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A Conserved α-Helical Motif Mediates the Binding of Diverse Nuclear Proteins to the SRC1 Interaction Domain of CBP

2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês

10.1074/jbc.m310188200

ISSN

1083-351X

Autores

Sachiko Matsuda, Janet C. Harries, Maria Viskaduraki, Philip J.F. Troke, Karin B. Kindle, Colm M. Ryan, David M. Heery,

Tópico(s)

Retinoids in leukemia and cellular processes

Resumo

CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the α-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERα/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the α-helix Aα1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic α-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP. CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the α-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERα/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the α-helix Aα1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic α-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP. CBP 1The abbreviations used are: CBP, CREB-binding protein; CREB, cAMP response element-binding protein; GST, glutathione S-transferase; UTR, untranslated region; PNT, pointed domain; HRP, horseradish peroxidase; AAD, acidic activation domain; HA, hemagglutinin; SRC, steroid receptor coactivator. and p300 interact with a wide range of DNA-binding transcription factors and their cofactors (1Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Google Scholar, 2Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577Crossref Google Scholar). Recruitment of CBP and associated factors permits acetylation and methylation of histones and other proteins at gene promoters, leading to chromatin remodeling, RNA polymerase II recruitment, and transcription. The ability of CBP and p300 to form contacts with multiple diverse factors assembled at gene promoters such as the IFN-β enhanceosome, facilitates synergistic activation of transcription (3Agalioti T. Lomvardas S. Parekh B. Yie J. Maniatis T. Thanos D. Cell. 2000; 103: 667-678Google Scholar). Conversely, competition between transcription factors for common binding sites on CBP/p300, which are in limiting concentrations in the nucleus, is likely to be important in negative cross-talk, as observed between nuclear receptors (NRs) and AP-1, NFκB, or STAT proteins (4Horvai A.E. Xu L. Korzus E. Brard G. Kalafus D. Mullen T.M. Rose D.W. Rosenfeld M.G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1074-1079Google Scholar, 5Kamei 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: 403-414Google Scholar) or hypoxia-inducible factor (HIF1α) and CITED2 (6Freedman S.J. Sun Z.Y. Kung A.L. France D.S. Wagner G. Eck M.J. Nat. Struct. Biol. 2003; 10: 504-512Google Scholar). Furthermore, CBP and p300 are important targets in viral infection, as they associate with viral proteins such as adenoviral E1A, SV40 large T antigen, and HTLV Tax (1Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Google Scholar, 2Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577Crossref Google Scholar). Thus, CBP and p300 act as molecular integrators of signal transduction pathways regulating cellular processes such as proliferation, differentiation, apoptosis, and the response to viral infection. The interaction of CBP/p300 with a large number of functionally diverse proteins is facilitated by a series of modular protein-binding domains. These include the cysteine/histidinerich domains CH1 and CH3, also known as TAZ1 and ZZ/TAZ2, which are major sites of protein interaction. The CH3/TAZ2 domain has a compact globular structure consisting of four α-helices and three HCCC zinc-binding motifs (7De Guzman R.N. Liu H.Y. