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NEMO oligomerization and its ubiquitin-binding properties

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 regulat...

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Autores principales: Ivins, Frank J., Montgomery, Mark G., Smith, Susan J. M., Morris-Davies, Aylin C., Taylor, Ian A., Rittinger, Katrin
Formato: Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708934/
https://www.ncbi.nlm.nih.gov/pubmed/19422324
http://dx.doi.org/10.1042/BJ20090427
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author Ivins, Frank J.
Montgomery, Mark G.
Smith, Susan J. M.
Morris-Davies, Aylin C.
Taylor, Ian A.
Rittinger, Katrin
author_facet Ivins, Frank J.
Montgomery, Mark G.
Smith, Susan J. M.
Morris-Davies, Aylin C.
Taylor, Ian A.
Rittinger, Katrin
author_sort Ivins, Frank J.
collection PubMed
description 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.
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spelling pubmed-27089342009-07-10 NEMO oligomerization and its ubiquitin-binding properties Ivins, Frank J. Montgomery, Mark G. Smith, Susan J. M. Morris-Davies, Aylin C. Taylor, Ian A. Rittinger, Katrin Biochem J Research Article 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. Portland Press Ltd. 2009-06-26 2009-07-15 /pmc/articles/PMC2708934/ /pubmed/19422324 http://dx.doi.org/10.1042/BJ20090427 Text en © 2009 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by-nc/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Ivins, Frank J.
Montgomery, Mark G.
Smith, Susan J. M.
Morris-Davies, Aylin C.
Taylor, Ian A.
Rittinger, Katrin
NEMO oligomerization and its ubiquitin-binding properties
title NEMO oligomerization and its ubiquitin-binding properties
title_full NEMO oligomerization and its ubiquitin-binding properties
title_fullStr NEMO oligomerization and its ubiquitin-binding properties
title_full_unstemmed NEMO oligomerization and its ubiquitin-binding properties
title_short NEMO oligomerization and its ubiquitin-binding properties
title_sort nemo oligomerization and its ubiquitin-binding properties
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708934/
https://www.ncbi.nlm.nih.gov/pubmed/19422324
http://dx.doi.org/10.1042/BJ20090427
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