A Conserved alpha-helical motif mediates the binding of diverse nuclear proteins to the SRC1 interaction domain of 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 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.
Mesh Terms:
Adenovirus E1A Proteins, Amino Acid Sequence, Amino Acid Substitution, Animals, Binding, Competitive, COS Cells, CREB-Binding Protein, Conserved Sequence, DNA-Binding Proteins, Histone Acetyltransferases, Humans, Kidney, Mice, Molecular Sequence Data, Nuclear Proteins, Nuclear Receptor Coactivator 1, Protein Structure, Secondary, Protein Structure, Tertiary, Proto-Oncogene Protein c-ets-2, Proto-Oncogene Proteins, Repressor Proteins, Trans-Activators, Transcription Factors, Transcriptional Activation, Two-Hybrid System Techniques
Adenovirus E1A Proteins, Amino Acid Sequence, Amino Acid Substitution, Animals, Binding, Competitive, COS Cells, CREB-Binding Protein, Conserved Sequence, DNA-Binding Proteins, Histone Acetyltransferases, Humans, Kidney, Mice, Molecular Sequence Data, Nuclear Proteins, Nuclear Receptor Coactivator 1, Protein Structure, Secondary, Protein Structure, Tertiary, Proto-Oncogene Protein c-ets-2, Proto-Oncogene Proteins, Repressor Proteins, Trans-Activators, Transcription Factors, Transcriptional Activation, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Apr. 02, 2004
PubMed ID: 14722092
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