Abstract
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.
Highlights
Among the signaling pathways known to date, the transcription factor NF-B is one of the most intensely studied regulators of gene expression, playing a crucial role in inflammatory responses, cell proliferation, and apoptosis [1,2,3,4]
To understand further the molecular role of NEMO in the activation of the IKK complex, we have studied the biochemical properties of purified murine NEMO recombinant protein as well as of a truncated C-terminal domain produced in Escherichia coli
Expression and Purification of Recombinant NEMO—Recombinant His-tagged NEMO was expressed in E. coli at 22 °C to prevent the formation of inclusion bodies and purified following the protocol under “Experimental Procedures.”
Summary
Materials—Octyl glucoside (OG) and dodecyl maltoside (DDM) were from Roche Molecular Biochemicals. An rNEMO sample was applied to a Resource Q column (6 ml, Amersham Biosciences) equilibrated in the buffer A and eluted with a 180-ml linear gradient 30 –500 mM KCl. Fractions containing rNEMO (according to the OD profile and to SDS-PAGE) were pooled (15 ml) and dialyzed against 1 liter of 10 mM potassium phosphate, pH 7.0, 1 mM dithioerythritol, and 1 mM DDM. In Vitro Binding Assay with Prokaryotic and Eukaryotic Hsp70s and Co-immunoprecipitation—Ni-NTA magnetic agarose beads suspensions (25 l, Qiagen) equilibrated with the buffer C (20 mM Tris-HCl, pH 8.0, 20 mM imidazole, 300 mM NaCl, 1 mM DDM, 5% glycerol, and 0.1 mM dithioerythritol) were incubated for 30 min at 4 °C with the purified His-tagged NEMO. The fluorescence yield was determined as described previously [26]
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