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 303: 243-253Google Scholar). A short sequence (TRAM) has been identified within CH3, which partly mediates the interaction of CBP with adenoviral E1A proteins (8O'Connor M.J. Zimmermann H. Nielsen S. Bernard H.U. Kouzarides T. J. Virol. 1999; 73: 3574-3581Google Scholar). Although recent studies have confirmed that the CH1 and CH3 domains are structurally similar (9Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Google Scholar, 10Freedman S.J. Sun Z.Y. Poy F. Kung A.L. Livingston D.M. Wagner G. Eck M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5367-5372Google Scholar), they appear to exhibit distinct, if overlapping, specificities for binding subsets of transcription factors. Some proteins such as E1A, p53 and Ets proteins, have been observed to interact with several discrete CBP domains (10Freedman S.J. Sun Z.Y. Poy F. Kung A.L. Livingston D.M. Wagner G. Eck M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5367-5372Google Scholar). The KIX domain undergoes a conformational change upon association with the phosphorylated form of the kinase-inducible domain (KID) of CREB (11Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Google Scholar). In addition, other protein-interaction domains in CBP/p300 include the bromodomain, which mediates interaction of CBP with the acetylated p53, MyoD and other proteins (12Polesskaya A. Naguibneva I. Duquet A. Bengal E. Robin P. Harel-Bellan A. Mol. Cell. Biol. 2001; 21: 5312-5320Google Scholar), and three LXXLL motifs that mediate interactions with NRs (13Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Google Scholar, 14Heery D.M. Hoare S. Hussain S. Parker M.G. Sheppard H. J. Biol. Chem. 2001; 276: 6695-6702Google Scholar). The p160 coactivators bind to a domain located near the C terminus of CBP and p300 (5Kamei 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: 403-414Google Scholar, 10Freedman S.J. Sun Z.Y. Poy F. Kung A.L. Livingston D.M. Wagner G. Eck M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5367-5372Google Scholar, 15Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Google Scholar, 17Voegel J.J. Heine M.J. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO J. 1998; 17: 507-519Google Scholar). We have characterized this sequence, termed the SRC1 interaction domain (SID), and mapped it to amino acids 2058–2130 of CBP (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar). The SID domain interacts with amino acids 926–960 of SRC1, the transcriptional activation domain AD1, which is conserved among the p160 proteins (5Kamei 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: 403-414Google Scholar, 15Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Google Scholar, 16McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullen T.M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Google Scholar, 17Voegel J.J. Heine M.J. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO J. 1998; 17: 507-519Google Scholar, 18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar). The solution structure of this region of CBP in complex with the AD1 domain of ACTR has revealed that SID and AD1 polypeptides undergo a synergistic fold giving a structure resembling a four-helix bundle (19Demarest S.J. Martinez-Yamout M. Chung J. Chen H. Xu W. Dyson H.J. Evans R.M. Wright P.E. Nature. 