Inhibition of transcription by B cell Leukemia 3 (Bcl-3) protein requires interaction with nuclear factor κB (NF-κB) p50.
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.
Department of Biochemistry, University College Cork, Cork, Ireland.
Patricia E
PE
Collins
Department of Life Sciences and the Materials and Surface Science Institute, University of Limerick, Limerick, Ireland.
Patrick A
PA
Kiely
Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow G12 8TA, Scotland, Glasgow, United Kingdom. Electronic address: ruaidhri.carmody@glasgow.ac.uk.
Ruaidhrí J
RJ
Carmody
D000595:Amino Acid Sequence; D000818:Animals; D000081242:B-Cell Lymphoma 3 Protein; D018360:Crystallography, X-Ray; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D052002:NF-kappa B p50 Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D000081242:B-Cell Lymphoma 3 Protein; C000631344:BCL3 protein, human; C000631345:Bcl3 protein, mouse; D052002:NF-kappa B p50 Subunit; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; C073701:Nfkb1 protein, mouse
Cytokine Induction; Inflammation; Mutagenesis; NF-κB transcription factor; Ubiquitination