Growth Factors Signal to Steroid Receptors through Mitogen-activated Protein Kinase Regulation of p160 Coactivator Activity

Abstract

Promoter-bound steroid receptors activate gene expression by recruiting members of the p160 family of coactivators. Many steroid receptors, most notably the progesterone and estrogen receptors, are regulated both by cognate hormone and independently by growth factors. Here we show that epidermal growth factor regulates the activities of the p160 GRIP1 through the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases. ERKs phosphorylate GRIP1 at a specific site, Ser-736, the integrity of which is required for full growth factor induction of GRIP1 transcriptional activation and coactivator function. We propose that growth factors signal to nuclear receptors in part by targeting the p160 coactivators. Promoter-bound steroid receptors activate gene expression by recruiting members of the p160 family of coactivators. Many steroid receptors, most notably the progesterone and estrogen receptors, are regulated both by cognate hormone and independently by growth factors. Here we show that epidermal growth factor regulates the activities of the p160 GRIP1 through the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases. ERKs phosphorylate GRIP1 at a specific site, Ser-736, the integrity of which is required for full growth factor induction of GRIP1 transcriptional activation and coactivator function. We propose that growth factors signal to nuclear receptors in part by targeting the p160 coactivators. estrogen receptor progesterone receptor ligand binding domain epidermal growth factor extracellular signal-regulated kinase mitogen-activated protein high pressure liquid chromatography green fluorescent protein mitogen-activated protein kinase/extracellular signal-regulated kinase kinase c-Jun NH2-terminal kinase glutathioneS-transferase luciferase DNA binding domain estrogen response element progesterone response element wild type transforming growth factor CREB-binding protein glucocorticoid receptor interacting protein 1 Nuclear receptors such as the estrogen receptor (ER)1 and progesterone receptor (PR) tether via their DNA binding domain to response elements in the promoter region of target genes and stimulate transcription. To do so the receptors must bind to coactivators that they recruit through transcriptional activation functions, the constitutive AF-1, found in the amino-terminal receptor domain, and the hormone-activated AF-2 in the carboxyl ligand binding domain (LBD) (for review, see Ref. 1Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (815) Google Scholar). Perhaps the most important of these coactivators is the p160 family, SRC-1 (N-CoA1), GRIP1 (TIF2/N-CoA2), and ACTR (pCIP/AIB1/RAC3). These bind to the LBD only in the presence of cognate hormone, and their binding is blocked by antagonist ligands. The mechanism of binding is now understood in atomic detail and involves the docking of coactivator nuclear receptor boxes, which have the motif LXXLL with a hydrophobic cleft that forms on the surface of the hormone-bound LBD (2Feng W. Ribeiro R.C. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. 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Feng W. Nguyen M.P. Chen D. Huang S.M. Subramanian S. McKinerney E. Katzenellenbogen B.S. Stallcup M.R. Kushner P.J. Mol. Endocrinol. 1998; 12: 1605-1618Crossref PubMed Scopus (0) Google Scholar, 8Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar). The p160s are complex proteins with multiple domains (Fig. 1). In addition to the nuclear receptor boxes they have two intrinsic transcriptional activation domains AD1 and AD2, whose activities may be monitored when the coactivators are directly tethered on DNA via fusion to a heterologous DNA binding domain (8Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar, 9Voegel J.J. Heine M.J. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO J. 1998; 17: 507-519Crossref PubMed Scopus (432) Google Scholar). AD1, which is essential for transcriptional mediation by p160s, is coextensive with the binding domain for the CBP/p300 family of coactivators. CBP/p300 complexes with the p160s and synergizes in coactivator function (10Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar,11Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1108) Google Scholar). In particular, CBP/p300s contain a potent acetyltransferase activity that can transfer acetate from acetyl-CoA to histones and also to other proteins in the complex on DNA (12Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1535) Google Scholar, 13Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. 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AD2 contributes to coactivation by p160s in some circumstances and does so in part by binding CARM1 and other proteins that have histone methyltransferase activity (19Chen D. Ma H. Hong H. Koh S.S. Huang S.M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2177Crossref PubMed Scopus (1006) Google Scholar). The coactivators are believed to mediate transcriptional activation by remodeling chromatin through their histone modification activities and also by direct effects on the transcriptional complex. In addition to regulating AF-2, hormones regulate steroid receptors (but not other nuclear receptors) in part by releasing the receptors from inhibitory complexes with heat shock proteins. The activity of steroid receptors is not regulated solely by hormones, however. Growth factors, such as EGF and insulin-like growth factor 1, can have profound and surprising effects on steroid receptors, even in the absence of cognate hormone (for review, see Ref. 20Weigel N.L. Zhang Y. J. Mol. Med. 1998; 76: 469-479Crossref PubMed Scopus (283) Google Scholar). In the most dramatic examples, EGF activates progesterone receptors almost as well as does hormone (21Bai W. Rowan B.G. Allgood V.E. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1997; 272: 10457-10463Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and will partly activate ER in the absence of hormone and enhance activity in the presence of hormone (22Kato S. Kitamoto T. Masuhiro Y. Yanagisawa J. Oncology. 1998; 55 Suppl. 1: 5-10Crossref PubMed Scopus (39) Google Scholar). This later activity has been traced in part to EGF initiation of a cascade through the ERK family of MAP kinases that ultimately phosphorylates the ER at Ser-118 in the AF-1 region (23Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1722) Google Scholar, 24Ignar-Trowbridge D.M. Pimentel M. Teng C.T. Korach K.S. McLachlan J.A. Environ. Health Perspect. 1995; 7: 35-38Google Scholar, 25Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (853) Google Scholar, 26Ignar T.D. Pimentel M. Parker M.G. McLachlan J.A. Korach K.S. Endocrinology. 1996; 137: 1735-1744Crossref PubMed Scopus (203) Google Scholar). Phosphorylation of Ser-118 leads to increased AF-1 activity by increasing binding of a p68 RNA helicase that is or becomes bound to CBP. This Ser-118-dependent link of AF-1 to CBP is in addition to the Ser-118-independent link with GRIP1 and thereby CBP (27Endoh H. Maruyama K. Masuhiro Y. Kobayashi Y. Goto M. Tai H. Yanagisawa J. Metzger D. Hashimoto S. Kato S. Mol. Cell. Biol. 1999; 19: 5363-5372Crossref PubMed Google Scholar). Although EGF-mediated phosphorylation of steroid receptors underlies part of EGF enhancement, it cannot underlie all of it. In particular, mutation of Ser-118 to glutamate in the ER AF-1 domain, while blocking phosphorylation, nonetheless preserves EGF enhancement of ER action (25Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (853) Google Scholar). Furthermore, careful mutation of sites of phosphorylation in the progesterone receptor again reveals a potent action of EGF in the absence of direct receptor phosphorylation (21Bai W. Rowan B.G. Allgood V.E. O'Malley B.W. Weigel N.L. J. Biol. Chem. 1997; 272: 10457-10463Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). These studies suggest the existence of a pathway of EGF action that targets a nonreceptor protein. We have explored the possibility that this unknown target is one of the p160 coactivators, particularly GRIP1. We present evidence that at least part of the signal from EGF to steroid receptor is conducted through the p160 coactivators. HeLa cells were transfected by electroporation as previously described (28Webb P. Lopez G.N. Greene G.L. Baxter J.D. Kushner P.J. Mol. Endocrinol. 1992; 6: 157-167Crossref PubMed Scopus (129) Google Scholar). Generally transfections included 5 μg of luciferase reporter plasmid, 1.0 μg of Gal-GRIP expression vectors, and 1.0 μg of β-actin-β-galactosidase expression vector for internal control. After electroporation, the cells were treated with vehicle, EGF (25 ng/ml), or TGF-α (15 ng/ml). Luciferase and chloramphenicol acetyltransferase values are the means and standard deviations of triplicate treatments from a single experiment, representative of at least three independent experiments. GST-GRIP fragments were expressed in bacteria (HB101) and partially purified by glutathione-Sepharose affinity column. Beads bearing the fusion proteins (1–3 μg of total protein) were subjected to in vitro phosphorylation by activated ERK2 according to the instructions of the supplier (Stratagene, La Jolla, CA). The phosphorylated products were extracted from the beads, resolved by 10% polyacrylamide gel electrophoresis, stained with Coomassie Blue to monitor expression, and subjected to autoradiography. Approximately 8.5 μg of total protein on beads were labeled in a 50-μl final reaction volume, extracted from the beads, and purified by 10% SDS-polyacrylamide gel electrophoresis. The gel was fixed, dried, and exposed to x-ray film to visualize the radiolabeled proteins. In-gel digestion of the protein with trypsin or endoproteinase Glu-C (Roche Diagnostics, Indianapolis, IN) was carried out as described previously (29Hellman U. Wernstedt C. Gonez J. Heldin C.H. Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (687) Google Scholar). The resultant peptides were separated using reversed phase HPLC on a microbore C8 column (Vydac, Hesperia, CA), and the collected fractions were subjected to scintillation counting. Individual peptides were subjected to covalent Edman degradation on a Sequelon AA membrane (PerSeptive Biosystems, Cambridge, MA) with a protein sequencer (model 492; Applied Biosystems, Foster City, CA). The anilinothiazolı́noneamino acids were extracted from the filter with neat trifluoroacetic acid and scintillation counted. Radioactive profiles for each sequencing run were compared with theoretical peptide sequences derived from the protein. To construct GalDBD-GRIP1, the GalDBD coding fragment from pGBT9·GRIPFL (32Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar) was removed withHindIII-EcoRI and ligated toSmaI-EcoRI-cut pBS (Stratagene) giving rise to pBS·Gal4DBD. An EcoRI fragment of GRIP1 (amino acids 5–1462) from pGBT9 vector was then inserted into pBS·Gal4DBD. The entire Gal4 DBD-GRIP1 coding segment was then removed withXbaI-EcoRV and subcloned into of vector has been described by J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). and for expression have been described by Webb (7Webb P. Nguyen P. Shinsako J. Anderson C. Feng W. Nguyen M.P. Chen D. Huang S.M. Subramanian S. McKinerney E. Katzenellenbogen B.S. Stallcup M.R. Kushner P.J. Mol. Endocrinol. 1998; 12: 1605-1618Crossref PubMed Scopus (0) Google Scholar). The and for expression were by reaction from vector a with expression vector was a of D. of expression vector has been described by S. K. Science. PubMed Scopus Google Scholar). was from (32Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar) using by reaction as described and are of vector and were as the were by and of two were carried has green fluorescent protein to the of expression vector and was a from of Health and was by a fragment from and it into sites of The mutation was by The is a of L. A. Metzger D. M. Chambon P. EMBO J. PubMed Scopus Google Scholar) and is described by Webb (7Webb P. Nguyen P. Shinsako J. Anderson C. Feng W. Nguyen M.P. Chen D. Huang S.M. Subramanian S. McKinerney E. Katzenellenbogen B.S. Stallcup M.R. Kushner P.J. Mol. Endocrinol. 1998; 12: 1605-1618Crossref PubMed Scopus (0) Google Scholar). Gal4 response elements of a promoter and has been previously described Lopez G. West B.L. E. Feng W. H. A. Apriletti J.W. Kushner P.J. Baxter J.D. M. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar). is of the as described by P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: PubMed Google Scholar). the estrogen response element from the promoter expression of the luciferase gene in a vector of the vector G. Webb P. Baxter J.D. Kushner P.J. Mol. Cell. Biol. PubMed Scopus Google Scholar) and has been previously described P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: PubMed Google Scholar). The reporter has three glucocorticoid response elements from the region of gene of the promoter to luciferase expression J.A. D. Mol. Cell. Biol. PubMed Scopus Google Scholar). HeLa cells were transfected with μg of or expression at exposed to EGF for and in and and The was resolved by polyacrylamide gel electrophoresis, to a membrane and with fluorescent protein or GRIP1 by the were by the After transfections cells to be by were on and a of cells was treated for luciferase were collected on a using a and and were collected using a and from to the in were collected at the and that growth factor of the transcriptional activation of CBP R. A. Biochem. 1996; PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) the domain of CBP that p160 binding (10Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar). We in transfected cells the p160 GRIP1 tethered to a reporter gene promoter by fusion to the heterologous Gal4 DNA binding domain activate in response to growth factors. EGF and TGF-α on reporter gene expression in the absence of GRIP1 but these of the EGF receptor each activated when GRIP1 was bound to the The GRIP1 response to EGF and TGF-α required the action of the ERK family of MAP kinases it was by of the MAP kinase by a that activation of and by a specific of activation (Fig. 1, and In EGF to stimulate reporter gene by a Gal4 fusion to the and as was inhibitory not GRIP1 transcriptional activation and ERKs are a of the pathway of To GRIP1 as a direct for we GRIP1 in and it with activated ERK2 in fragments that the entire two fragments from acids and were phosphorylated by MAP kinase (Fig. was and fragment was GRIP1 as a MAP kinase in and the of phosphorylation acids most and The of action of ERK MAP kinase was by using protein and Edman of the of these a single labeled peptide on and the was on the acid of the peptide