UC San Diego

Nuclear factor kappa B (NF-kB) plays a crucial role in the regulation of inflammation and immune response. NF-kB is composed to five family members, RelA, RelB, cRel, p52, and p50, which form combinatorial homo- and heterodimers. In resting cells, NF-kB dimers remain inactive and bound to an inhibitor of NF-kB (IkB) protein. IkB Kinase (IKK) phosphorylates IkB leading to its degradation and release of NF-kB into the nucleus. The function of the ternary IKK complex (IKK1, IKK2, NEMO) and the mechanism of NF-kB activation is not known. In an effort to understand the structure, the IKK complex has been expressed and isolated in insect, E. coli, and mammalian cells. However, full length wild type IKK proteins do not form a stable complex post-overexpression and are difficult to purify. This suggests that aberrant post-translational modifications in the overexpression system might alter the stability of the native complex.

B-cell lymphoma 3-encoded protein (Bcl3) is an atypical IkB which binds to DNA:p52 and DNA:p50 binary complexes ultimately repressing or activating DNA transcription. The phosphorylation sites which affect DNA binding are not well known. The phosphorylation sites Ser33, Ser114, and Ser446 known to be crucial for DNA binding were analyzed through EMSA and wound healing assay. Results indicate that Ser114 is the critical residue in forming ternary complexes with DNA:p52 and DNA:p50 binary complexes. Our results elucidate the mechanism of how Bcl3 acts as a transcriptional regulator.