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ABSTRACT: The Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death, whose amplification has been correlated with tumor progression. Due to the presence of a RING domain, IAP proteins are classed as ubiquitin ligases and regulate cell survival by orchestrating a variety of ubiquitin modifications. Ubiquitin protein modification is fundamental in cell signaling and different ubiquitin modifications may label proteins for destruction, relocalization or provide a recruitment platform for ubiquitin binding proteins. Ubiquitin performs a myriad of different functions because it can be conjugated to a large range of target proteins through numerous different types of ubiquitin linkages. Despite the fact that ubiquitin is extremely versatile, the E3s such as the IAPs provide an important level of control due to their specificity for certain substrates. Several recent reviews have discussed the role of IAPs in regulating immune signaling so we have therefore focused our review on the interplay between IAPs and ubiquitin and discussed the importance of this relationship for the regulation of themselves, specific substrates and various cell death and survival signaling pathways.
International Union of Biochemistry and Molecular Biology Life 02/2012; 64(5):411-8. · 3.51 Impact Factor
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Maryline Moulin,
Holly Anderton,
Anne K Voss,
Tim Thomas,
Wendy Wei-Lynn Wong,
Aleksandra Bankovacki, Rebecca Feltham,
Diep Chau,
Wendy D Cook,
John Silke,
David L Vaux
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ABSTRACT: Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X-linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid-embryonic lethality. In contrast, Xiap(-/-)cIap2(-/-) mice were viable. The death of cIap2(-/-)cIap1(-/-) double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor-interacting protein kinase 1 (Ripk1) allowed Xiap(-/-)cIap1(-/-) double mutants to survive past birth, and prolonged cIap2(-/-)cIap1(-/-) embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid-gestation defect of cIap2(-/-)cIap1(-/-) embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.
The EMBO Journal 02/2012; 31(7):1679-91. · 9.20 Impact Factor
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Rebecca Feltham,
Bodhi Bettjeman,
Rhesa Budhidarmo,
Peter D Mace,
Sarah Shirley,
Stephen M Condon,
Srinivas K Chunduru,
Mark A McKinlay,
David L Vaux,
John Silke,
Catherine L Day
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ABSTRACT: The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.
Journal of Biological Chemistry 03/2011; 286(19):17015-28. · 4.77 Impact Factor
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ABSTRACT: Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.
Journal of Biological Chemistry 03/2010; 285(23):17525-36. · 4.77 Impact Factor
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Tobias L Haas,
Christoph H Emmerich,
Björn Gerlach,
Anna C Schmukle,
Stefanie M Cordier,
Eva Rieser, Rebecca Feltham,
James Vince,
Uwe Warnken,
Till Wenger,
Ronald Koschny,
David Komander,
John Silke,
Henning Walczak
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ABSTRACT: TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.
Molecular cell 12/2009; 36(5):831-44. · 14.61 Impact Factor
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James E. Vince,
Delara Pantaki, Rebecca Feltham,
Peter D. Mace,
Stephanie M. Cordier,
Anna C. Schmuckle,
Angelina J. Davidson,
Bernard A. Callus,
Wendy Wei-Lynn Wong,
Ian E. Gentle,
Holly Carter,
Erinna F. Lee,
Henning Walczak,
Catherine L. Day,
David L. Vaux,
John Silke
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ABSTRACT: TNF receptor associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of
several members of the TNF receptor superfamily (TNFRSF), including the TNF-TNFR1 ligand-receptor complex. Here we define
a cIAP1/2 interacting motif (CIM) within the TRAF-N domain of TRAF2, and use TRAF2 CIM mutants to determine the role of TRAF2
and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1 dependent signaling. We show that both the TRAF2 RING
domain and the TRAF2 CIM are required to regulate NF-κB inducing kinase (NIK) stability and suppress constitutive non-canonical
NF-κB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM mutated TRAF2 showed reduced canonical NF-κB
activation and TNF induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions.
However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF induced apoptosis. These results
show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-κB signaling and that efficient activation
of NF-κB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.
Journal of Biological Chemistry 10/2009; · 4.77 Impact Factor
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James E Vince,
Delara Pantaki, Rebecca Feltham,
Peter D Mace,
Stephanie M Cordier,
Anna C Schmukle,
Angelina J Davidson,
Bernard A Callus,
Wendy Wei-Lynn Wong,
Ian E Gentle,
Holly Carter,
Erinna F Lee,
Henning Walczak,
Catherine L Day,
David L Vaux,
John Silke
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ABSTRACT: Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-kappaB-inducing kinase stability and suppress constitutive noncanonical NF-kappaB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-kappaB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-kappaB signaling and that efficient activation of NF-kappaB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.
Journal of Biological Chemistry 10/2009; 284(51):35906-15. · 4.77 Impact Factor
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ABSTRACT: Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo.
Journal of Biological Chemistry 10/2008; 283(46):31633-40. · 4.77 Impact Factor
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James E Vince,
W Wei-Lynn Wong,
Nufail Khan, Rebecca Feltham,
Diep Chau,
Afsar U Ahmed,
Christopher A Benetatos,
Srinivas K Chunduru,
Stephen M Condon,
Mark McKinlay,
Robert Brink,
Martin Leverkus,
Vinay Tergaonkar,
Pascal Schneider,
Bernard A Callus,
Frank Koentgen,
David L Vaux,
John Silke
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ABSTRACT: XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.
Cell 12/2007; 131(4):682-93. · 32.40 Impact Factor
