Transglutaminase 2 mediates polymer formation of I-kappaBalpha through C-terminal glutamine cluster.
Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-kappaB (NF-kappaB) independently of I-kappaB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-kappaBalpha (I-kappaBalpha). 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-kappaBalpha 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-kappaBalpha (amino acids 266-268) that appeared to play a key role in the formation of I-kappaBalpha 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-kappaBalpha. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266-268 reversed I-kappaBalpha polymerization in vitro. Furthermore, the depletion of free I-kappaBalpha in EcR/TG cells was completely rescued in vivo by transfection of mutant I-kappaBalphas 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-kappaB.
Park
Sung-Soo
SS
Molecular Oncology Branch, Division of Basic Sciences, Research Institute, National Cancer Center, 809 Madu-1-dong, Ilsandong-gu, Goyang, Gyeonggi-do 411-769, Republic of Korea.
Kim
Jung-Mo
JM
Kim
Dae-Seok
DS
Kim
In-Hoo
IH
Kim
Soo-Youl
SY
D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry* / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000378:metabolism*
D001704:Biopolymers; D020672:I-kappa B Proteins; D016328:NF-kappa B; D005973:Glutamine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins