The nuclear IκB protein IκBζ specifically binds NF-κB p50 homodimers and forms a ternary complex on κB DNA

Although they share sequence homology to classical cytoplasmic I kappa B inhibitors of transcription factor NF-kappa B, the proteins I kappa B zeta, Bcl-3, and I kappa BNS function in the nucleus as factors that influence NF-kappa B-dependent gene expression profiles. Through the use of purified recombinant proteins and by comparison with the classical I kappa B protein I kappa B alpha, we have discovered mechanistic details of the interaction between I kappa B zeta and functional NF-kappa B dimers. Whereas I kappa B alpha and other classical I kappa B proteins bind tightly to NF-kappa B dimers that possess the p65 subunit, I kappa B zeta binds preferentially to NF-kappa B p50 homodimers. This altered specificity is particularly interesting in light of the fact that both NF-kappa B subunits exhibit high sequence and structural homology, while the I kappa B alpha and I kappa B zeta proteins are also conserved in primary amino acid sequence. We further show that I kappa B zeta is capable of forming a stable ternary complex with the NF-kappa B p50 homodimer and kappa B DNA. Again, this is a stark contrast from I kappa B alpha, which inhibits NF-kappa B p65 homodimer binding to NF-kappa B target DNA sequences. Removal of the DNA sequences flanking the NF-kappa B binding site does not directly affect the interaction of p50 and I kappa B zeta. Rather, we have discovered that the carboxy-terminal glycine-rich region of the NF-kappa B p50 homodimer is involved in mediating high-affinity binding of I kappa B zeta and NF-kappa B p50. We conclude that the NF-kappa B p50 homodimer functions as a legitimate activator of gene expression through formation of a ternary complex between itself, I kappa B zeta, and DNA. The requirement for formation of this complex could explain why the nuclear I kappa B protein I kappa B zeta is absolutely required for expression of the pluripotent pro-inflammatory cytokine interleukin-6 in peritoneal macrophages. (C) 2008 Elsevier Ltd. All rights reserved.