|14 September 2000, Volume 19, Number 39, Pages 4491-4499|
|Table of contents Previous Article Next [PDF]
|Activation of death-inducing signaling complex (DISC) by pro-apoptotic C-terminal fragment of RIP|
|Jin Woo Kim1,2, Eui-Ju Choi2 and Cheol O Joe1|
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea
2National Creative Research Initiative for Cell Death, Graduate School of Biotechnology, Korea University, Seoul 156-701, Korea
Correspondence to: C O Joe, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea
The two opposite signaling pathways that stimulate NF-B 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-B activation. The uncleavable RIPD324A mutant was less apoptotic, but its ability to activate NF-B activation was greater than the wild type counterpart. Ectopic expression of the pro-apoptotic C-terminal fragment of RIP inhibited TNF-induced NF-B activation by suppressing the activity of I-B kinase (IKK) which phosphorylates I-kB, an inhibitor of NF-B, 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-B activation, and thereby amplifies the activation of caspase-8 which initiates the downstream apoptotic events. Oncogene (2000) 19, 4491-4499
RIP; caspase-8; apoptosis; NF-B activation; I-B kinase
PKC, protein kinase C; PAK, p21-activated protein kinase; MEKK1, MEK kinase 1; TNF, tumor necrosis factor; TNFR, TNF receptor; FADD, Fas associated death domain; TRADD, TNF receptor associated death domain; RIP, receptor interacting protein; DISC, death inducing signal complex; NF-B, nuclear factor B; TRAF, TNF receptor associating factor; JNK, c-Jun N-terminal kinase; KD, kinase domain; ID, intermediate domain; DD, death domain; NTF, N-terminal fragment; CTF, C-terminal fragment; IKK, I-B kinase
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 (Cardone 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 effector 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 (Scaffidi et al., 1998).
While Fas specifically mediates apoptosis, TNFRs mediate cell survival as well as apoptosis through the activation of transcription factor NF-B (Beg and Baltimore, 1996). NF-B 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-B (McCarthy et al., 1998; Sun et al., 1999). The indispensable role of RIP on NF-B activation was supported by the studies showing a severe reduction in TNF-induced NF-B activation but normal JNK activation in RIP deficient mice (Kelliher et al., 1998; Yeh et al., 1997). Positively charged amino acid residues in ID were known to be required for NF-B activation (Ting et al., 1996). Cellular function of RIP on NF-B activation appeared to be independent of TRAF2, since TRAF2 knock-out mice exhibited almost intact TNF-induced NF-B activation but severe reduction in JNK activation (Yeh et al., 1997).
Studies have described, the anti-apoptotic effects of RIP on TNF-induced apoptosis through the activation of NF-B. 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-B activation and mediates downstream apoptotic events induced by TNF. The cleavage of RIP blocks TNF-induced NF-B activation by inhibiting the activity of IKK. In addition, the C-terminal fragment of RIP contributes to apoptosis by activating caspase-8 through the recruitment of FADD to TNFR1 complex.
RIP is cleaved by caspase-8 during apoptosis
TNF-induced apoptotic signal transduction pathway is known to be mediated by the recruitment of intracellular death domain proteins such as FADD, TRADD, and RIP into the death receptors (Nagata, 1997). Cellular levels of death domain proteins were measured during the course of UV-induced apoptosis in HEK293 cells (Figure 1a). UV irradiation was reportedly known to induce Fas and FasL expression (Kasibhatla et al., 1998). However, the expression of FADD or TRADD was not changed during UV-induced apoptosis. Interestingly, a 39 kDa fragment of RIP was generated during UV-induced apoptosis (Figure 1a). The RIP cleavage was also observed during Fas- or TNF-induced apoptosis (Figure 1b,c; upper panels). The RIP cleavage during apoptosis seems to be mediated by caspases since the treatment of HEK293 cells with zVAD-fmk, a general caspase inhibitor, suppressed the RIP cleavage induced by Fas antibody or TNF (Figure 1b,c; lower panels).
