The Ku Antigen-Recombination Signal-binding Protein Jκ Complex Binds to the Nuclear Factor-κB p50 Promoter and Acts as a Positive Regulator of p50 Expression in Human Gastric Cancer Cells

Joo Weon Lim, Hye Young Kim, Kyung Hwan Kim

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Abstract

The p50 subunit of NF-κB is a transcription factor that regulates the expression of a variety of genes. Previously, we showed that the expression of Ku antigen, a DNA repair protein, is mediated by NF-κB in gastric cancer AGS cells (Lim, J. W., Kim, H., and Kim, K. H. (2002) J. Biol. Chem. 277, 46093-46100). In this study, we report that the inhibition of Ku activity reduced both p50 expression and nuclear NF-κB activity in AGS cells. A co-immunoprecipitation experiment demonstrated that Ku antigen interacted with recombination signal-binding protein Jκ (RBP-Jκ), a DNA-binding protein. Ku antigen, RBP-Jκ, and p50 were found to bind to the DNA region containing the κB element in the p50 promoter. Supershift and competition experiments demonstrated that Ku antigen and RBP-Jκ bound sequence-specifically to downstream elements of κB at GCTTC and TGGGGG. mRNA expression and de novo synthesis of p50 were inhibited in cells transfected with the mutant gene expression constructs for IκBα, Ku8O, and RBP-Jκ. A reporter assay demonstrated that p50 transcription was positively mediated by NF-κB, Ku antigen, and RBP-Jκ and that the binding elements for these proteins were required for optimal p50 expression. The interaction of Ku antigen with RBP-Jκ and NF-κB p50 may act as a positive regulator of p50 expression in gastric cancer AGS cells.

Original languageEnglish
Pages (from-to)231-237
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number1
DOIs
Publication statusPublished - 2004 Jan 2

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Immunoglobulin J Recombination Signal Sequence-Binding Protein
Stomach Neoplasms
Carrier Proteins
Cells
Antigens
DNA
DNA-Binding Proteins
Transcription
Immunoprecipitation
Protein Binding
Gene expression
DNA Repair
Ku Autoantigen
Assays
Proteins
Repair
Transcription Factors
Genes
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "The Ku Antigen-Recombination Signal-binding Protein Jκ Complex Binds to the Nuclear Factor-κB p50 Promoter and Acts as a Positive Regulator of p50 Expression in Human Gastric Cancer Cells",
abstract = "The p50 subunit of NF-κB is a transcription factor that regulates the expression of a variety of genes. Previously, we showed that the expression of Ku antigen, a DNA repair protein, is mediated by NF-κB in gastric cancer AGS cells (Lim, J. W., Kim, H., and Kim, K. H. (2002) J. Biol. Chem. 277, 46093-46100). In this study, we report that the inhibition of Ku activity reduced both p50 expression and nuclear NF-κB activity in AGS cells. A co-immunoprecipitation experiment demonstrated that Ku antigen interacted with recombination signal-binding protein Jκ (RBP-Jκ), a DNA-binding protein. Ku antigen, RBP-Jκ, and p50 were found to bind to the DNA region containing the κB element in the p50 promoter. Supershift and competition experiments demonstrated that Ku antigen and RBP-Jκ bound sequence-specifically to downstream elements of κB at GCTTC and TGGGGG. mRNA expression and de novo synthesis of p50 were inhibited in cells transfected with the mutant gene expression constructs for IκBα, Ku8O, and RBP-Jκ. A reporter assay demonstrated that p50 transcription was positively mediated by NF-κB, Ku antigen, and RBP-Jκ and that the binding elements for these proteins were required for optimal p50 expression. The interaction of Ku antigen with RBP-Jκ and NF-κB p50 may act as a positive regulator of p50 expression in gastric cancer AGS cells.",
author = "Lim, {Joo Weon} and Kim, {Hye Young} and Kim, {Kyung Hwan}",
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AU - Lim, Joo Weon

AU - Kim, Hye Young

AU - Kim, Kyung Hwan

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N2 - The p50 subunit of NF-κB is a transcription factor that regulates the expression of a variety of genes. Previously, we showed that the expression of Ku antigen, a DNA repair protein, is mediated by NF-κB in gastric cancer AGS cells (Lim, J. W., Kim, H., and Kim, K. H. (2002) J. Biol. Chem. 277, 46093-46100). In this study, we report that the inhibition of Ku activity reduced both p50 expression and nuclear NF-κB activity in AGS cells. A co-immunoprecipitation experiment demonstrated that Ku antigen interacted with recombination signal-binding protein Jκ (RBP-Jκ), a DNA-binding protein. Ku antigen, RBP-Jκ, and p50 were found to bind to the DNA region containing the κB element in the p50 promoter. Supershift and competition experiments demonstrated that Ku antigen and RBP-Jκ bound sequence-specifically to downstream elements of κB at GCTTC and TGGGGG. mRNA expression and de novo synthesis of p50 were inhibited in cells transfected with the mutant gene expression constructs for IκBα, Ku8O, and RBP-Jκ. A reporter assay demonstrated that p50 transcription was positively mediated by NF-κB, Ku antigen, and RBP-Jκ and that the binding elements for these proteins were required for optimal p50 expression. The interaction of Ku antigen with RBP-Jκ and NF-κB p50 may act as a positive regulator of p50 expression in gastric cancer AGS cells.

AB - The p50 subunit of NF-κB is a transcription factor that regulates the expression of a variety of genes. Previously, we showed that the expression of Ku antigen, a DNA repair protein, is mediated by NF-κB in gastric cancer AGS cells (Lim, J. W., Kim, H., and Kim, K. H. (2002) J. Biol. Chem. 277, 46093-46100). In this study, we report that the inhibition of Ku activity reduced both p50 expression and nuclear NF-κB activity in AGS cells. A co-immunoprecipitation experiment demonstrated that Ku antigen interacted with recombination signal-binding protein Jκ (RBP-Jκ), a DNA-binding protein. Ku antigen, RBP-Jκ, and p50 were found to bind to the DNA region containing the κB element in the p50 promoter. Supershift and competition experiments demonstrated that Ku antigen and RBP-Jκ bound sequence-specifically to downstream elements of κB at GCTTC and TGGGGG. mRNA expression and de novo synthesis of p50 were inhibited in cells transfected with the mutant gene expression constructs for IκBα, Ku8O, and RBP-Jκ. A reporter assay demonstrated that p50 transcription was positively mediated by NF-κB, Ku antigen, and RBP-Jκ and that the binding elements for these proteins were required for optimal p50 expression. The interaction of Ku antigen with RBP-Jκ and NF-κB p50 may act as a positive regulator of p50 expression in gastric cancer AGS cells.

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