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.

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-Binducing kinase stability and suppress constitutive noncanonical NF-B activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-B 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-B signaling and that efficient activation of NF-B by TNFR1 requires the recruitment of cIAP1/2 by TRAF2. The inhibitor of apoptosis (IAP)7 family is composed of baculoviral IAP repeat-containing proteins, several of which also bear a RING domain that is capable of acting as a ubiquitin E3 ligase (1). cIAP1 and cIAP2 (cIAP1/2) are two RING-containing IAPs whose amplification or genetic mutation has been associated with cancers and may promote tumor cell survival (2-8). These highly conserved IAPs were initially identified as components of a TRAF2-containing complex bound to the cytoplasmic domain of TNFR2 (9), and they have subsequently been implicated in the regulation of signaling by several more receptors of the TNF superfamily (10 -21). Although the BIR1 domain of cIAP1/2 mediates binding to TRAF2 (17,22,23), the complementary binding region of TRAF2 is unknown, and the relative roles of TRAF2 and cIAP1/2 E3 ligase activity in TNF superfamily signaling remain unclear.Genetic deletion of TRAF2 in the mouse results in early postnatal lethality that is caused by increased NF-B-mediated TNF production and increased cellular sensitivity to TNF killing (12,24,25). Most TRAF family members, including TRAF2, bear RING E3 ubiquitin ligase domains that are believed to catalyze 27). One of the proposed targets of TRAF2 RING E3 ligase activity is RIPK1 (28, 29), which is modified with Lys-63-linked polyubiquitin chains upon TNFR1 activation. This modified TNFR1-complexed RIPK1 has been proposed to serve as a platform for the recruitment and/or activation of TAB2-TAB3-TAK1 and NEMO-IKK␣-IKK␤ kinase complexes (30 -32), which target the NF-B inhibitor IB for Lys-48-linked polyubiquitin-mediated proteasomal degradation, to initiate canonical p65-dependent NF-B gene transcription.As in the case of TRAF2, genetic deletion of cIAP1, or IAP antagonist-induced loss of cIAP1/2, also sensitizes cells to TNF killing (16,19,(33)(34)(35)(36). Also like TRAF2, both ...

Genetic screens in Drosophila have revealed that the serine/threonine kinase Hippo (Hpo) and the scaffold protein Salvador participate in a pathway that controls cell proliferation and apoptosis. Hpo most closely resembles the pro‐apoptotic mammalian sterile20 kinases 1 and 2 (Mst1 and 2), and Salvador (Sav) has a human orthologue hSav (also called hWW45). Here we show that Mst and hSav heterodimerize in an interaction requiring the conserved C‐terminal coiled‐coil domains of both proteins. hSav was also able to homodimerize, but this did not require its coiled‐coil domain. Coexpression of Mst and hSav led to phosphorylation of hSav and also increased its abundance. In vitro phosphorylation experiments indicate that the phosphorylation of Sav by Mst is direct. The stabilizing effect of Mst was much greater on N‐terminally truncated hSav mutants, as long as they retained the ability to bind Mst. Mst mutants that lacked the C‐terminal coiled‐coil domain and were unable to bind to hSav, also failed to stabilize or phosphorylate hSav, whereas catalytically inactive Mst mutants that retained the ability to bind to hSav were still able to increase its abundance, although they were no longer able to phosphorylate hSav. Together these results show that hSav can bind to, and be phosphorylated by, Mst, and that the stabilizing effect of Mst on hSav requires its interaction with hSav but is probably not due to phosphorylation of hSav by Mst.

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.