To identify the caspase isozyme responsible for the cleavage of RIP, in vitro translated [35S]RIP was incubated with E. coli cell lysates containing the active recombinant caspase-1, -3, or -8 in the presence or absence of zVAD-fmk. Only caspase-8 was able to cleave RIP into the fragments of 39 kDa and 34 kDa along with a nonspecific 12 kDa fragment (Figure 2a). In order to identify the domain containing the cleavage site, the genetic template of each RIP fragment was produced using PCR and cloned into pcDNA3-HA (Figure 2b). RIP fragments produced by in vitro translation were incubated with E. coli lysate containing caspase-8. The data reveal that the cleavage by caspase-8 occurred in the intermediate domain (ID) of RIP. The ID contains several potential cleavage sites for caspase-8: D300, D324, and D380. The RIPD324A mutant, in which Asp at 324 was substituted to Ala, was resistant to caspase-8-catalysed cleavage, while RIPD300A and RIPD380A mutants remained sensitive to caspase-8 (Figure 2c). The cellular cleavage of HA-tagged RIP or RIPD324A expressed in HEK293 cells was also examined during TNF-induced apoptosis. The data indicate that RIP is cleaved in cells at D324 by caspase-8 in response to TNF (Figure 2d).
The C-terminal cleavage product of RIP is apoptotic
The RIP cleavage by caspase-8 produces two fragments: a 34 kDa N-terminal fragment (NTF) containing the Ser/Thr kinase domain and a 39 kDa C-terminal fragment (CTF) containing the intermediate domain (ID) and the death domain (DD). Normal or RIP(-) Jurkat cells (Ting et al., 1996) were transfected with RIP, NTF, CTF or RIPD324A, and the roles of RIP cleavage products on apoptosis were evaluated in transfected cells (Figure 3a,b). Ectopic expression of RIP or CTF induced apoptosis in both normal and RIP(-) Jurkat cells, but the expression of NTF did not induce apoptosis (Figure 3b). RIPD324A mutant was less apoptotic than wild type RIP. The ability of RIP or CTF to induce apoptosis was abrogated if the viral caspase inhibitor, crmA, was expressed.
TNF-induced NF-B activation is abrogated by RIP cleavage
RIP has been shown to play a role in TNF-induced NF-B activation (Kelliher et al., 1998). In Figure 4, we examined the effects of the RIP cleavage on TNF-induced NF-B activation in RIP(-) Jurkat cells. The ectopic expression of RIP or RIPD324A stimulated TNF-induced NF-B activation in transfected cells while CTF expression suppressed TNF-induced NF-B activation. However, TNF-induced NF-B activation was not affected by the expression of NTF. The uncleavable RIPD324A displayed stronger stimulatory effects on NF-B activation than the wild type counterpart did (Figure 4a). If the cleavage of RIP was blocked by the co-expression of crmA, RIP induced almost same folds of NF-B activation as RIPD324A (Figure 4b).
Since NF-B activity has been shown to be directly regulated by the level of I-B, an inhibitor of NF-B (DiDonato et al., 1996), the cellular levels of I-B in RIP(-) Jurkat cells over-expressing RIP or its cleavage products were examined (Figure 4c). Cellular levels of I-kB were down-regulated by the expression of RIP, but the expression of the RIP cleavage products, CTF and NTF, did not influence the cellular I-kB level. I-B was almost undetectable in TNF-treated cells over-expressing RIPD324A. Studies have shown that the down-regulation of I-B is mediated by phosphorylation of I-B by IKK (Mercurio et al., 1997; Zandi et al., 1997). TNF-induced activation of IKK was known to be mediated by the regulatory subunit IKK (Rothwarf et al., 1998), and RIP was shown to associate with IKK (Zhang et al., 2000). In Figure 4d, we examined the roles of RIP and its cleavage products on IKK activity. The expression of RIP or its cleavage products in RIP(-) Jurkat cells did not affect TNF-induced activation of IKK. In contrast, expression of RIP or RIPD324A induced an enhancement of IKK activity, while IKK activity was inhibited by the expression of NTF or CTF suggesting that the activation of IKK requires the action of uncleaved RIP.
The C-terminal cleavage product of RIP stimulates the association of FADD with TNFR1 complex and subsequent activation of caspase-8
Death domain proteins such as FADD, TRADD, and RIP form a signaling complex to transmit apoptotic signals downstream from Fas and TNFR1. In Figure 5a, we evaluated the apoptotic function of RIP cleavage products by monitoring the recruitment of death domain proteins to TNFR1 in RIP(-) Jurkat cells. HA-RIP fragments, TNFR1, and crmA were co-expressed along with FLAG-TRAF2, Myc-TRADD, or GFP-FADD in RIP(-) Jurkat cells, and the association of TNFR1 with TRADD, FADD, TRAF2 or RIP fragments was examined (Figure 5a). The expression of RIP resulted in an increase in the TNFR1 interaction with TRADD, FADD or TRAF2. TNFR1 interaction with TRADD or FADD was also enhanced by the co-expression of CTF, while the association between TNFR1 and TRAF2 was enhanced by the NTF expression. The data suggest that CTF alone can induce apoptosis by enhancing recruitment of FADD and TRADD to TNFR1. The recruitment of death domain proteins to TNFR1 has been shown to activate caspase-8 and triggers a caspase cascade during the course of apoptosis (Varfolomeev et al., 1998). Thus, we measured cellular caspase activities in RIP(-) Jurkat cells expressing RIP fragments by using colorimetric peptide substrates (Figure 5b). The activation of caspase-8 and caspase-3 was observed in RIP(-) Jurkat cells expressing RIP or CTF, while the activities of caspase-9 were marginally affected by the expression of the death domain of RIP. The levels of caspase-8 and caspase-3 activities in cells expressing uncleavable RIPD324A were lower than those in cells expressing wild type RIP. This result suggests that the proteolytic cleavage of RIP contributes to apoptosis by activating the caspase-8 and the subsequent activation of caspase-3.
