A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-κB (nuclear factor κB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-κB-inducing kinase) and regulate the non-canonical NF-κB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys48- and Lys63-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain–ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile44 of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.
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Research Article|
December 12 2008
Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP21
John W. Blankenship;
John W. Blankenship
2
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Eugene Varfolomeev;
Eugene Varfolomeev
3
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Tatiana Goncharov;
Tatiana Goncharov
3
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Anna V. Fedorova;
Anna V. Fedorova
3
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Donald S. Kirkpatrick;
Donald S. Kirkpatrick
†Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Anita Izrael-Tomasevic;
Anita Izrael-Tomasevic
†Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Lilian Phu;
Lilian Phu
†Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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David Arnott;
David Arnott
†Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Mariam Aghajan;
Mariam Aghajan
4
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Kerry Zobel;
Kerry Zobel
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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J. Fernando Bazan;
J. Fernando Bazan
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Wayne J. Fairbrother;
Wayne J. Fairbrother
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
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Kurt Deshayes;
Kurt Deshayes
5
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
5Correspondence may be addressed to either of these authors (email deshayes@gene.com or domagoj@gene.com).
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Domagoj Vucic
Domagoj Vucic
5
*Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, U.S.A.
5Correspondence may be addressed to either of these authors (email deshayes@gene.com or domagoj@gene.com).
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Publisher: Portland Press Ltd
Received:
September 15 2008
Revision Received:
October 17 2008
Accepted:
October 22 2008
Accepted Manuscript online:
October 22 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 417 (1): 149–165.
Article history
Received:
September 15 2008
Revision Received:
October 17 2008
Accepted:
October 22 2008
Accepted Manuscript online:
October 22 2008
Connected Content
This is a commentary on:
Inhibitors of apoptosis catch ubiquitin
Citation
John W. Blankenship, Eugene Varfolomeev, Tatiana Goncharov, Anna V. Fedorova, Donald S. Kirkpatrick, Anita Izrael-Tomasevic, Lilian Phu, David Arnott, Mariam Aghajan, Kerry Zobel, J. Fernando Bazan, Wayne J. Fairbrother, Kurt Deshayes, Domagoj Vucic; Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2. Biochem J 1 January 2009; 417 (1): 149–165. doi: https://doi.org/10.1042/BJ20081885
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