Activation of death-inducing signaling complex (DISC) by pro-apoptotic C-terminal fragment of RIP Jin Woo Kim 1,2 , Eui-Ju Choi 2 and Cheol O Joe* ,1 1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea; 2 National Creative Research Initiative for Cell Death, Graduate School of Biotechnology, Korea University, Seoul 156-701, Korea The two opposite signaling pathways that stimulate NF- kB activation and apoptosis are both mediated by tumor necrosis factor receptor 1 (TNFR1) and its cytosolic associated proteins. In this study, we demonstrate that the proteolytic cleavage of receptor interacting protein (RIP) by caspase-8 during TNF-induced apoptosis abrogates the stimulatory role of RIP on TNF-induced NF-kB activation. The uncleavable RIP D324A mutant was less apoptotic, but its ability to activate NF-kB activation was greater than the wild type counterpart. Ectopic expression of the pro-apoptotic C-terminal fragment of RIP inhibited TNF-induced NF-kB activa- tion by suppressing the activity of I-kB kinaseb (IKKb) which phosphorylates I-kB, an inhibitor of NF-kB, and triggers its ubiquitin-mediated degradation. The C- terminal fragment of RIP also enhanced the association between TNFR1 and death domain proteins including TNFR1 associated death domain (TRADD) and Fas associated death domain (FADD), resulting in the activation of caspase-8 and stimulation of apoptosis. The present study suggest that the C-terminal fragment of RIP produced by caspase-8 activates death-inducing signaling complex (DISC), attenuates NF-kB activation, and thereby ampli®es the activation of caspase-8 which initiates the downstream apoptotic events. Oncogene (2000) 19, 4491 ± 4499. Keywords: RIP; caspase-8; apoptosis; NF-kB activa- tion; I-kB kinase Introduction CED-3 and its mammalian homologous caspase are required for the execution of apoptosis (Cryns and Yuan, 1998). Numerous cellular proteins have been reported to be cleaved by caspases during the course of apoptosis. Among the caspase substrates, protein kinase C (PKC), p21 activated protein kinase (PAK), and MEK kinase 1 (MEKK1) are transformed into pro-apoptotic executors by proteolytic cleavage (Car- done et al., 1997; Datta et al., 1997; Rudel and Bokoch, 1997). In addition, caspases destroy death antagonists such as Bcl-2 (Cheng et al., 1997) and DFF45/ICAD (Enari et al., 1998; Liu et al., 1999). Caspase-8, a member of a mammalian caspase family, has been reported to be activated after recruitment to death receptors such as Fas/CD95 and TNF receptor-1 (TNFR1) (Muzio et al., 1996). These receptors recruit death domain proteins such as Fas associated death domain (FADD) and TNFR1 associated death domain (TRADD) to propagate the apoptotic signals (Nagata, 1997). The association of FADD with the death domain of Fas results in the formation of the death-inducing signaling complex (DISC) (Kischkel et al., 1995). Upon the induction of apoptosis, caspase-8 is recruited to DISC through the interaction between the death eector domain (DED) of caspase-8 and FADD. The recruitment of caspase-8 leads to the activation of caspase-8, an initiator of the downstream apoptotic events (Li et al., 1998). The activated caspase-8 directly mediates the downstream caspase cascade by activating caspase-3 through proteolytic cleavage, as well as triggering the mitochondrial damage through the cleavage of BID, a death agonist member of the Bcl-2 family (Scadi et al., 1998). While Fas speci®cally mediates apoptosis, TNFRs mediate cell survival as well as apoptosis through the activation of transcription factor NF-kB (Beg and Baltimore, 1996). NF-kB activation by TNF is mediated by the TNF receptor associated proteins, including the death domain kinase receptor interacting protein (RIP) (Hsu et al., 1996; Stanger et al., 1995) and the TNFR associating factors (TRAFs) (Rothe et al., 1994, 1995). RIP interacts with the proteins containing the death domains such as Fas, TNFR1, FADD, and TRADD, through its C-terminal death domain (DD), while TRAF2 interacts with the N-terminal kinase domain (KD) and the intermediate domain (ID) of RIP (Hsu et al., 1996; Varfolomeev et al., 1996). Recently, RIP2 and RIP3 containing the conserved kinase domain and the variable C-terminal domains have been isolated and proved to activate NF-kB (McCarthy et al., 1998; Sun et al., 1999). The indispensable role of RIP on NF-kB activation was supported by the studies showing a severe reduction in TNF-induced NF-kB activation but normal JNK activation in RIP de®cient mice (Kelliher et al., 1998; Yeh et al., 1997). Positively charged amino acid residues in ID were known to be required for NF-kB activation (Ting et al., 1996). Cellular function of RIP on NF-kB activation appeared to be independent of TRAF2, since TRAF2 knock-out mice exhibited almost intact TNF-induced NF-kB activation but severe reduction in JNK activation (Yeh et al., 1997). Studies have described, the anti-apoptotic eects of RIP on TNF-induced apoptosis through the activation of NF-kB. However, there are studies suggesting that RIP mediates apoptosis as well as cell survival (Hsu et al., 1996; Stanger et al., 1995). The present study demonstrates that the proteolytic cleavage of RIP by caspase-8 abrogates TNF-induced NF-kB activation and mediates downstream apoptotic events induced by Oncogene (2000) 19, 4491 ± 4499 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: CO Joe Received 23 February 2000; revised 2 May 2000; accepted 11 July 2000