cIAP1 and cIAP2 Facilitate Cancer Cell Survival by Functioning as E3 Ligases that Promote RIP1 Ubiquitination

Abstract

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.

NIK promotes tissue destruction independently of the alternative NF-κB pathway through TNFR1/RIP1-induced apoptosis

“…Previous studies reported that, at least in certain cell types, Tweak-and SM-induced cell death relied on the NIK-dependent neosynthesis of endogenous TNFα via induction of the classical and/or the alternative NF-κB pathway. 11,26,27,33 We can rule out this possibility in our in vitro and in vivo genetic models. Indeed, we showed that Tweak-, LTβR agonist-or SM-induced NIK stabilization in MEFs is by itself not sufficient to induce cell death and that NIK deficiency protected the MEFs from TNFR1-mediated death induced by exogenous TNFα.…”

“…MEF cell extracts were made in NP-40-containing buffer as previously described. 11 Cell extracts were either used directly for western blotting or for co-immunoprecipitation experiments. For caspase-8/RIP1 complex analysis, 20 μl of protein G-Agarose beads (Santa Cruz) were incubated O/N with 2 μg of anti-murine caspase-8 antibody at 4°C.…”

“…[5][6][7][8][9] c-IAP1/2 and LUBAC target RIP1 for K11, K63 and linear polyubiquitination priming RIP1 for the recruitment of two kinase-containing complexes, namely, TAK1/TAK1-binding protein 1 (TAB1)/TAB2 and NEMO/ inhibitor of NF-κB kinase α (IKKα)/IKKβ. 5,[10][11][12][13] Ultimately, activation of IKKβ leads to the phosphorylation and proteasomal degradation of IκBα and the nuclear translocation of NF-κB, a pathway defined as the classical, or the canonical, NF-κB pathway. [14][15][16] Besides its role in the activation of NF-κB, the dissociation of complex I from TNFR1 triggers the recruitment of Fas-associated protein with death domain (FADD) and caspase-8 to form a cytosolic death complex, initially defined as complex II.…”

“…[21][22][23][24][25] Synthetic small molecules mimicking Smac (Smac mimetics (SMs)) emerged as tools to induce c-IAP1/2 depletion and were consequently shown to sensitize particular cancer cell lines to TNFR1-mediated RIP1-dependent death. 11,[26][27][28] Apart from these chemicals, a set of biological ligand of the TNF family, such as lymphotoxin LTα 1 β 2 , CD40L or Tweak, also appeared to target c-IAP1/2 toward the proteasomal and/ or lysosomal degradative pathways. [29][30][31][32][33][34] c-IAP1/2 depletion leads to NIK (NF-κB-inducing kinase) stabilization and the phosphorylation of both IKKα and NF-κB2/p100 prior to the processing of p100 into p52, a pathway defined as the alternative, or non-canonical, NF-κB pathway.…”

New frontiers in promoting tumour cell death: targeting apoptosis, necroptosis and autophagy

“…Upon TNF binding, TNFR1 undergoes a conformational change that allows the recruitment of various signalling molecules such as TRADD, TRAF2, cellular inhibitor of apoptosis 1 (cIAP1), cIAP2 and RIP1, to form a complex referred to as complex I (Micheau and Tschopp, 2003;Vandenabeele et al, 2010). cIAP1 and cIAP2, both E3 ligases, causes the polyubiquitylation of proteins in complex I, including RIP1, which serves as a platform to dock additional signalling molecules (that is, the IKK complex) that proceed to activate the nuclear factor (NF)-kB survival or inflammatory pathways (Devin et al, 2000;Bertrand et al, 2008;Bianchi and Meier, 2009).…”