induced Ser118phosphorylation. We propose that the hormone-induced change in ER conformation exposes Ser118 for phosphorylation by a constitutively active kinase. Phosphorylation of Ser118 of human estrogen receptor α (ER) enhances ER-mediated transcription and is induced by hormone binding and by activation of the mitogen-activated protein kinase (MAPK) pathway. We discovered that phosphorylation of Ser118 reduces the electrophoretic mobility of the ER. Using this mobility shift as an assay, we determined the in vivo stoichiometry and kinetics of Ser118 phosphorylation in response to estradiol, ICI 182,780, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA). In human breast cancer MCF-7 cells, estradiol induced a steady state phosphorylation of Ser118 within 20 min with a stoichiometry of 0.67 mol of phosphate/mol of ER. Estradiol did not activate p42/p44 MAPK, and basal p42/p44 MAPK activity was not sufficient to account for phosphorylation of Ser118 in response to estradiol. In contrast, both EGF and PMA induced a rapid, transient phosphorylation of Ser118 with a stoichiometry of ~0.25, and the onset of Ser118 phosphorylation correlated with the onset of p42/p44 MAPK activation by these agents. Either the EGF- or PMA-induced Ser118 phosphorylation could be inhibited without influencing estradiol-induced Ser118phosphorylation. The data suggest that a kinase other than p42/p44 MAPK is involved in the estradiol-induced Ser118phosphorylation. We propose that the hormone-induced change in ER conformation exposes Ser118 for phosphorylation by a constitutively active kinase. Estrogen receptor α (ER) 1The abbreviations used are: ER, estrogen receptor; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate; AF-1, hormone-independent transcriptional activation function; AF-2, hormone-dependent transcriptional activation function; CFBS, charcoal-dextran-treated fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; mAb, monoclonal antibody; cdk2, cyclin-dependent kinase 2; PP1, protein phosphatase 1; ERE, estrogen response element.1The abbreviations used are: ER, estrogen receptor; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate; AF-1, hormone-independent transcriptional activation function; AF-2, hormone-dependent transcriptional activation function; CFBS, charcoal-dextran-treated fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; mAb, monoclonal antibody; cdk2, cyclin-dependent kinase 2; PP1, protein phosphatase 1; ERE, estrogen response element. is a member of a superfamily of nuclear receptors that act as transcription factors when bound to specific lipophilic hormones (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6002) Google Scholar). In common with other steroid hormone receptors, the ER has an N-terminal domain with a hormone-independent transcriptional activation function (AF-1), a central DNA-binding domain, and a C-terminal ligand-binding domain with a hormone-dependent transcriptional activation function (AF-2) (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6002) Google Scholar, 2Weigel N. Biochem. J. 1996; 319: 657-667Crossref PubMed Scopus (321) Google Scholar). In addition to being activated by binding to its ligand, estradiol, the ER can also be activated as a transcription factor by several nonsteroidal agents (2Weigel N. Biochem. J. 1996; 319: 657-667Crossref PubMed Scopus (321) Google Scholar). Phosphorylation of the ER increases in response to estradiol (2Weigel N. Biochem. J. 1996; 319: 657-667Crossref PubMed Scopus (321) Google Scholar). A major site of estradiol-induced phosphorylation is serine 118 (Ser118), which is located in AF-1 (3Le Goff P. Montano M.M. Schodin D.J. Katzenellenbogen B.S. J. Biol. Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar, 4Ali S. Metzger D. Bornert J.-M. Chambon P. EMBO J. 1993; 12: 1153-1160Crossref PubMed Scopus (376) Google Scholar, 5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar). In a number of cell types, mutation of Ser118 to Ala causes a reduction in estradiol-dependent transcriptional activation (4Ali S. Metzger D. Bornert J.