TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1–TRAF2 complex to sensitize tumor cells to TNFα

Vince, James E.; Chau, Diep; Callus, Bernard A.; Wong, Wendy Wei-Lynn; Hawkins, Christine J.; Schneider, Pascal; McKinlay, Mark A.; Benetatos, Christopher A.; Condon, Stephen M.; Chunduru, Srinivas K.; Yeoh, George; Brink, Robert; Vaux, David L.; Silke, John

doi:10.1083/jcb.200801010

Cited by 227 publications

(200 citation statements)

References 56 publications

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“…Receptor engagement results in lysosomal mediated degradation of TRAF2 and cIAP1 and activation of non-canonical NF-B resulting from NIK stabilization and subsequent processing of p100 to the active NF-B subunit p52 (7,10,11). In support of this idea, TRAF2 or cIAP1 knock-out MEFs were shown to have high constitutive levels of p52 and low levels of p100 compared with wild-type cells (6,12).…”

Section: Cellular Inhibitors Of Apoptosis (Ciaps)mentioning

confidence: 89%

“…Generation of MEFs-Generation of MEFs has been described in detail elsewhere (6,10). Briefly, primary MEFs were generated from embryos using standard protocols and then infected with SV40 large T antigen expressing lentivirus to generate immortal cell lines.…”

Section: Methodsmentioning

confidence: 99%

“…TWEAK Induces Degradation of cIAP1 RING Mutants-The TNFSF ligand TWEAK triggers lysosomal degradation of cIAP1 and TRAF2, whereas IAP antagonists induce proteasomal degradation of cIAP1 (4,6,7,10,15,30). It was therefore interesting to ask whether the RING activity of cIAP1 was also required for TWEAK-induced lysosomal degradation of cIAP1.…”

mentioning

confidence: 99%

“…Both forms of the ligand, however, drive cIAP1 and TRAF2 into a Triton X-100-insoluble fraction (10,31). We therefore further examined whether recruitment of TRAF2 to the insoluble fraction was dependent on cIAP1 RING dimerization.…”

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Tumor Necrosis Factor (TNF) Signaling, but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation, Requires cIAP1 RING Dimerization and E2 Binding

Feltham

Moulin

Vince

et al. 2010

Journal of Biological Chemistry

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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-B. 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-B, however, delayed activation of NF-B 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-B 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. Cellular inhibitors of apoptosis (cIAPs)5 were identified as proteins that bound directly to the adaptor protein TRAF2, which in turn binds to tumor necrosis factor receptor 2 (TNF-R2) (1). cIAPs have three baculoviral IAP repeat domains and interaction of cIAP1 with TRAF2 is dependent on residues within the N-terminal, baculoviral IAP repeat 1 (BIR1) (2, 3). cIAP1 and cIAP2 also bear a C-terminal RING E3 ligase domain, and it is this domain that is required for IAP antagonist compound (IAC)-induced autoubiquitylation and proteasomal degradation (4 -7). cIAPs control activation of the NF-B family of transcription factors in both unstimulated and cytokinetreated cells. In the absence of cytokines, together with TRAF2 and TRAF3, cIAPs ubiquitylate and promote proteasomal degradation of NF-B inducing kinase (NIK), thereby preventing NIK from activating NF-B2. Consistent with a central role for cIAPs in these processes, antagonism of IAPs by IACs is sufficient to activate the non-canonical NF-B pathway through NIK stabilization and processing of p100 to p52 (6 -9).TNF-like weak inducer of apoptosis (TWEAK) binding to its receptor Fn14 is a physiological activator of NF-B. Receptor engagement results in lysosomal mediated degradation of TRAF2 and cIAP1 and activation of non-canonical NF-B resulting from NIK stabilization and subsequent processing of p100 to the active NF-B subunit p52 (7,10,11). In...

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Section: Cellular Inhibitors Of Apoptosis (Ciaps)mentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Tumor Necrosis Factor (TNF) Signaling, but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation, Requires cIAP1 RING Dimerization and E2 Binding

Feltham

Moulin

Vince

et al. 2010

Journal of Biological Chemistry

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“…Generation of MEFs-Generation of MEFs and lentiviral particles has been described recently (18,32). Briefly, MEFs were generated from embryos in accordance with standard procedures and were infected with SV40 large T antigen-expressing lentivirus.…”

