NEMO oligomerization and its ubiquitin-binding properties

Abstract

The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

“…1). The preference for linear ubiquitin chains is in agreement with previous studies, which showed that the NEMO-UBAN has an up to 100ϫ higher affinity to linear di-ubiquitin over Lys-63-, or Lys-48-linked di-ubiquitin (14,16,17). This might suggest that in a cellular environment where different ubiquitin chain types exist, e.g.…”

Nuclear initiated NF-κB signaling: NEMO and ATM take center stage

“…Indeed, NEMO was reported to exist predominantly as a monomer in IKKα and IKKβ double knockout MEFs [92]. In contrast, a recent biochemical analysis indicates that an "apo" form of NEMO 1-355 missing the C-terminal ZF domain forms a dimer and a weaker tetramer, arising from self-association of the N-terminal domain, with an apparent K d of 25.6 µM (dimer-tetramer equilibrium) [93]. Moreover, addition of an IKK-derived NEMO-binding peptide eliminates the NEMO tetramer formation in vitro, thus suggesting that IKK prevents NEMO tetramerization.…”

“…Since (i) ATM usually (although not absolutely) requires the S/ T-Q motif in its substrates for phosphorylation [94] and (ii) Q86N and Q86A point mutations of NEMO completely abrogate NF-κB activation by DNA-damaging agents without affecting activation by LPS [54], it can be concluded that the ATM accessibility of both S85 and Q86 residues is required to mediate S85 phosphorylation and NF-κB activation. Ivins et al [93] demonstrated that binding of diubiquitin at the NOA/NUB/UBAN domain induced a modest but measurable structural alteration in the N-terminal CC1 domain that is located ~200 aa away. This suggests the possibility that a binding event in a region away from the S85-Q86 motif may cause a conformational shift to permit efficient ATM accessibility to the SQ motif.…”

“…However, the ZF domain is not required for NEMO association with the E3 ligase PIASy, nor is it required for PIASy-mediated SUMOylation in vitro [60]. The ZF is suggested to destabilize the dimeric coil-coil structure of full-length NEMO [93]. Cordier et al [96] demonstrated that the NEMO ZF is a ubiquitin-binding module.…”

“…Moreover, addition of an IKK-derived NEMO-binding peptide eliminates the NEMO tetramer formation in vitro, thus suggesting that IKK prevents NEMO tetramerization. The presence of the ZF domain destabilizes the dimer configuration, causing NEMO to aggregate [93]. In cell extracts prepared from HeLa or HEK293 cells, an IKK-free NEMO fraction elutes at [100 kDa in gel filtration chromatography [15].…”