Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 13 converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAFI and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

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Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor κB (NF-κB) signaling, and sensitize cells to tumor necrosis factor α (TNFα). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1–Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1–TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-κB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFα-induced death occurs. TWEAK-induced loss of the cIAP1–TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFα-induced death, whereas primary cells remain resistant. Conversely, cIAP1–TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFα sensitization. Lysosomal degradation of cIAP1–TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.

A central issue in the control of apoptosis is whether its essential mediators Bax and Bak must be restrained by Bcl-2-like prosurvival relatives to prevent their damaging mitochondria and unleashing apoptosis. The issue is particularly vexed for Bax, which is largely a cytosolic monomer in unstressed cells. To determine whether Bax regulation requires its binding by prosurvival relatives, we replaced a conserved aspartate in its BH3 interaction domain with arginine. Bax D68R functioned and behaved like wild-type Bax in localization and activation but had greatly impaired binding to the prosurvival family members. Nevertheless, Bcl-x L remained able to block apoptosis induced by Bax D68R. Whereas cells with sufficient Bcl-x L tolerated expression of Bax D68R, it provoked apoptosis when Bcl-x L was absent, downregulated, or inactivated. Moreover, Bax D68R rendered membrane bound by a C-terminal anchor mutation overwhelmed endogenous Bcl-x L and killed cells. These unexpected results suggest that engagement of Bax by its prosurvival relatives is a major barrier to its full activation. We propose that the Bcl-2-like proteins must capture the small proportion of Bax molecules with an exposed BH3 domain, probably on the mitochondrial membrane, to prevent Bax-imposed cell death, but that Bcl-x L also controls Bax by other mechanisms.