-M. Chambon P. EMBO J. 1993; 12: 1153-1160Crossref PubMed Scopus (376) Google Scholar, 6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). Ser118 is located within the sequence PPQLSPFLQ which has a high degree of homology with the optimum peptide substrate identified for p44 MAPK (9Songyang Z. Lu K.P. Kwon Y.T. Tsai L.H. Filhol O. Cochet C. Brickey D.A. Soderling T.R. Bartleson C. Graves D.J. DeMaggio A.J. Hoekstra M.F. Blenis J. Hunter T. Cantley L.C. Mol. Cell. Biol. 1996; 16: 6486-6493Crossref PubMed Scopus (484) Google Scholar). Ser118 is also phosphorylated in response to EGF and PMA, possibly via p42/p44 MAPK (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar). Transcriptional activation of the ER by EGF is diminished when Ser118 is mutated to Ala (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). Thus both estradiol binding and activation of the MAPK pathway lead to phosphorylation of Ser118, and this phosphorylation is involved in regulating ER-mediated transcription. The evidence that p42/p44 MAPK directly phosphorylates Ser118 is based on in vitro observations that activated p42/p44 MAPK phosphorylated the N-terminal domain of the ER but not a mutant of this construct in which Ser118 was replaced with Ala (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar). Additional supporting evidence is provided by the observation that in vivo a constitutively activated MAPK kinase (MAPKK) stimulated phosphorylation of the N-terminal domain of the ER but did not stimulate phosphorylation of a mutant of this construct in which Ser118 was replaced with Ala (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar). However, it has still not been established whether p42/p44 MAPK directly phosphorylates Ser118 in the context of the full-length ER in vivo. We demonstrate that the onset of Ser118 phosphorylation correlates with the onset of p42/p44 MAPK activation in response to EGF and PMA. These data are consistent with the hypothesis that p42/p44 MAPK phosphorylates Ser118. However, our data demonstrate that p42/p44 MAPK is not responsible for the observed Ser118 phosphorylation in response to estradiol binding by the ER. Our results raise the possibility that Ser118 may be the site of phosphorylation by either two or more kinases in response to various stimuli. ICI 182,780 was a gift from Zeneca Pharmaceuticals, Cheshire, United Kingdom. Pierre Chambon (Unité 184 de Biologie Moléculaire et de Génétique de I'INSERM, Strasbourg Cédex, France) kindly provided pSG5-ER (the human estrogen receptor cDNA (10Green S. Walter P. Kumar V. Krust A. Bornert J.-M. Argos P. Chambon P. Nature. 1986; 320: 134-139Crossref PubMed Scopus (1938) Google Scholar)). The mutants S118A-ER, S104/106/118A-ER, S154A-ER, and S167A-ER were created by site-directed mutagenesis of pSG5-ER using the megaprimer method (11Barik S. White B.A. Methods in Molecular Biology. Humana Press, Totowa, NJ1993: 277-286Google Scholar). The luciferase reporter plasmid, 2ERE-MpGL2, has been described (8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). pSI-β-galactosidase contains the β-galactosidase gene in the pSI vector from Promega. COS-1, MCF-7, T-47D, and NMuMG cell lines were obtained from American Type Culture Collection, Manassas, VA. All media used in this study were phenol-red free, and all fetal bovine serum used was charcoal-dextran-treated (CFBS). COS-1, T-47D, and NMuMG cells were maintained in DMEM/Ham's Nutrient Mix F-12 supplemented with 10% CFBS. COS-1 media contained 50 units/ml penicillin and 50 μg/ml streptomycin. T-47D and NMuMG cell media contained antibiotic-antimycotic (Life Technologies, Inc.). MCF-7 cells were maintained in DMEM supplemented with 5% CFBS, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were incubated 24 h in media without serum prior to treatment with steroids or other agents unless indicated otherwise. COS-1 cells were transfected by the diethylaminoethyl-dextran method as modified by Reese and Katzenellenbogen (12Reese J.C. Katzenellenbogen B.S. Nucleic Acids Res. 1991; 19: 6595-6602Crossref PubMed Scopus (114) Google Scholar). Each 6-cm plate was transfected with 0.5 μg of pSG5-ER or pSG5-ER mutant plus 2 μg of pGL2-Basic Vector (Promega) as carrier DNA. Following dimethyl sulfoxide treatment, the cells were incubated for 24 h in serum-free medium before treatment with estradiol or PMA and harvesting. Following treatment with steroids or agents as indicated in the figure legends, the cells (on 6-cm plates) were washed with warm PBS and 0.5 ml of 2× SDS-PAGE sample buffer (0.12 m Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 0.2 m DTT, 0.008% bromphenol blue) near 100 °C was then added to the plate. The cells were scraped into a 1.5-ml centrifuge tube in a heat block at 100 °C. The tubes were heated for 10 min, cooled on ice, and stored at −20 °C until samples were analyzed on SDS-PAGE. At the time of electrophoresis, samples were reheated for 5 min at 100 °C, and 30-μl aliquots were layered on 7% SDS-PAGE and electrophoresed until the 43-kDa marker reached the bottom of the gel. Proteins on the gel were transferred to nitrocellulose membranes in 25 mm Tris-HCl, pH 8.3, 192 mm glycine, and 20% methanol. For detection of the ER, immunoblotting was performed with monoclonal antibody α78–1-C6 (mAb 78) (13Traish A.M. Al-Fadhli S.M. Klinge C.M. Quick T.C. 76th Annual Meeting of The Endocrine Society. 1994; (, Anaheim, CA): 319Google Scholar) for COS-1 extracts or monoclonal antibody EVG F9 (mAb F9) (14Traish A.M. Pavao M. Steroids. 1996; 61: 549-556Crossref PubMed Scopus (13) Google Scholar) for MCF-7, T-47D, or NMuMG extracts. For detection of activated p42/p44 MAPK, immunoblotting was performed with anti-ACTIVE MAPK (Promega). For detection of total p42/p44 MAPK, immunoblotting was performed with ANTI-pan ERK (Transduction Laboratories). All immunoblots used either anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase as a secondary antibody. Antigen-antibody interactions were detected with NEN Life Science Products Western chemiluminescence reagent. Varying exposures of the film were taken. A Molecular Dynamics Personal Densitometer with ImageQuant software was used to determine the relative amount of ER in the immunoblot bands. To ensure that a linear response over the range of ER concentrations obtained from the experiments was achieved, a standard curve relating the amount of ER to the densitometric reading was performed. The relative intensities of the ER bands were quantitated from various exposures of the film. MCF-7 cells were treated for 38 min with 1 nm estradiol and then rinsed with cold PBS. The cells were lysed in buffer A (50 mm Tris-Cl, pH 8, 0.15 mNaCl, 1% Nonidet P-40 (Nonidet P-40), 20 mmNa2MoO4, 20 mm NaF, 2 mm Na3VO4, 20 mmβ-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml each of pepstatin, leupeptin, and aprotinin, 200 nmokadaic acid, 10 mm MgCl2, and 100 units/ml DNase 1 (Life Technologies, Inc.)). The ER was immunoprecipitated with mAb α78 as described (5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar). The immunoprecipitates were washed with protein phosphatase 1 (PP1) buffer (New England Biolabs), and two aliquots were suspended in a final volume of 30 μl of PP1 buffer with 1 mm MnCl2. One tube received 1 unit of PP1, and both tubes were incubated for 5 min at 30 °C. Reactions were stopped by addition of SDS-PAGE sample buffer and heating for 5 min at 100 °C. Sample proteins were analyzed on SDS-PAGE, electrotransferred, and immunoblotted as described above. For transcriptional analysis, MCF-7 cells were seeded at a density of 5 × 105 cells per 6-cm dish in DMEM containing 5% CFBS and antibiotic-antimycotic the day before transfection. Cells were transfected by 15 μl of lipofectAMINE in 2 ml of DMEM according to the instructions provided by Life Technologies, Inc. with 0.5 μg of 2ERE-MpGL2 (luciferase reporter plasmid), 1.0 μg of pSI-β-galactosidase (for normalizing luciferase data), and 1.5 μg of pGL2-Basic Vector (carrier DNA). The cells were left overnight in the incubator and washed once with PBS before the addition of DMEM with the appropriate concentration of estradiol, modulators, or vehicle. After treatment for 5.5 h, the cells were lysed with Reporter Lysis Buffer (Promega). Luciferase and β-galactosidase activity were measured as described previously (5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar). For the in-gel kinase assay, MCF-7 cells were washed with PBS and lysed with ice-cold extraction buffer (20 mm Tris-Cl, pH 7.5, 5 mm EGTA, 0.5% Triton X-100, 6 mm DTT, 50 mm β-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 1 mmNa3VO4, 10 μg/ml aprotinin and leupeptin, and 2 μg/ml pepstatin). The supernatant was cleared by centrifugation, and SDS-PAGE sample buffer was added. The extracts (10 μg of protein) were electrophoresed on 7% SDS-PAGE with 0.05 mg/ml myelin basic protein polymerized in the gel. Following electrophoresis, the proteins were denatured and renatured as described (15Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (434) Google Scholar). The gels were incubated for 30 min at 22 °C in 40 mm Hepes-NaOH, pH 8.0, 2 mm DTT, 0.1 mm EGTA, 5 mmMgCl2, 25 μm ATP (40 μCi/ml [γ-32P]ATP). The gels were washed as described (15Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (434) Google Scholar), dried, and autoradiographed. Many proteins migrate more slowly on SDS-PAGE due to phosphorylation of particular sites. Estradiol addition to cells causes the ER to migrate as a doublet on SDS-PAGE (5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar, 16Washburn T. Hocutt A. Brautigan D.L. Korach K.S. Mol. Endocrinol. 1991; 5: 235-242Crossref PubMed Scopus (63) Google Scholar). We showed that in human ER expressed in COS-1 cells the major difference in the phosphorylation of the upper and lower bands was phosphorylation of Ser118 in the upper band. Furthermore, a mutant ER in which Ser118 was replaced with Ala was phosphorylated at a level only 20% that of the wild type (5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar) and did not upshift when cells were treated with estradiol (5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar). Thus, phosphorylation of Ser118appeared to be required for the electrophoretic upshift. This electrophoretic upshift could be used to monitor the phosphorylation of Ser118, greatly facilitating the characterization of kinases that phosphorylate Ser118. Therefore, we wanted to confirm that only the phosphorylation of Ser118 was required for the electrophoretic upshift. Phosphorylation of the ER occurs only on Ser residues (3Le Goff P. Montano M.M. Schodin D.J. Katzenellenbogen B.S. J. Biol. Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar, 17Denton R.R. Koszewski N.J. Notides A.C. J. Biol. Chem. 1992; 267: 7263-7268Abstract Full Text PDF PubMed Google Scholar, 18Lahooti H. Thorsen T. Aakwaag A. Mol. Cell. Endocrinol. 1990; 74: 21-32Crossref PubMed Scopus (8) Google Scholar) with the exception of a low level of phosphorylation on Tyr537in the human ER (19Arnold S.F. Obourn J.D. Jaffe H. Notides A.C. Mol. Endocrinol. 1995; 9: 24-33Crossref PubMed Google Scholar). Le Goff et al. (3Le Goff P. Montano M.M. Schodin D.J. Katzenellenbogen B.S. J. Biol. Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar) reported that a mutant of the ER lacking the A/B region (amino acids 1–175) showed no estradiol-induced phosphorylation, indicating that the hormone-induced phosphorylation sites are predominantly in the A/B region. To determine whether phosphorylation of another Ser in the A/B region in addition to Ser118 is required for the electrophoretic upshift, we prepared mutants in which other Ser residues in the A/B region known or suspected to be phosphorylated were replaced by Ala residues (2Weigel N. Biochem. J. 1996; 319: 657-667Crossref PubMed Scopus (321) Google Scholar). Ser-104 and Ser-106 were replaced with Ala residues to create the mutant S104/106A-ER. Ser-118, Ser-154, and Ser-167 were individually mutated to Ala to create the mutants S118A-ER, S154A-ER, and S167A-ER. The wild type and mutant ERs were transfected into COS-1 cells. Cell lysates were immunoblotted with an anti-ER antibody. Data displayed in Fig. 1, lanes 1–4 show that after subtraction of the control, estradiol converted 30% of the wild type ER to the upshifted form, PMA converted about 65% of the receptor, and estradiol plus PMA converted 83% of the receptor. In contrast, Fig. 1, lanes 5–8 show that neither estradiol nor PMA caused a change in mobility with the mutant S118A-ER. All of the other mutants, S167A-ER (lanes 9–12), S154A-ER (lanes 13–16), and S104/106A-ER (lanes 17–20) responded to estradiol and PMA similarly to the wild type. Thus, the change in mobility of the ER did not require phosphorylation of Ser residues 104, 106, 154, or 167. To determine whether estradiol causes phosphorylation of Ser118 in endogenous ER in mammary cell lines, MCF-7 (human breast carcinoma), NMuMG (mouse mammary gland normal epithelium), and T-47D (human breast duct carcinoma, pleural effusion), cells were incubated for 30 min with estradiol or vehicle. Estradiol induced an electrophoretic upshift of the ER in all three mammary cell lines (Fig.2 A,immunoblot). To confirm that the upshift was due to phosphorylation, MCF-7 cells were treated with estradiol, and the ER was immunoprecipitated. Aliquots of the immunoprecipitate were incubated with and without PP1. ER incubated without PP1 (Fig. 2 B, lane 1) had 35% of the ER upshifted, whereas only 3% of the ER was upshifted (lane 2) in ER incubated with PP1. The amount of ER in thelower band in lane 2 is equivalent to the amount of ER in the two bands in lane 1 combined, indicating that dephosphorylation of the ER in the upper band converted it into the lower band. The fraction of ER recovered in the upper band following immunoprecipitation was less than when the cells were treated directly with hot SDS sample buffer (Fig. 2 A), presumably due to the action of phosphatases during immunoprecipitation. These results together with the data with mutants confirm that Ser118 was phosphorylated in endogenous ER in human breast cells and most likely on the analogous Ser122 in mouse mammary cells. To determine the dose response of Ser118 phosphorylation to estradiol in vivo, MCF-7 cells were treated with increasing concentrations of estradiol. Estradiol-induced phosphorylation of Ser118 was maximal at approximately 1 nm estradiol, with an average of 67% of the receptor phosphorylated on Ser118 (Fig.3). Thus the stoichiometry of Ser118 phosphorylation is higher in cells that normally express ER as compared with heterologously expressed ER in COS-1 cells. To determine whether ICI 182,780 induces Ser118phosphorylation, MCF-7 cells were treated with various concentrations of ICI 182,780. Interestingly, although ICI 182,780 is a pure ER antagonist (20Gibson M.K. Nemmers L.A. Beckman Jr., W.C. Davis V.L. Curtis S.W. Korach K.S. Endocrinol. 1991; 129: 2000-2010Crossref PubMed Scopus (182) Google Scholar), 30 nm of this compound induced phosphorylation of Ser118 to the same extent as estradiol (Fig. 3). We found that ICI 182,780 caused a rapid decrease in total ER protein, an observation in agreement with reports for ICI 164,384 (20Gibson M.K. Nemmers L.A. Beckman Jr., W.C. Davis V.L. Curtis S.W. Korach K.S. Endocrinol. 1991; 129: 2000-2010Crossref PubMed Scopus (182) Google Scholar, 21Dauvois S. Danielian P.S. White R. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4037-4041Crossref PubMed Scopus (423) Google Scholar). Treatment of MCF-7 cells with 10 nm estradiol for 1 h did not significantly alter the total amount of ER protein, whereas treatment with 10 nm ICI 182,780 for 1 h reduced the total amount of ER protein by 70% compared with the control level (data not shown). Ser118 is also phosphorylated in response to EGF and PMA, possibly via p42/p44 MAPK (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). Therefore, we wanted to compare the time course and stoichiometry of Ser118 phosphorylation in response to both EGF and PMA as compared with estradiol. Within 5 min of estradiol treatment, phosphorylation of Ser118 was already noticeable (Fig.4 A) and was maximal within 20 min, remaining at that maximal level at 60 min. In response to EGF, approximately 24% of the receptor was phosphorylated on Ser118, and this maximal level was reached within 5 min after addition of ligand (Fig. 4 B). In response to PMA, approximately 26% of the receptor was phosphorylated on Ser118, and phosphorylation reached a maximum level within 10 min (Fig. 4 C). Thus, the extent and time course of induction of Ser118 phosphorylation differed considerably between estradiol, EGF, and PMA. Estradiol causes a rapid but prolonged phosphorylation, whereas EGF and PMA cause rapid and transient phosphorylation of Ser118. Next, to ensure that we were studying Ser118phosphorylation under conditions in which EGF and PMA influence ER-mediated transcription, we determined the effect of both EGF and PMA on ER-dependent transcription in MCF-7 cells. These cells were cotransfected with a reporter construct that contained a luciferase gene regulated by two palindromic estrogen response elements (ERE) and a plasmid that constitutively expresses β-galactosidase. This latter construct allows the luciferase data to be normalized for transfection efficiency. In parallel experiments, the cells were cotransfected with a reporter construct that did not contain the EREs. These experiments were necessary to eliminate transcriptional responses that were independent of the ERE. Following transfection, the cells were treated for 5.5 h with PMA, EGF, or vehicle with or without estradiol. PMA and EGF caused a small steroid-independent activation of transcription from the ERE-containing reporter plasmid of 1.9- and 1.8 times, respectively (Fig. 5). Estradiol alone stimulated transcription by 5.5 times. Adding PMA and estradiol together resulted in 11.7 times activation, indicating that their effects are synergistic. A combination of EGF and estradiol modestly increased transcription by 1.2 times compared with estradiol alone. Thus ligand-dependent and -independent activation of ER-dependent transcription occurs in MCF-7 cells. It has been reported that estradiol can stimulate signal transduction pathways that lead to p42/p44 MAPK activation in MCF-7 cells (22Migliaccio A. Di Domenico M. Castoria G. de Falco A. Bontempo P. Nola E. Aurricchio F. EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (857) Google Scholar). To investigate the possibility that p42/p44 MAPK was responsible for Ser118 phosphorylation in response to estradiol, serum-starved MCF-7 cells were treated with either estradiol, EGF, or PMA, and Ser118 phosphorylation and p42/p44 MAPK activation were determined. As Fig.6 A shows, the onset of Ser118 phosphorylation paralleled the onset of activation of p42/p44 MAPK by EGF. The activated form of p42/p44 MAPK was detected by using an antibody that specifically recognizes the dually phosphorylated form of p42/p44 MAPK (23Anderson N.G. Maller J.L. Tonks N.K. Sturgill T.W. Nature. 1990; 343: 651-653Crossref PubMed Scopus (791) Google Scholar). The rapid onset of phosphorylation of Ser118 also paralleled the onset of p42/p44 MAPK activation by PMA. However, activation of p42/p44 MAPK was not observed in response to estradiol. The immunoblot with an antibody that recognizes both activated and unactivated forms of p42/p44 MAPK demonstrated that approximately equal levels of p42/p44 MAPK were present in each lane. In other experiments, extended exposure of the anti-active MAPK immunoblot demonstrated the presence of basal p42/p44 MAPK activity. However, this low level of activity was not enhanced by estradiol (Fig. 7 A).Figure 7The inhibitors PD98059, staurosporine, and GF109203X prevent Ser118 phosphorylation induced by either PMA or EGF but not that induced by estradiol. Serum-starved MCF-7 cells were used in all experiments. In panel A,cells were pretreated with 100 μm PD98059 or vehicle for 30 min. Then either 1 nm estradiol or vehicle was added for 20 min, or 100 ng/ml EGF or vehicle was added for 5 min. Inpanel B, cells were pretreated 30 min with 5 μm staurosporine or vehicle. Then 1 nmestradiol, 100 nm PMA, or vehicle was added for 15 min. Inpanel C, cells were pretreated for 30 min with 10 μm GF109203X or vehicle. Then 1 nm estradiol, 100 nm PMA, or vehicle was added for 15 min. Lysates were analyzed as described in Fig. 6. The percent upshift of the ER was determined from immunoblots by densitometry. The means ± S.D. are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As an alternative approach to determine whether estradiol activates p42/p44 MAPK, an in-gel kinase assay was performed using extracts from MCF-7 cells that had been treated with estradiol, PMA, EGF, or vehicle. The substrate embedded in the gel was myelin basic protein. Both PMA and EGF stimulated a protein kinase of ~42 kDa (Fig. 6 B,lanes 1, 3, and 5 versus 4 and6), which indicates that p42/p44 MAPK was active in the in-gel kinase assay. However, estradiol did not stimulate p42/p44 MAPK or any other protein kinases to phosphorylate myelin basic protein (Fig. 7 B, lanes 1, 3, and 5 versus 2). Thus two different approaches both indicated that estradiol did not activate p42/p44 MAPK. The above results suggest that activation of p42/p44 MAPK may be involved in the phosphorylation of Ser118 in response to EGF and PMA but not in response to estradiol. To further test this hypothesis we compared the effects of various inhibitors on either the EGF- or PMA-induced Ser118phosphorylation versus the estradiol-induced Ser118 phosphorylation. Fig. 7 A shows that PD98059, an inhibitor of MAPK-activating enzyme (MEK1/2) (24Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2582) Google Scholar), diminished the EGF-induced Ser118 phosphorylation but did not affect the estradiol-induced Ser118 phosphorylation. Extended exposure of the anti-active MAPK immunoblot showed faint bands corresponding to active p42 MAPK and p44 MAPK. This basal activity of p42/p44 MAPK diminishes in extracts taken from cells pretreated with PD98059 before addition of estradiol or EGF. Estradiol did not increase p42/p44 MAPK activity above the basal level. Importantly, even though PD98059 was able to diminish basal p42/p44 MAPK activity, PD98059 did not effect estradiol-induced Ser118 phosphorylation. It has been suggested that there are two MEK1/2-independent pathways for activating p42/p44 MAPK; one pathway involves PKC, and the other pathway involves phosphatidylinositol 3′-kinase (25Grammer T.C. Blenis J. Oncogene. 1997; 14: 1635-1642Crossref PubMed Scopus (176) Google Scholar). Both staurosporine and GF109203X, inhibitors of PKC (26Meijer L. Trends Cell. Biol. 1996; 6: 393-397Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perrett T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar), completely prevented phosphorylation of Ser118 in response to PMA but had no effect on Ser118 phosphorylation induced by estradiol (Fig. 7, B and C). Pretreatment of MCF-7 cells for 30 min with 100 nm wortmannin, conditions shown to inhibit phosphatidylinositol 3′-kinase (28Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar), did not reduce estradiol-induced Ser118 phosphorylation (data not shown). An inhibitor combination of GF109203X, PD98059, and wortmannin also did not affect estradiol-induced Ser118 phosphorylation (data not shown). Pretreatment of MCF-7 cells for 30 min with 20 μm SB203580, conditions shown to inhibit the activation of p38 MAPK (29Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS. 1995; 364: 229-233Crossref PubMed Scopus (1971) Google Scholar), had no effect on the estradiol-induced Ser118 phosphorylation (data not shown). These results provide additional evidence that estradiol does not activate a signal transduction pathway that leads to p42/p44 MAPK or p38 MAPK activation. Of interest to note is that Trowbridge et al. (30Trowbridge J.M. Rogatsky I. Garabedian M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10132-10137Crossref PubMed Scopus (80) Google Scholar) have suggested that the cyclin A·cyclin-dependent kinase 2 (cdk2) complex phosphorylates the ER. However, staurosporine, which inhibits a number of kinases including PKC and cdk2 at similar concentrations (26Meijer L. Trends Cell. Biol. 1996; 6: 393-397Abstract Full Text PDF PubMed Scopus (189) Google Scholar), had no affect on estradiol-induced phosphorylation (Fig. 7 C). Moreover, pretreatment of MCF-7 cells for 30 min with 100 μm olomoucine, a condition that inhibits cdk1 and cdk2 (31VeselyÖ J. Havliček L. Strnad M. Blow J.J. Donella-Deana A. Pinna L. Letham D.S. Kato J.-Y. Detivaud L. Leclerc S. Meijer L. Eur. J. Biochem. 1994; 224: 771-786Crossref PubMed Scopus (608) Google Scholar), also did not affect estradiol-induced Ser118phosphorylation (data not shown). These data do not support a model in which cdk2 is the kinase responsible for Ser118phosphorylation in response to estradiol binding by the ER. Our results demonstrate that p42/p44 MAPK is not activated in response to estradiol. It could be argued, however, that the estradiol-bound receptor is an extremely good substrate for p42/p44 MAPK and that even the activity of p42/p44 MAPK in the presence of PD98059 is sufficient for 67% of the ER molecules to be phosphorylated in response to estradiol. To test this hypothesis, we determined whether increasing p42/p44 MAPK activity would enhance estradiol-induced Ser118 phosphorylation. Increasing amounts of EGF were added to MCF-7 cells that had been pretreated with a high concentration of estradiol for an extended period of time. This pretreatment was performed to ensure that ER binding to estradiol was not rate-limiting for Ser118 phosphorylation. The lowest dose of EGF did not effect estradiol-induced Ser118 phosphorylation even though p42/p44 MAPK activity was significantly increased over the basal level (Fig. 8). Higher levels of EGF which dramatically enhanced p42/p44 MAPK activity only increased Ser118 phosphorylation to the same extent as EGF alone and did not induce 100% of the estradiol-bound ER molecules to be phosphorylated (Fig. 8). Phosphorylation of Ser118 is involved in regulating the transcriptional activity of AF-1 in response to either estradiol or EGF (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). Although Ser118 had been identified as a putative substrate for p42/p44 MAPK, no information concerning the stoichiometry and kinetics of Ser118 phosphorylation in response to estrogen agonists or antagonists, EGF, or PMA was known. We developed a convenient and quantitative electrophoretic mobility assay to monitor specifically the in vivo phosphorylation of Ser118. Using this assay, we provide the first evidence that phosphorylation of Ser118 occurs in human breast cancer cells, a physiologically relevant target tissue. The rapidity of Ser118 phosphorylation in response to estradiol suggests that the phosphorylation is a direct response to hormone binding. Le Goff et al. (3Le Goff P. Montano M.M. Schodin D.J. Katzenellenbogen B.S. J. Biol. Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar) have shown that ICI 164,384 increases phosphorylation of the ER but they did not determine the sites of phosphorylation. Interestingly, we have found that both estradiol and ICI 182,780 induce Ser118 phosphorylation to a similar extent. The receptor is known to have a different conformation after it binds agonist as opposed to antagonist (32Beekman J.M. Allan G.F. Tsai S.Y. Tsai M.-J. O'Malley B.W. Mol. Endocrinol. 1993; 7: 1266-1274PubMed Google Scholar). However, the conformational difference induced by estradiol and ICI 182,780 does not seem to affect the N-terminal region of the receptor. Although EGF and PMA only transiently induce Ser118phosphorylation, these agents are able to stimulate ER-mediated transcription. In agreement with published reports, we observed that EGF and PMA with or without estradiol enhanced ER-mediated transcription (7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar, 33Ignar-Trowbridge D.M. Teng C.T. Ross K.A. Parker M.G. Korach K.S. McLachlan J.A. Mol. Endocrinol. 1993; 7: 992-998Crossref PubMed Scopus (304) Google Scholar, 34Cho H. Katzenellenbogen B.S. Mol. Endocrinol. 