To delineate the role of CTF on the activation of caspases through the recruitment of FADD to TNFR1, GFP-FADD was co-expressed with RIP fragments in HEK293 cells and the subcellular location of GFP-FADD was monitored. Exposure of cells to TNF resulted in the translocation of GFP-FADD from cytoplasm to cell membrane (Figure 6). The translocation of GFP-FADD to cell membrane was also observed in cells over-expressing RIP or CTF. NTF did not affect the subcellular localization of GFP-FADD. These findings suggest that CTF stimulates the recruitment of FADD to the transmembrane death receptors leading to the formation of DISC and subsequent activation of caspase-8.
The proinflammatory cytokine TNF triggers two distinct cellular events, apoptosis and NF-B activation. TNF-induced apoptosis is mediated by the recruitment of FADD to TRADD followed by the activation of caspase-8 or 10 (Nagata, 1997). Activated caspase-8 elicits apoptotic signals by cleaving the downstream caspases or other cellular substrates such as Bid (Li et al., 1998). On the other hand the recruitment of other cytoplasmic adapter proteins such as RIP and TNFR associated factors (TRAF) into the death domain proteins, contributes to TNF-induced NF-B activation (Hsu et al., 1996; Rothe et al., 1994, 1995; Stanger et al., 1995). The TNF signaling pathway leading to NF-B activation is thought to contribute to cell survival by transcriptional activation of anti-apoptotic gene products such as caspase inhibitor survivin and cIAP (Shu et al., 1996; Tamm et al., 1998). However, the link between these two contradictory cellular events induced by TNF is unknown.
Although RIP protects cells from TNF-induced apoptosis in animal studies (Kelliher et al., 1998), over-expression of RIP has been shown to induce apoptosis in T lymphocytes (Hsu et al., 1996; Stanger et al., 1995). As shown in Figure 1, RIP cleavage is as early an event as PARP cleavage during apoptosis induced by Fas, TNF or UV irradiation. Caspase-8 cleaves RIP at Asp324 in the intermediate domain (ID), and generates two RIP cleavage products: an N-terminal fragment containing KD and a C-terminal fragment containing ID and DD (Figure 2). Results in Figure 3 show that CTF is responsible for the stimulation of apoptosis in RIP(-) Jurkat cells. The caspase resistant RIPD324A mutant was less apoptotic than the wild type RIP, supporting the pro-apoptotic function of the C-terminal cleavage product of RIP.
While CTF sensitizes cells to apoptosis, RIP was known to participate in the NF-B activation as well as cell survival (Kelliher et al., 1998). The caspase-8-mediated RIP cleavage attenuated the stimulatory function of RIP on NF-B activation (Figure 4a). Ting et al. (1996) have reported that the positively charged amino acids between residues 391 and 422 in intermediate domain (ID) of RIP are essential for the stimulation of NF-B activation. The data in Figure 4 indicate that the uncleaved RIP is required for NF-B activation. Since CTF containing these positively charged amino acid residues, 391-422, lacks the ability to stimulate TNF-induced NF-B activation.
A recent report by Lin et al. (1999) also showed the cleavage of RIP by caspase-8 during apoptosis, suggesting the abrogation of NF-B activation by RIP cleavage. However, the biochemical mechanism for the abrogation of TNF-induced NF-B activation by RIP cleavage remains ambiguous. NF-B activation by TNFR1 is known to be mediated by down-regulating the activity of I-B, which is an inhibitory protein of NF-B (DiDonato et al., 1996). The phosphorylation of S32 and S36 targets I-B and I-B for ubiquitination and subsequent ubiquitin-mediated degradation (DiDonato et al., 1996). Recent studies have shown that IKKs phosphorylate I-B (Zandi et al., 1998), and RIP contributes to NF-B activation by stimulating IKK activity (Sanz et al., 1999; Zhang et al., 2000). As shown in Figure 4c, intact RIP induced NF-B activation through IKK activation, while the cleavage products of RIP failed to activate IKK.