Section: Methodsmentioning

confidence: 99%

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization

Feltham

Bettjeman

Budhidarmo

et al. 2011

Journal of Biological Chemistry

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146

152

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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.The attachment of ubiquitin and ubiquitin-like molecules to intracellular proteins is a key process that regulates many cellular events (1). A hierarchical multienzyme cascade brings about the attachment of ubiquitin to substrate proteins. Ubiquitin is first activated by the ubiquitin-activating enzyme (E1) and then is transferred to the active site cysteine of the ubiquitin-conjugating enzyme (E2). Subsequently, ubiquitin-protein ligases (E3s) promote the transfer of ubiquitin from the E2 to lysine residues in target proteins. The RING 4 domain-containing E3 ligases, which are prevalent in mammals (Ͼ300), do not directly interact with ubiquitin; instead, the RING domain binds to the E2 and promotes the transfer of ubiquitin from the E2ϳubiquitin (E2ϳUb) thioester conjugate to the target protein (2). The mechanism by which the RING domain promotes ubiquitin transfer is not obvious because the RING-binding site on the E2 is distant from the active site. It has been proposed that the RING domain simply brings the E2ϳUb conjugate into close proximity with the substrate and that the increased availability of E2s promotes transfer of ubiquitin to the substrate. However, interactions between RING domains and E2s are generally transient and have modest affinity, and tighter binding does not always correlate with increased activity. In addition, not all RING-E2 complexes promote transfer, demonstrating that proximity alone is insufficient (3). Notably, the RING domains of Brca1 and c-Cbl interact with UbcH7 and UbcH5b, but only interaction with UbcH5b results in ubiquitin transfer (3, 4). Others have suggested that an allosteric mechanism is important whereby interaction of the RING domain with the E2 leads to changes at its active site that promote release of ubiquitin (5, 6). Consistent with this, binding of the G2BR domain of the E3 gp78...

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Oxyphenisatin acetate (NSC59687) triggers a cell starvation response leading to autophagy, mitochondrial dysfunction, and autocrineTNFα‐mediated apoptosis

et al. 2013

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Oxyphenisatin (3,3-bis(4-hydroxyphenyl)-1H-indol-2-one) and several structurally related molecules have been shown to have in vitro and in vivo antiproliferative activity. This study aims to confirm and extend mechanistic studies by focusing on oxyphenisatin acetate (OXY, NSC 59687), the pro-drug of oxyphenisatin. Results confirm that OXY inhibits the growth of the breast cancer cell lines MCF7, T47D, HS578T, and MDA-MB-468. This effect is associated with selective inhibition of translation accompanied by rapid phosphorylation of the nutrient sensing eukaryotic translation initiation factor 2α (eIF2α) kinases, GCN2 and PERK. This effect was paralleled by activation of AMP-activated protein kinase (AMPK) combined with reduced phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6K and 4E-BP1. Microarray analysis highlighted activation of pathways involved in apoptosis induction, autophagy, RNA/protein metabolism, starvation responses, and solute transport. Pathway inhibitor combination studies suggested a role for AMPK/mTOR signaling, de novo transcription and translation, reactive oxygen species (ROS)/glutathione metabolism, calcium homeostasis and plasma membrane Na+/K+/Ca2+ transport in activity. Further examination confirmed that OXY treatment was associated with autophagy, mitochondrial dysfunction, and ROS generation. Additionally, treatment was associated with activation of both intrinsic and extrinsic apoptotic pathways. In the estrogen receptor (ER) positive MCF7 and T47D cells, OXY induced TNFα expression and TNFR1 degradation, indicating autocrine receptor-mediated apoptosis in these lines. Lastly, in an MCF7 xenograft model, OXY delivered intraperitoneally inhibited tumor growth, accompanied by phosphorylation of eIF2α and degradation of TNFR1. These data suggest that OXY induces a multifaceted cell starvation response, which ultimately induces programmed cell death.The mechanistic basis for oxyphenisatin acetate anti-cancer activity remains unresolved. This study demonstrates that exposure is associated with an acute nutrient deprivation response leading to translation inhibition, induction of autophagy, transient estrogen receptor (ER) stress and mitochondrial dysfunction. Ultimately these effects promote apoptosis induction, which in ER+ breast cancer cells is mediated by autocrine TNFα production. This is the first study implicating a nutrient deprivation response as central to the downstream effects of oxyphenisatin acetate.

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TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1–TRAF2 complex to sensitize tumor cells to TNFα

Cited by 227 publications

References 56 publications

Tumor Necrosis Factor (TNF) Signaling, but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation, Requires cIAP1 RING Dimerization and E2 Binding

Tumor Necrosis Factor (TNF) Signaling, but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation, Requires cIAP1 RING Dimerization and E2 Binding

Smac Mimetics Activate the E3 Ligase Activity of cIAP1 Protein by Promoting RING Domain Dimerization

Oxyphenisatin acetate (NSC59687) triggers a cell starvation response leading to autophagy, mitochondrial dysfunction, and autocrineTNFα‐mediated apoptosis

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