Abstract
Transcription factor CCAAT/enhancer-binding protein (C/EBP)-beta is crucial for regulating transcription of genes involved in a number of diverse cellular processes, including those involved in some cytokine-induced responses. However, the mechanisms that contribute to its diverse transcriptional activity are not yet fully understood. To gain an understanding into its mechanisms of action, we took a proteomic approach and identified cellular proteins that associate with C/EBP-beta in an interferon (IFN)-gamma-dependent manner. Transcriptional mediator (Mediator) is a multisubunit protein complex that regulates signal-induced cellular gene transcription from enhancer-bound transcription factor(s). Here, we report that the Med1 subunit of the Mediator as a C/EBP-beta-interacting protein. Using gene knock-out cells and mutational and RNA interference approaches, we show that Med1 is critical for IFN-induced expression of certain genes. Med1 associates with C/EBP-beta through a domain located between amino acids 125 and 155 of its N terminus. We also show that the MAPK, ERK1/2, and an ERK phosphorylation site within regulatory domain 2, more specifically the Thr(189) residue, of C/EBP-beta are essential for it to bind to Med1. Last, an ERK-regulated site in Med1 protein is also essential for up-regulating IFN-induced transcription although not critical for binding to C/EBP-beta.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Animals
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Apoptosis Regulatory Proteins / genetics
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Apoptosis Regulatory Proteins / metabolism
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CCAAT-Enhancer-Binding Protein-beta / deficiency
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CCAAT-Enhancer-Binding Protein-beta / genetics
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CCAAT-Enhancer-Binding Protein-beta / metabolism*
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Calcium-Calmodulin-Dependent Protein Kinases / genetics
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Line
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Death-Associated Protein Kinases
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Endodeoxyribonucleases / chemistry
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Endodeoxyribonucleases / genetics
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Endodeoxyribonucleases / metabolism*
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Gene Expression Regulation / drug effects
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Humans
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Interferon-Stimulated Gene Factor 3, gamma Subunit / metabolism
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Interferon-gamma / pharmacology*
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Mice
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Mice, Knockout
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3 / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutation / genetics
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Promoter Regions, Genetic / genetics
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Protein Binding
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Protein Structure, Tertiary
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Recombinant Proteins
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Transcription, Genetic / drug effects*
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Transcription, Genetic / genetics*
Substances
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Apoptosis Regulatory Proteins
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CCAAT-Enhancer-Binding Protein-beta
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Interferon-Stimulated Gene Factor 3, gamma Subunit
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Isgf3g protein, mouse
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Protein Subunits
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Recombinant Proteins
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Interferon-gamma
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Death-Associated Protein Kinases
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Endodeoxyribonucleases
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MBD4 protein, human
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Mbd4 protein, mouse