B cell leukemia 3 (Bcl-3) is an essential negative regulator of NF-κB during Toll-like receptor and TNF receptor signaling. Bcl-3 also interacts with a number of transcriptional regulators, including homodimers of the NF-κB p50 subunit. Deletion of Bcl-3 results in increased NF-κB p50 ubiquitination and proteasomal degradation and increased inflammatory gene expression. We employed immobilized peptide array technology to define a region of p50 required for the formation of a Bcl-3·p50 homodimer immunosuppressor complex. Our data demonstrate that amino acids 359-361 and 363 of p50 are critical for interaction with Bcl-3 and essential for Bcl-3-mediated inhibition of inflammatory gene expression. Bcl-3 is unable to interact with p50 when these amino acids are mutated, rendering it incapable of inhibiting the transcriptional activity of NF-κB. Bcl-3 interaction-defective p50 is hyperubiquitinated and has a significantly reduced half-life relative to wild-type p50. Nfkb1(-/-) cells reconstituted with mutated p50 precursor p105 are hyperresponsive to TNFα stimulation relative to wild-type p105, as measured by inflammatory gene expression. Mutant p105 recapitulates a Bcl3(-/-) phenotype. This study demonstrates that interaction with p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3 and further highlights the importance of p50 homodimer stability in the control of NF-κB target gene expression.
Keywords: Cytokine Induction; Inflammation; Mutagenesis; NF-κB transcription factor; Ubiquitination.