A Conserved α-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 ␣-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 ER␣/ 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 cotransfection 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 ␣-helix A␣1 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 ␣-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP. CBP1 and p300 interact with a wide range of DNA-binding transcription factors and their cofactors (1, 2). 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-␤ enhanceosome, facilitates synergistic activation of transcription (3). 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, NFB, or STAT proteins (4, 5) or hypoxia-inducible factor (HIF1␣) and CITED2 (6). 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 (1, 2). 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/TA...

Structural Diversity in p160/CREB-binding Protein Coactivator Complexes

“…We have shown previously that the LLXXLXXXL sequence motif within ␣1 is conserved in other SID-binding proteins (see Fig. 4A), and that disruption of this sequence in Ets2 and E1A abrogates their binding to the CBP SID (22). Fusion of the SRC1 AD1 sequence to a GAL4 DNA binding domain permits very potent activation of a GAL reporter gene in mammalian cells, through recruitment of endogenous CBP and p300 (12).…”

“…4A). The adenoviral oncoprotein E1A revealed also sequences essential for CBP binding, which may be functionally and structurally equivalent to SRC1 AD1 S␣1 and S␣3 (22) (Fig. 4A).…”

“…In addition to binding p160s, the SID also facilitates interactions of CBP with activation domains in other nuclear proteins, including the transcription factors Ets1, Ets2, p53, and IRF3, and viral activators such as E1A, KSHV IRF1, and Tax (19 -22). Indeed, it has been shown that competition between such proteins for binding to the SID contributes to the negative cross-talk observed between different signaling pathways (22). Similarly, binding of viral proteins to the SID and other CBP/p300 domains not only facilitates viral gene transcription but may also down-regulate expression of host defense genes, through exclusion of host factors from CBP-p300 complexes.…”

MOZ–TIF2-mediated destruction of CBP/p300 is blocked by calpain inhibitor 2

“…3 This interaction requires a C-terminal activation domain (AD1) present in TIF2. 6,7 The AD1 sequence forms an amphipathic a-helix that is conserved in proteins such as Ets2, E1A, p160s and IRFs, 6 and which docks with the SID (SRC1 interaction domain) of CBP/p300 to form a stable fourhelix bundle. 7 Mutations in the AD1 a-helix render MOZ-TIF2 incapable of transforming haematopoietic cells and unable to induce leukaemia in a mouse model, 2,3 indicating that recruitment of CBP/p300 is essential for cell transformation.…”

“…Cell transfections, reporter assays, western blots and immunocytochemistry were performed essentially as described previously. 3,6 YFP-positive cells were sorted using a BD FacsAria cell sorter. Inhibitors or vehicle were added to cell cultures 8 h before sorting, fixation or harvesting for the above assays.…”