The Ku antigen-recombination signal-binding protein Jkappa complex binds to the nuclear factor-kappaB p50 promoter and acts as a positive regulator of p50 expression in human gastric cancer cells.
The p50 subunit of NF-kappaB 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-kappaB 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-kappaB activity in AGS cells. A co-immunoprecipitation experiment demonstrated that Ku antigen interacted with recombination signal-binding protein Jkappa (RBP-Jkappa), a DNA-binding protein. Ku antigen, RBP-Jkappa, and p50 were found to bind to the DNA region containing the kappaB element in the p50 promoter. Supershift and competition experiments demonstrated that Ku antigen and RBP-Jkappa bound sequence-specifically to downstream elements of kappaB at GCTTC and TGGGGG. mRNA expression and de novo synthesis of p50 were inhibited in cells transfected with the mutant gene expression constructs for IkappaBalpha, Ku80, and RBP-Jkappa. A reporter assay demonstrated that p50 transcription was positively mediated by NF-kappaB, Ku antigen, and RBP-Jkappa and that the binding elements for these proteins were required for optimal p50 expression. The interaction of Ku antigen with RBP-Jkappa and NF-kappaB p50 may act as a positive regulator of p50 expression in gastric cancer AGS cells.
Lim
Joo Weon
JW
Department of Pharmacology and the Institute of Gastroenterology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
Kim
Hyeyoung
H
Kim
Kyung Hwan
KH
D034961:Antigens, Nuclear / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D000072200:Ku Autoantigen; D016328:NF-kappa B / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics; D045522:Recombinases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013274:Stomach Neoplasms; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D016328:NF-kappa B; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D021122:Protein Subunits; C497570:RBPJ protein, human; D045522:Recombinases; D014157:Transcription Factors; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen