Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347.
NEMO is an essential regulatory component of the IkappaB kinase (IKK) complex, which controls activation of the NF-kappaB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H(2)O(2)) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H(2)O(2) decreases TNFalpha-induced IKK activity in NEMO-reconstituted cells, and that TNFalpha has a diminished ability to activate NF-kappaB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.
Herscovitch
Melanie
M
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Comb
William
W
Ennis
Thomas
T
Coleman
Kate
K
Yong
Sheila
S
Armstead
Brinda
B
Kalaitzidis
Demetrios
D
Chandani
Sushil
S
Gilmore
Thomas D
TD
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051550:I-kappa B Kinase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004220:Disulfides; D047908:Intracellular Signaling Peptides and Proteins; C514981:NEMO protein, mouse; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D006861:Hydrogen Peroxide; D051550:I-kappa B Kinase; D003545:Cysteine