DIFFERENTIAL PP40IKB-BETA INHIBITION OF DNA-BINDING BY REL PROTEINS

Regulation of gene expression by members of the NF-kappaB/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-kappaB/rel transcription factor family are regulated by association with a family of inhibitor (IkappaB) proteins. To address the importance of the association between rel and IkappaB proteins for oncogenesis by rel proteins, we characterized rel-IkappaB interactions in chicken embryo fibroblasts (CEF) infected with retroviral vectors encoding the avian c-rel (p68c-rel), v-rel (p59v-rel), and IkappaB-beta (pp40IkappaB-beta) proteins. In these experiments, the p59v-rel:pp40IkappaB-beta ratio in coinfected CEF was nearly identical to the p59v-rel:pp40IkappaB-beta ratio in v-rel-transformed cells. The avian IkappaB-beta protein, pp40IkappaB-beta, was able to associate with both the nononcogenic p68c-rel and the oncogenic p59v-rel. Association of p68c-rel with pp40IkappaB-beta in coinfected CEF resulted in inhibition of the DNA-binding activity of p68c-rel. Anti-pp40IkappaB-beta serum was able to restore DNA binding to p68c-rel in the presence of high levels of pp40IkappB-beta, indicating that pp40IkappaB-beta functions in a trans-acting manner to inhibit DNA binding by p68c-rel. In contrast, sequence-specific DNA binding by the oncogenic v-rel protein, p59v-rel, was not abolished by pp40IkappaB-beta in coinfected CEF. Anti-pp40IkappaB-beta serum did not immunoprecipitate the p59v-rel-DNA adduct or after the electrophoretic mobility of the p59v-rel-DNA adduct, consistent with the idea that pp40IkappaB-beta 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 pp40IkappaB-beta-mediated inhibition of DNA binding by p59v-rel. Loss of pp40IkappaB-beta-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 pp40IkappaB-beta-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.