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-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.