NEMO trimerizes through its coiled-coil C-terminal domain.
NEMO/IkappaB kinase (IKK) gamma is the regulatory component of the IKK complex comprising the two protein kinases, IKKalpha and IKKbeta. To investigate the self-assembly properties of NEMO and to understand further the mechanism of activation of the IKK complex, we purified wild-type and mutant NEMO expressed in Escherichia coli. In the absence of its IKK partners, recombinant NEMO (rNEMO) is a metastable functional monomer correctly folded, according to its fluorescence and far-UV CD spectra, which is binding specifically to the IKK complex. A minor fraction of rNEMO was found tightly associated with DnaK (E. coli Hsp70). We also examined the interaction of NEMO with prokaryotic and eukaryotic Hsp70, and we showed that the Hsp70-NEMO complex forms a supramolecular structure probably corresponding to an assembly intermediate. In vivo cross-linking experiments indicate that native NEMO in association with IKK is in equilibrium between a dimeric and a trimeric form. Similarly to native NEMO, a NEMO mutant deleted from its IKK binding N-terminal domain (residues 242-388) forms a stable trimeric coiled-coil, suggesting that the association of NEMO with IKK or with Hsp70 prevents incorrect interdomain pairing reactions that could lead to aggregation or to an non-native oligomeric state of rNEMO. We propose a model in which the activation of the IKK complex occurs through the trimerization of NEMO upon binding to a not yet identified upstream activator.
Agou
Fabrice
F
Unité de Régulation Enzymatique des Activités Cellulaires, Paris Cedex 15, France. fagou@pasteur.fr
Ye
Fei
F
Goffinont
Stéphane
S
Courtois
Gilles
G
Yamaoka
Shoji
S
Israël
Alain
A
Véron
Michel
M
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D051550:I-kappa B Kinase; D051379:Mice; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; C063842:dnaK protein, E coli