and the of the peptide of these that the of phosphorylation of GRIP1 is (Fig. which is ERK To the of the site, the phosphorylation were with wild type and in which or it a MAP kinase site, was to Phosphorylation was on GRIP1 on (Fig. The was phosphorylated as as wild In a MAP kinase with phosphorylated all three We that is a target for phosphorylation of GRIP1 by ERK but not in To the of in we studies of EGF activation of tethered GRIP1 using both wild type and EGF transcriptional activation by wild type GRIP1 or the but was on the (Fig. this of the was well expressed as by and by to as in a (Fig. These that GRIP1 bound directly to the promoter for full activation by We then the for when GRIP1 as a coactivator for the activated by EGF We a reporter gene with three response elements from the promoter which the activates in the presence of EGF (Fig. type GRIP1 as well as the of the reporter gene activation with the and constitutive activity of the were as is for nuclear receptors with coactivators not The was only as as wild type in In with hormone-activated glucocorticoid which does not to both wild type and GRIP1 receptor action to not that the of GRIP1 full function in some GRIP1 wild type GRIP1 to of this may a for activation of ERK or some other kinase on the of GRIP1 on even in the absence of EGF not other see C.A. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). GRIP1 full to as a coactivator in some circumstances but is in coactivator function on the receptor activation of from a reporter gene regulated by a is by EGF to on cells and We the of ER in the of phosphorylation, for response to EGF with GRIP1. EGF or on transcriptional activation by ER in the absence or presence of estrogen EGF both and ER action the GRIP1 was to EGF The ER AF-2 which can be as a fusion of the ER LBD to the Gal4 DNA binding domain tethered on a Gal4 response does not to EGF (Fig. The was in to function as a coactivator for ER full coactivator function with the ER LBD but is in coactivator function for ER GRIP1 is also bound to and synergizes with CBP/p300 (10Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar, J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1108) Google Scholar, H. T.E. Onate S.A. Jenster G. Tsai S.Y. Tsai M.J. O'Malley B.W. 1997; Google Scholar). is that EGF regulates GRIP1 coactivator activities by the of this it was that activated increased the of CBP and the p160 J. M. Mol. Cell. Biol. PubMed Scopus Google Scholar). Although expression of activated to phosphorylation of the was not We the of wild type and GRIP1 to by CBP. We tethered CBP directly to a promoter by fusion to a heterologous Gal4 DNA binding In the absence of GRIP1 the CBP domain activates of wild type GRIP1 or the which becomes to induction by The of GRIP1 in to activate tethered and the EGF response is To that the of the are not to of or nuclear we both wild type and GRIP1 to and monitored GRIP1 expression and nuclear wild type and were well expressed and both to the (Fig. expression of wild type and (Fig. to wild type GRIP1 full coactivator action with tethered CBP and but the was (Fig. the mutation does not GRIP1 expression or has a specific on coactivator function. The that ERK MAP kinase the transcriptional activation of GRIP1. Furthermore, activated ERKs phosphorylate GRIP1 on in of to the of GRIP1 to enhance of the and of the and the GRIP1 is to function as a coactivator for the and for the ER LBD AF-2, receptors that do not to it that the of is and may be specific for GRIP1 coactivator function with steroid receptors or other factors. In these suggest that ERKs coactivator of GRIP1 through a pathway the integrity and most the phosphorylation of this was in it was that of the p160 family, a transcriptional function that was activated by with constitutive a potent of ERKs J. M. Mol. Cell. Biol. PubMed Scopus Google Scholar). by was independent of the domain of that AD1 was fragments of that contain AD1 and the nuclear receptor boxes are phosphorylated in that the target of activation is in this region and may AD1 for a of AD1 of GRIP1 the EGF response with ER and not by was also to the binding of CBP with fragments that contain the AD1 studies this in that we in the the p160 binding domain of CBP tethered to DNA by fusion to the Gal4 DNA binding domain and GRIP1. The is by the a direct for this in the of and GRIP1 that the is not must the of the in and their The EGF pathway to GRIP1 and to factors that recruit GRIP1 may a means to EGF effects at factors and their target has been well that EGF a signal phosphorylation cascade that leads to factors such as and phosphorylation of the factors is required for wild type and ER response to are that in the absence of direct phosphorylation, such as when the ER Ser-118 is to EGF response we suggest that for signal cascade to be the cascade may to target both the factor and the This may from activation of the EGF We and for and