Abstract
Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. We discovered this phosphorylation-augmented interaction in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain were required for the interaction. Minimal regions that were competent for induced CBP/p300 binding in vitro also supported MAPK-enhanced transcription in vivo. CBP coexpression potentiated MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interacted in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, our findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetyltransferases / metabolism
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Amino Acid Sequence
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Baculoviridae / genetics
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Blotting, Western
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CREB-Binding Protein
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Cell Cycle Proteins / metabolism
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Chromatography, Affinity
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Genes, Reporter
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Genes, ras*
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HeLa Cells
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Histone Acetyltransferases
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Humans
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Luciferases / metabolism
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Mitogen-Activated Protein Kinases / metabolism*
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Models, Biological
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Models, Molecular
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Nuclear Magnetic Resonance, Biomolecular
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Nuclear Proteins / metabolism*
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Phosphorylation
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Protein Structure, Tertiary
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Proto-Oncogene Protein c-ets-1
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Proto-Oncogene Protein c-ets-2
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Proto-Oncogene Proteins / chemistry
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-ets
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Recombinant Proteins / metabolism
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Signal Transduction*
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Silver Staining
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Trans-Activators / chemistry
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Trans-Activators / genetics
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Trans-Activators / metabolism*
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Transcription Factors / chemistry
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcriptional Activation
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p300-CBP Transcription Factors
Substances
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Cell Cycle Proteins
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ETS1 protein, human
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ETS2 protein, human
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Nuclear Proteins
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Proto-Oncogene Protein c-ets-1
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Proto-Oncogene Protein c-ets-2
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-ets
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Recombinant Proteins
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Trans-Activators
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Transcription Factors
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Luciferases
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Acetyltransferases
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CREB-Binding Protein
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CREBBP protein, human
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Histone Acetyltransferases
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p300-CBP Transcription Factors
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p300-CBP-associated factor
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Mitogen-Activated Protein Kinases