NEMO oligomerization in the dynamic assembly of the IkappaB kinase core complex.
NF-kappaB essential modulator (NEMO) plays an essential role in the nuclear factor kappaB (NF-kappaB) pathway as a modulator of the two other subunits of the IkappaB kinase (IKK) complex, i.e. the protein kinases, IKKalpha and IKKbeta. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation, we observed stimulus-dependent dynamic IKK complex assembly. In wild-type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350-kDa high-molecular-mass complex. In response to constitutive NF-kappaB stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high-molecular-mass complex of 440 kDa which displayed increased IKK activity. This stimulus-dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin-1beta. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax-activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKKalpha and IKKbeta, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKKbeta dimers are present that are less stable than IKKalpha dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKKalpha-NEMO and IKKbeta-NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKKalpha-IKKbeta heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKKalpha and IKKbeta.
Fontan
Elisabeth
E
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 2185, Institut Pasteur, Paris, France.
Traincard
François
F
Levy
Samuel G
SG
Yamaoka
Shoji
S
Véron
Michel
M
Agou
Fabrice
F
D000818:Animals; D002499:Centrifugation, Density Gradient; D005347:Fibroblasts / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D051550:I-kappa B Kinase / Q000096:biosynthesis*; D053583:Interleukin-1beta / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D051379:Mice; D016328:NF-kappa B / Q000502:physiology; D020836:Protein Structure, Quaternary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
C520609:Ikbkg protein, rat; D053583:Interleukin-1beta; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D051550:I-kappa B Kinase