Phosphorylation-induced flexibility of proto-oncogenic Bcl3 regulates transcriptional activation by NF-κB p52 homodimer.

The overexpression of proto-oncogene Bcl3 is observed in various cancers. Bcl3 is extensively phosphorylated and associates with homodimers of NF-κB p50 and p52 to regulate transcription. Through cellular and biochemical assays, we observed that phospho-mimetic Glu substitution at Ser366 in addition to previously studied Ser33, 114 and 446 is necessary to switch Bcl3 from an IκB-like inhibitor to a transcriptional activator. To study interactive features of p52 and Bcl3, and phosphorylation- mediated changes in Bcl3 that regulate DNA-binding by p52, we performed HDX-MS of both Bcl3 and p52 within various complexes. Nature of interactions within Bcl3:(p52:p52) complex in presence and absence of DNA, differential flexibility of Bcl3, and allosteric changes in Bcl3 upon phospho-modifications revealed why a facile accommodation of DNA requires phosphorylation. The inhibitory nature of unphosphorylated Bcl3 on DNA binding by p52:p52 also relieved by a C-terminal deletion of Bcl3. Overall, this study revealed mechanistic bases of how Bcl3 phosphorylation regulates transcriptional potential of NF-κB and intricate cell physiology, a dysregulation of which can lead to cancers.