Abstract
ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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CCAAT-Enhancer-Binding Proteins / genetics
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CCAAT-Enhancer-Binding Proteins / metabolism
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Cell Line
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Cell Membrane / enzymology
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Cell Membrane / metabolism
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Cell Nucleus / enzymology
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Cell Nucleus / metabolism
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Chloramphenicol O-Acetyltransferase / metabolism
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Dogs
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Epithelial Cells / cytology
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Epithelial Cells / enzymology*
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Epithelial Cells / metabolism*
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Gene Expression
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Genes, Reporter
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Glutathione Transferase / metabolism
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Guanylate Kinases
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Membrane Proteins / chemistry
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Membrane Proteins / metabolism*
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Nucleoside-Phosphate Kinase / metabolism*
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Promoter Regions, Genetic
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-fos / genetics
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Proto-Oncogene Proteins c-fos / metabolism
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Proto-Oncogene Proteins c-jun / genetics
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Proto-Oncogene Proteins c-jun / metabolism
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Recombinant Fusion Proteins / metabolism
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Tight Junctions / enzymology
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Tight Junctions / metabolism
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Transcription Factor AP-1 / metabolism
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcription, Genetic
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Zonula Occludens-2 Protein
Substances
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CCAAT-Enhancer-Binding Proteins
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Membrane Proteins
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Proto-Oncogene Proteins c-fos
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Proto-Oncogene Proteins c-jun
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Recombinant Fusion Proteins
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Transcription Factor AP-1
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Transcription Factors
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Zonula Occludens-2 Protein
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Chloramphenicol O-Acetyltransferase
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Glutathione Transferase
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Nucleoside-Phosphate Kinase
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Guanylate Kinases