Differential pp40^IκB-β Inhibition of DNA Binding by rel Proteins

Abstract

Regulation of gene expression by members of the NF-κB/rel transcription factor family is a central component of signal transduction pathways utilized by many cellular processes, including lymphocyte activation, embryonic development, and oncogenesis. The members of the NF-κB/rel transcription factor family are regulated by association with a family of inhibitor (IκB) proteins (IκB) proteins. To address the importance of the association between rel and IκB proteins for oncogenesis by rel proteins, we characterized rel-IκB interactions in chicken embryo fibroblasts (CEF) infected with retroviral vectors encoding the avian c-rel (p68^c-rel), v-rel (p59^v-rel), and IκB-β (pp40^IκB-β) proteins. In these experiments, the p59^v-rel:pp40^IκB-β ratio in coinfected CEF was nearly identical to the p59^v-rel:pp40^IκB-β ratio in v-rel-transformed cells. The avian IκB-β protein, pp40IκB-β, was able to associate with both the nononcogenic p68^c-rel and the oncogenic p59^v-rel. Association of p68^c-rel with pp40^IκB-β in coinfected CEF resulted in inhibition of the DNA-binding activity of p68^c-rel. Anti-pp40^IκB-β serum was able to restore DNA binding to p68^c-rel in the presence of high levels of pp40^IκB-β, indicating that pp40^IκB-β functions in a trans-acting manner to inhibit DNA binding by p68^c-rel. In contrast, sequence-specific DNA binding by the oncogenic v-rel protein, p59^v-rel, was not abolished by pp40^IκB-β in coinfected CEF. Anti-pp40^IκB-β serum did not immunoprecipitate the p59^v-rel-DNA adduct or alter the electrophoretic mobility of the p59^v-rel-DNA adduct, consistent with the idea that pp40^IκB-β and DNA are competitive inhibitors for the same or overlapping domains on rel proteins. Internal v-rel-derived sequences were identified that are responsible for loss of pp40^IκB-β-mediated inhibition of DNA binding by p59^v-rel. Loss of pp40^IκB-β-mediated inhibition of DNA binding by recombinant v/c-rel proteins was not sufficient for oncogenic activation of c-rel. Instead, removal of C-terminal c-rel-derived sequences in addition to loss of pp40^IκB-β-mediated inhibition of DNA binding was required for oncogenic activation of c-rel. These results demonstrate the presence of an interaction between internal and C-terminal regions of the c-rel protein that is important for the ability of c-rel to regulate the proliferation of lymphoid cells.