2002; 415: 549-553Google Scholar). In addition to binding p160 proteins, recent reports have demonstrated that other factors can interact with the SID including IRF-3, E1A, p53, Tax, Ets-2, and KSHV IRF-1 (20Lin C.H. Hare B.J. Wagner G. Harrison S.C. Maniatis T. Fraenkel E. Mol. Cell. 2001; 8: 581-590Google Scholar, 21Livengood J.A. Scoggin K.E. Van Orden K. McBryant S.J. Edayathumangalam R.S. Laybourn P.J. Nyborg J.K. J. Biol. Chem. 2002; 277: 9054-9061Google Scholar, 22Scoggin K.E.S. Ulloa A. Nyborg J.K. Mol. Cell. Biol. 2001; 21: 5520-5530Google Scholar). In this study we describe experiments which indicate that diverse nuclear proteins such as SRC1, E1A, and Ets-2 display mutually exclusive binding to the CBP SID both in vitro and in vivo. We show that these proteins share a sequence motif similar to an amphipathic α-helix in the AD1 domain of p160s, which is important for their interactions with the CBP SID. Plasmid Expression Vectors—The following plasmids used in this study have been described previously; DBD LexA-CBP series 1982–2163, 1982–2130, 1982–2111, 1982–2100, 1982–2080, 2000–2163, 2017–2163, 2041–2163, 2041–2130, 2058–2163, 2058–2130, and 2073–2163; DBD LexA-CBP SID (2058–2130) mutant series, L2071A/L2072A/L2075A, Q2082R, F2101P, K2103A, K2103P, K2108A, and Q2117A/P2118A; GST-SID wild-type (CBP 2058–2130) and mutant series L2071A/L2072A/L2075A, L2071P/L2072P, L2071A/L2072A, Q2082P/Q2084P/Q2085G, Q2082R, F2101P, K2103A, K2103P, K2108A, Q2113P/G2115P and Q2117A/P2118A; pGEX-DMH, pSG5 CBP, GAL4-AD1, pSG5 FLAG-hSRC1e (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar) and pSG5 hSRC1e (29Kalkhoven E. Valentine J.E. Heery D.M. Parker M.G. EMBO J. 1998; 17: 232-243Google Scholar). The following plasmids were generous gifts and have been described previously; pcDNA3 FLAG-Ets-2, pcDNA3 FLAG-Ets-2 (T72A) and UPA-Luc reporter (25Smith J.L. Schaffner A.E. Hofmeister J.K. Hartman M. Wei G. Forsthoefel D. Hume D.A. Ostrowski M.C. Mol. Cell. Biol. 2000; 20: 8026-8034Google Scholar, 26Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Google Scholar), pGEX-CBP full-length (12Polesskaya A. Naguibneva I. Duquet A. Bengal E. Robin P. Harel-Bellan A. Mol. Cell. Biol. 2001; 21: 5312-5320Google Scholar), pGEX CBP-N (1–596), pGEX-TRAM (CBP 1808–1826) (8O'Connor M.J. Zimmermann H. Nielsen S. Bernard H.U. Kouzarides T. J. Virol. 1999; 73: 3574-3581Google Scholar), pASV3, pASV3-mouse embryonic cDNA library (23Le Douarin B. Heery D. Gaudon C. vom Baur E. Losson R. Methods in Molecular Biology, Steroid Receptor Methods: Protocols and Assays. 176. Humana Press Inc., Totowa, NJ2001: 227-248Google Scholar), pMT-MOR (ERα) (40White R Sjoberg M. Kalkhoven E. Parker M.G. EMBO J. 1997; 16: 1427-1435Google Scholar), pcDNA3 p53 (41Nie Y. Li H.H. Bula C.M. Liu X. Mol. Cell. Biol. 2000; 20: 741-748Google Scholar), pCI E1A12S, pCI E1A12S Δ2–36 (32Liu S.L. Rand A. Kelm R.J. Getz M.J. Oncogene. 2000; 19: 3352-3362Google Scholar), p3ERE-TATA-Luc (42Legler J. van den Brink C.E. Brouwer A. van der Saag P.T. Vethaak A.D. van der Burg B. Toxicol. Sci. 1999; 48: 55-66Google Scholar), pJ7-lacZ (E. Kalkhoven). cDNA sequences flanked by appropriate restriction enzyme sites were generated by PCR using Elongase (Invitrogen) and cloned in frame into a modified version of the pASV3 vector (23Le Douarin B. Heery D. Gaudon C. vom Baur E. Losson R. Methods in Molecular Biology, Steroid Receptor Methods: Protocols and Assays. 176. Humana Press Inc., Totowa, NJ2001: 227-248Google Scholar) to generate VP16-acidic activation domain (AAD) fusion proteins. Full-length FLAG-Ets-2 cDNA was amplified from pcDNA3 FLAG-Ets-2 and cloned into a modified pSG5 vector to create pSG5 FLAG-Ets-2. The FLAG-Ets-2 ΔPNT (deletion of amino acids 116–161) and FLAG-Ets-2 ΔH5 (deletion of amino acids 154–161) were constructed by PCR methods. All constructs generated in this study were verified by sequence analysis. Yeast Methods—The yeast two-hybrid screen was carried out essentially as described previously (23Le Douarin B. Heery D. Gaudon C. vom Baur E. Losson R. Methods in Molecular Biology, Steroid Receptor Methods: Protocols and Assays. 176. Humana Press Inc., Totowa, NJ2001: 227-248Google Scholar). Saccharomyces cerevisiae L40 cells carrying DBD-LexA-CBP (1982–2163) were transformed with the pASV3 mouse embryo cDNA library using a modified lithium acetate transformation method (23Le Douarin B. Heery D. Gaudon C. vom Baur E. Losson R. Methods in Molecular Biology, Steroid Receptor Methods: Protocols and Assays. 176. Humana Press Inc., Totowa, NJ2001: 227-248Google Scholar). Transformants were initially selected for l-leucine and l-tryptophan prototrophy on dropout medium resulting in the recovery of 1.5 × 106 library clones. The library was then replated (10-fold) onto dropout medium with selection for l-leucine, l-tryptophan, and l-histidine prototrophy, and resistance to either 10 mm or 20 mm 3-aminotriazole (3-AT), to select bait-interacting clones. Colonies capable of growth on 3-AT and showing strong activation of the secondary reporter (β-galactosidase) were subjected to further analysis. The 331 putative positives from the first round of selection were cultured in the presence of l-tryptophan to eliminate the bait plasmid, followed by rescue of pASV3 library (LEU2) plasmids in E. coli HB101 (leuB-) by complementation on M9 minimal medium containing 100 μg/ml ampicillin, and lacking l-leucine (23Le Douarin B. Heery D. Gaudon C. vom Baur E. Losson R. Methods in Molecular Biology, Steroid Receptor Methods: Protocols and Assays. 176. Humana Press Inc., Totowa, NJ2001: 227-248Google Scholar). Bona fide-positive clones were selected by retransformation of fresh L40 pBTM116-CBP 1982–2163 and testing for 3-AT resistance and β-galactosidase activity. Quantitative β-galactosidase assays were carried out in duplicate, in three separate experiments, as previously described and reporter activities are expressed as nmol of substrate transformed/min/mg protein extract (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar, 23Le Douarin B. Heery D. Gaudon C. vom Baur E. Losson R. Methods in Molecular Biology, Steroid Receptor Methods: Protocols and Assays. 176. Humana Press Inc., Totowa, NJ2001: 227-248Google Scholar). The expression of fusion proteins in cell-free extracts was monitored by Western blotting using antibodies (Autogen BioClear) directed against VP16 (sc-7545), or LexA (sc-7544) or FLAG-M2 (Sigma) epitopes. Cell Culture and Transient Transfection—COS-1 and HEK293 cells were maintained in Dulbecco's Modified Eagle's Medium, (DMEM, Invitrogen) supplemented with 10% fetal calf serum, (Invitrogen). For reporter assays, COS-1 cells were seeded at a density of 1 × 105 cells per ml in 6-well dishes in phenol red-free DMEM supplemented with dextran charcoal-stripped fetal calf serum (5%), 24 h prior to transfection. Cells were transfected by calcium phosphate co-precipitation (Clontech) or by Transfast (Promega) according to the manufacturer's instructions. The transfected DNAs were used at the following concentrations (per well) unless otherwise indicated; pJ7-lacZ control plasmid (500 ng), p3ERE-TATA-Luc, pUPA-Luc reporter plasmids (1 ug), pMT-MOR (ERα) expression plasmid (5 ng), pSG5 hSRC1e (100 ng), pcDNA3 FLAG-Ets-2 (250 ng), pSG5 pGAL4-AD1 (250 ng), pGAL-E1B-Luc (500 ng), pCI E1A12S (50 ng), pCI E1A12S Δ2–36 (50 ng) and pSG5-CBP (100 ng). The amount of DNA per well was equilibrated with empty pSG5 or the pcDNA3 vector as appropriate. After 24 h, fresh medium containing either vehicle or 10-8m 17β-estradiol (E2) was added. After a further 24 h, the cells were harvested; extracts were assayed for luciferase activity using the luciferase assay system (Promega); and the data were normalized relative to β-galactosidase activities in the same extracts, as determined using the Galacto-Light chemiluminescence assay (Tropix). All measurements were done in triplicate, and each experiment repeated at least twice. Reporter activities are expressed as fold induction of the normalized luciferase activity relative to that