Pan, W.; Biswas, T.; Shahabi, S.; Suryajaya, W.; Vasquez, A.; Du, Q.; Ghosh, G.; Wang, V. Y.-F.

The overexpression of proto-oncogenic protein Bcl3 is observed in various cancers. Bcl3 is extensively phosphorylated and associates with NF-{kappa}B p50 and p52 homodimers to regulate transcription. Through cellular and biochemical assays, we observed that phospho-mimetic Glu substitution of Bcl3 Ser366 in addition to previously studied Ser33, 114 and 446 is necessary to switch it from an I{kappa}B-like inhibitor to a transcriptional activator. To study interactive features of p52 homodimer and Bcl3, and phospho-modification mediated changes in Bcl3 that regulates 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, and differential flexibility and allosteric changes in Bcl3 upon phospho-modifications revealed why a facile accommodation of DNA requires phosphorylations. The inhibitory nature of unphosphorylated Bcl3:(p52:p52) complex for DNA binding was also relieved by deletion of Bcl3 C-terminal 28 residues. Overall, this study revealed mechanistic bases of how phospho-modification of Bcl3 regulate transcriptional potential of NF-{kappa}B and intricate cell physiology, a dysregulation of which can lead to cancers.