Transglutaminase 2 mediates polymer formation of I-κBα through C-terminal glutamine cluster

Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-κB (NF-κB) independently of I-κB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-κBα(I-κBα). TGase 2 catalyzes covalent isopeptide bond formation between the peptide bound-glutamine and the lysine residues. Using matrix-assisted laser desorption ionization time-of-flight mass spectra analysis of I-κBα polymers cross-linked by TGase 2, as well as synthetic peptides in an in vitro competition assay, we identified a glutamine cluster at the C terminus of I-κBα (amino acids 266-268) that appeared to play a key role in the formation of I-κBα polymers. Although there appeared to be no requirement for specific lysine residues, we found a considerably higher preference for the use of lysine residues at positions 21, 22, and 177 in TGase 2-mediated cross-linking of I-κBα. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266-268 reversed I-κBα polymerization in vitro. Furthermore, the depletion of free I-κBα in EcR/TG cells was completely rescued in vivo by transfection of mutant I-κBαs in glutamine sites (Q266G, Q267G, and Q313G) as well as in a lysine site (K177G). These findings provide additional clues into the mechanism by which TGase 2 contributes to the inflammatory process via activation of NF-κB.