due to reporters alone (or reporters alone in the absence of ligand for ERα). In Vitro and In Vivo Interactions—In vitro translated 35S-labeled proteins were generated from pSG5, pT7 or pcDNA3 expression vectors, using the T7/SP6-coupled reticulocyte lysate system (Promega). GST fusion proteins were expressed in Escherichia coli DH5α or BL21 and purified on glutathione-Sepharose beads (Amersham Biosciences). The expression of GST proteins was monitored on (SDS)-polyacrylamide gels (12%) by Coomassie Blue staining, and equivalent amounts of proteins were used in the pull-down assays, which were performed as described previously (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar). Competition assays (38Kurokawa R. Kalafus D. Ogliastro M.H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. Science. 1998; 279: 700-703Google Scholar) were carried out as follows; a fixed amount of 35S-labeled test protein (close to saturation of the beads) relative to increasing amounts of a competitor protein (also 35S-labeled) or AD1 peptide (SRC1 925–958), were premixed before addition to the protein-loaded glutathione-Sepharose beads. For co-immunoprecipitation experiments, HEK293 cells were seeded at a density of 2 × 105 cells per ml in 10-cm dishes, and transfected with 15 μg of either pcDNA3-FLAG vector (Control), pcDNA3 FLAG-Ets-2 or pSG5 FLAG-SRC1e in addition to 15 μg of pSG5 CBP, pCMV HA-p300, or empty vectors. After 40 h, nuclear extracts were prepared as follows: cell pellets were washed twice in 0.5 ml of cold phosphate-buffered saline and lysed in 1 ml of Extraction Buffer I (50 mm Tris-HCl, pH 8.0; 150 mm NaCl; 0.5% Nonidet P-40), and protease inhibitors at 0 °C for 15 min. The cell nuclei were pelleted and resuspended in 1 ml of Extraction Buffer II (50 mm Tris-HCl, pH 8.0; 420 mm KCl; 10% glycerol; 2 mm dithiothreitol; with protease inhibitors). After 15 min at 0 °C, the lysate was centrifuged at 10 krpm, 4 °C, for 10 min, and precleared for 30 min at 4 °C with 20 μl of protein-A/G PLUS-agarose beads (Autogen Bioclear, sc-2003). FLAG-tagged proteins were purified using 20 μl of packed volume α-FLAG M2-agarose affinity gel (Sigma, A-2220) on a rotating wheel overnight at 4 °C, followed by three washes in 0.5 ml of Extraction Buffer II. Proteins bound to the beads were separated by SDS-PAGE (8% acrylamide for p300 or CBP, 12% acrylamide for FLAG-tagged proteins), transferred to nitrocellulose and detected by Western blotting. FLAG-tagged proteins were detected using a 1:500 dilution of α-FLAG M2 monoclonal antibody (Sigma, F-3165) followed by a 1:2000 dilution of secondary α-mouse IgG-HRP (Autogen Bioclear, sc-2954). For detection of co-purified HA-p300, a 1:500 dilution of α-HA (Autogen Bioclear, sc-7392) antibody was used, in conjunction with 1:2000 dilution of the α-mouse IgG-HRP. Similarly for CBP detection, a 1:500 dilution (mix) of CBP (A-22) and CBP (C-20) rabbit polyclonals (Autogen Bioclear, sc-583 and sc-369, respectively) was used, in conjunction with a 1:2000 dilution of anti-rabbit IgG-HRP (Autogen Bioclear, sc-2004). Bound peroxidase-coupled antibodies were revealed using the ECL Plus system (Amersham Biosciences). The PNT Domain of Ets-2 Mediates Its Interaction with CBP SID—To isolate proteins that interact with the CBP SID domain, we carried out a yeast two-hybrid screen of a VP16 AAD-fused mouse embryonic cDNA library (43Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Google Scholar), using a DBD LexA-CBP 1982–2163 clone as bait (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar). From two million individual clones screened, five bona fide positive clones were identified. Sequence analysis and data base searches revealed that one of these (clone 1–01) encoded the N terminus (amino acids 1–273) of the mouse Ets-2 protein, with an additional 54 codons of in-frame sequence upstream of the proposed start codon derived from 5′-UTR sequence. This comprises the entire transactivation domain of human Ets-2 including the conserved PNT domain (amino acids 60–170). Removal of the codons derived from the 5′-UTR sequence (AAD-Ets-2 (1–273)) did not affect the interaction with the bait sequence, confirming that this sequence was not required for the interaction with CBP (Fig. 1A). As the boundaries of the bait sequence (1982–2163) were extended beyond the minimal SID, we used a series of LexA-CBP fusion proteins to define the minimal sequences capable of binding Ets-2 (1–273). As shown in Fig. 1B, the minimal CBP sequence required to bind to the AAD-Ets-2 (1–273) corresponded to the previously characterized SID domain (CBP 2058–2130). A previous study reported interactions of Ets-2 with the CH1/TAZ1 domain and also a large C-terminal portion of CBP (amino acids 1678–2370) containing the CH3/ZZ-TAZ2 and SID domains (24Jayaraman G. Srinivas R. Duggan C. Ferreira E. Swaminathan S. Somasundaram K. Williams J. Hauser C. Kurkinen M. Dhar R. Weitzman S. Buttice G. Thimmapaya B. J. Biol. Chem. 1999; 274: 17342-17352Google Scholar). We assessed the binding of in vitro translated full-length Ets-2 to GST-CBP fusion proteins GST-CBP-N (1–596) or GST-SID (2058–2130). Similar amounts of the GST fusion proteins and control (GST alone) were used as indicated (Fig. 2A, lower panel). Ets-2 was found to bind strongly to the CBP SID domain, whereas a weaker interaction was detected with CBP N terminus containing CH1 under similar conditions (Fig. 2A, upper panel). This result indicates a strong interaction between Ets-2 and the SID domain of CBP in vitro. To determine the boundaries of the Ets-2 sequence required for binding to the CBP SID domain, we generated a series of VP16 AAD-Ets-2 fusion constructs. Reporter assays were carried out on extracts from yeast L40 cells co-expressing LexA-CBP-SID with AAD-Ets-2 proteins. As shown in Fig. 2B, the minimal region of Ets-2 necessary for SID binding mapped to amino acids 60–170, containing the PNT domain (residues 88–170). This domain is conserved in a subset of Ets family proteins, and has previously been shown to contain a conserved threonine residue adjacent to the PNT domain, which is phosphorylated by the kinases MAPK and Akt (25Smith J.L. Schaffner A.E. Hofmeister J.K. Hartman M. Wei G. Forsthoefel D. Hume D.A. Ostrowski M.C. Mol. Cell. Biol. 2000; 20: 8026-8034Google Scholar, 26Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Google Scholar). Previous studies have shown that substitution of this residue with alanine (T72A) results in a loss of MAPK signaling via Ets-2 (26Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Google Scholar). This raised the possibility that phosphorylation might be required to stabilize the interaction of Ets-2 with coactivators such as CBP, as previously observed for the interaction of the kinase inducible domain of CREB with the CBP KIX domain (11Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Google Scholar). However, as shown in Fig. 2B, the T72A mutation did not markedly impair the interaction of PNT with SID in yeast two-hybrid experiments, suggesting that phosphorylation of Thr-72 is not essential for the physical association of Ets-2 and CBP. This result was confirmed by in vitro binding studies, as we observed that wild-type and T72A Ets-2 proteins displayed equally strong binding to GST-SID (Fig. 2C). Thus, the association of Ets-2 protein with CBP can occur via an interaction between the PNT and SID domains, which does not require phosphorylation of Thr-72 in yeast two-hybrid or in vitro binding assays. To confirm the interaction of Ets-2 with CBP in vivo, we