title: A Conserved alpha-helical motif mediates the binding of diverse nuclear proteins to the SRC1 interaction domain of CBP.
abstract: 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 alpha-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 ERalpha/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 alpha-helix Aalpha1 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 alpha-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP.
authors.affiliation: Department of Biochemistry, University of Leicester, University Road Leicester LE1 7RH, United Kingdom.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Matsuda
authors.forename: Janet C
authors.initials: JC
authors.lastname: Harries
authors.forename: Maria
authors.initials: M
authors.lastname: Viskaduraki
authors.forename: Philip J F
authors.initials: PJ
authors.lastname: Troke
authors.forename: Karin B
authors.initials: KB
authors.lastname: Kindle
authors.forename: Colm
authors.initials: C
authors.lastname: Ryan
authors.forename: David M
authors.initials: DM
authors.lastname: Heery
mesh_terms: D017500:Adenovirus E1A Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D050882:CREB-Binding Protein; D017124:Conserved Sequence; D004268:DNA-Binding Proteins; D051548:Histone Acetyltransferases; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D056920:Nuclear Receptor Coactivator 1; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494069:ETS2 protein, human; C494070:Ets2 protein, mouse; D009687:Nuclear Proteins; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1