1993; 7: 441-452Crossref PubMed Scopus (187) Google Scholar, 35Ignar-Trowbridge D.M. Pimentel M. Parker M.G. McLachlan J.A. Korach K.S. Endocrinol. 1996; 137: 1735-1744Crossref PubMed Scopus (0) Google Scholar). Phosphorylation of Ser118appears to be important for ER-mediated transcription since a mutant ER in which Ser118 is replaced with Ala has a diminished transcriptional ability compared with the wild type ER (4Ali S. Metzger D. Bornert J.-M. Chambon P. EMBO J. 1993; 12: 1153-1160Crossref PubMed Scopus (376) Google Scholar, 6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). One possible model to explain the mechanism by which EGF and PMA can enhance ER-mediated transcription is that transient Ser118phosphorylation is sufficient to induce a cascade of events that eventually lead to transcriptional activation. Of interest to note is that a combination of EGF and estradiol additively enhance ER-mediated transcription, whereas a combination of PMA and estradiol synergistically effect transcription. It is possible that EGF only enhances Ser118 phosphorylation, whereas PMA can also influence another step in ER-mediated transcription. Thus the effects of EGF and estradiol are additive because both agents are influencing the same event, whereas the effects of PMA and estradiol are synergistic because these agents are influencing multiple steps that are involved in ER-mediated transcription. Our results with EGF and PMA are consistent with Ser118being a substrate for activated p42/p44 MAPK (6Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Susaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1694) Google Scholar, 7Bunone G. Briand P.-A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (842) Google Scholar). In contrast to our results with EGF and PMA, we obtained three different lines of evidence that p42/p44 MAPK is not the kinase responsible for the estradiol-induced Ser118 phosphorylation. First, activation of p42/p44 MAPK activity was not detectable in serum-starved MCF-7 cells treated with estradiol. These results are in disagreement with the work of Miagliaccio et al. (22Migliaccio A. Di Domenico M. Castoria G. de Falco A. Bontempo P. Nola E. Aurricchio F. EMBO J. 1996; 15: 1292-1300Crossref PubMed Scopus (857) Google Scholar). Second, basal level of p42/p44 MAPK is not sufficient to account for the estradiol-induced Ser118 phosphorylation. This reasoning is based on the observation that decreasing the basal p42/p44 MAPK activity with PD98059 had no effect on estradiol-induced Ser118phosphorylation. Furthermore, if basal p42/p44 MAPK activity was responsible for the estradiol-induced Ser118phosphorylation, then elevation of p42/p44 MAPK activity should result in 100% of the estradiol-bound ER molecules being phosphorylated. This result was not observed. Third, inhibitors that diminished either the EGF or PMA enhancement of p42/p44 MAPK activity and induction of Ser118 phosphorylation did not affect the estradiol-induced phosphorylation. Taken together, our results strongly suggest thatin vivo Ser118 of the human ER is a target for two or more kinases that are regulated differentially by second messengers. Trowbridge et al. (30Trowbridge J.M. Rogatsky I. Garabedian M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10132-10137Crossref PubMed Scopus (80) Google Scholar) reported that cdk2 enhances both ligand-dependent and -independent transcription by ER. Anin vitro kinase assay demonstrated that Ser104and Ser106 of the human ER were the likely targets of cdk2 activity. However, we did not observe any effect of the inhibitors staurosporine and olomoucine which inhibit cdk2 on estradiol-induced Ser118 phosphorylation. Like Ser118, Ser104 and Ser106 are adjacent to proline residues. In our 32P-labeling experiments, Ser104 and Ser106 were minor sites of phosphorylation compared with Ser118 (5Joel P.B. Traish A.M. Lannigan D.A. Mol. Endocrinol. 1995; 9: 1041-1052Crossref PubMed Google Scholar, 8Joel P. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (308) Google Scholar). This paper provides the first evidence that more than one kinase regulates Ser118 phosphorylation of the human ER. One simple model to explain the estradiol-induced phosphorylation of Ser118 is that, upon hormone binding, the ER undergoes a conformational change that makes the ER a better substrate for a constitutively active kinase. Phosphorylation of Ser118 may allow the ER to integrate signals from hormone binding and activation of the MAPK pathway. We thank Danielle Desrosiers for excellent technical assistance. We thank David L. Brautigan, Ian G. Macara, and T. W. Sturgill for helpful discussions.
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