performed co-immunoprecipitation experiments. HEK293 cells were transiently co-transfected with expression plasmids for FLAG-Ets-2 or FLAG-SRC1e along with CBP or HA-p300 expression plasmids, or empty vector. Nuclear extracts were prepared post-transfection, and FLAG-tagged proteins purified on anti-FLAG M2 Sepharose beads were detected by Western blotting (Fig. 2D, left panel). Co-immunoprecipitated CBP and HA-p300 proteins were detected using anti-CBP (middle panel) or anti-HA antibodies (right panel), respectively. Our results show that both CBP and p300 proteins co-precipitated with FLAG-Ets-2, but not a negative control (FLAG vector), indicating that these proteins can form a complex in vivo. The amount of HA-p300 detected with each of these proteins was comparable to that obtained with a positive control protein, FLAG-SRC1e (Fig. 2D, right panel). Effects of Amino Acid Substitutions in the CBP SID on Recruitment of SRC1, Ets-2, and E1A—In addition to binding p160 proteins and Ets-2, the SID domain has been reported to interact with a variety of other cellular and viral nuclear proteins including the adenoviral E1A 12S protein (20Lin C.H. Hare B.J. Wagner G. Harrison S.C. Maniatis T. Fraenkel E. Mol. Cell. 2001; 8: 581-590Google Scholar, 22Scoggin K.E.S. Ulloa A. Nyborg J.K. Mol. Cell. Biol. 2001; 21: 5520-5530Google Scholar). To determine whether these functionally diverse proteins make similar contacts with the CBP SID domain, we examined the impact of amino acid substitutions in the CBP SID domain on its interaction with SRC1, Ets-2, and E1A. Full-length 35S-labeled SRC1e, Ets-2 and E1A proteins were generated by in vitro translation and assessed for binding to a panel of GST-SID mutants (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar). Amino acid substitutions within the SID structure (19Demarest S.J. Martinez-Yamout M. Chung J. Chen H. Xu W. Dyson H.J. Evans R.M. Wright P.E. Nature. 2002; 415: 549-553Google Scholar) are depicted schematically in Fig. 3A. As shown in Fig. 3B, and shown previously for SRC1, (18Sheppard H.M. Harries J.C. Hussain S. Bevan C. Heery D.M. Mol. Cell. Biol. 2001; 21: 39-50Google Scholar), substitution of the conserved leucine residues in helix H1 (L2071A/L2072A/L2075A, L2071P/L2072P, and L2071A/L2072A) only weakly reduced binding of E1A and SRC1 to the SID. However, Ets-2 binding to the SID was more sensitive to mutations in H1 (Fig. 3B). In addition, the F2101P and K2103P substitutions, which are predicted to disrupt helix H3 formation, ablated or strongly reduced the interaction of all three proteins with the SID (Fig. 3B). In contrast, amino acid substitutions that replace Lys-2103 or Lys-2108 with alanine, which would not be expected to disrupt H3 formation, had little effect on E1A or SRC1 binding, and only partly reduced the binding of Ets-2 to the SID. Similarly, amino acid replacements in the polyglutamine rich sequence (Q-loop) between helices H1 and H2 (Q2082R) or the QPG(M/L) repeat region (Q2113P/G2115P or Q2117A/P2118A) had little or no deleterious effect on interactions with SRC1 or E1A, although substitution of three of the four glutamines in the region between H1 and H2 (Q2082P/Q2084P/Q2085G) resulted in reduced Ets-2 binding (Fig. 3B). These results suggest that SRC1, Ets-2, and E1A recognize a similar surface of the CBP SID domain, for which the integrity of H3 is critical. However, H1 appears to be specifically important for the docking of the Ets-2 protein. To assess whether the SRC1 AD1 domain (900–970) and Ets-2 PNT domain (60–170) displayed similar sensitivities to SID mutations as the full-length proteins, we assessed the abilities of AAD fusion proteins containing these sequences to interact with a panel of LexA-CBP-SID

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