Several studies have presented the pro-apoptotic function of CTF in different types of cells (Hsu et al., 1996; Lin et al., 1999; Stanger et al., 1995). However, the biochemical mechanism for the pro-apoptotic function of CTF has not been clarified. Our data in Figure 5a suggest the recruitment of cytoplasmic FADD into the transmembrane TNFR1 in cells over-expressing CTF. The interaction of FADD with TNFR1 was known to induce the activation of caspase-8 (Muzio et al., 1996). It was then necessary to determine whether the expression of CTF, which enhances the interaction between FADD and TNFR1, activates caspase-8 and initiates a caspase cascade. Our data demonstrate an increase in the activities of caspase-8 and -3 in cells expressing CTF (Figure 5b). The death domain motifs have a tendency to self-aggregate. It has been shown that the self-aggregation of the death domain of RIP by itself is sufficient to elicit apoptosis (Grimm et al., 1996; Varfolomeev et al., 1996). Self-association of RIP might induce the formation of DISC and activate caspase-8. Activated caspase-8, in turn, cleaves RIP, abrogating the stimulatory function of RIP on NF-B activation. The present study suggests that caspase-8 induces the RIP cleavage and the C-terminal fragment of RIP, a RIP cleavage product by caspase-8, mediates the intracellular signaling that leads to apoptosis.
Materials and methods
Reagents and antibodies
A caspase inhibitor, zVAD-fmk and the caspase substrates, Ac-DEVD-pNA, Ac-IETD-pNA and Ac-LEHD-pNA were purchased from Enzyme System Products (USA). Monoclonal antibody against hemaglutinin (HA) was supplied by Boehringer-Manheim (Germany) and anti-FLAG antibody (M2) was from Sigma (USA). Anti-human Fas IgM (CH-11) was obtained from Oncor (USA). Monoclonal antibodies against RIP (G322-2), TRADD (B36-2), and FADD (A66-2) were from Pharmingen (USA). Anti-TNFR1 (H-271), TRAF-2 (H-10), and I-B (H-4) antibody were from Santa Cruz Inc. (USA). Recombinant human TNF was purchased from R&D Systems (USA).
Cell lines and analysis of cell death
Human embryonic kidney (HEK) 293 cells were grown in DMEM (GIBCO-BRL) supplemented with 10% fetal bovine serum. Jurkat and RIP(-) Jurkat cells (gift from Dr Seed, B. Harvard Medical School, USA) (Ting et al., 1996) were grown in RPMI1640 with 10% fetal bovine serum. After induction of apoptosis, cells were harvested by centrifugation at 700 g for 5 min and resuspended in PBS containing 20 mM EDTA. Cells were immediately fixed with ice-cold 70% (final) ethanol and incubated on ice for 1 h. Fixed cells were treated with RNase A (2 units/ml) and stained with PI (50 g/ml). Apoptosis was determined by the percentage of hypoploid cells by FACS. Flow cytometric quantification of apoptosis was determined by using CellQuest software (Becton Dickinson, USA).
Vectors and transfection
Fragments of human RIP cDNA (Hsu et al., 1996) were generated by PCR (Figure 2b) and cloned into BamHI/XhoI site of pcDNA3-HA (Invitrogen, USA). UncleavableRIPD324A was produced by PCR mutagenesis kit (Stratagene, USA). Vector constructs were transfected into HEK293 cells (5´106) using LipoFECTAMINE reagent (GIBCO-BRL, USA) and into RIP(-) Jurkat cells (5´106) by electrophoration at 0.4 kV and 960 F using electrophorator (Bio-Rad, USA). To assess the activation of NF-B, pcDNA3-3X-B-Luc and pcDNA3--galactosidase were co-transfected into cells with various vector constructs, and luciferase activity was measured using Luciferase activity assay kit (Promega, USA). Luciferase activity was normalized relative to the co-expressed -galactosidase activity.
In vitro cleavage of RIP
Recombinant human [35S]RIP protein was produced from pcDNA3-HA-RIP using T7 Quick-Coupled Transcription/Translation System (Promega, USA). The cleavage of [35S]RIP by caspases was examined as described previously (Muzio et al., 1996). E. coli BL21(DE3) was transformed with pET21a-caspase-1, pMal-caspase-3, or pET15b-caspase-8, and the expression of recombinant caspases was induced by 1 mM IPTG. The [35S]RIP were incubated in 2 g of E. coli BL21(DE3) cell lysates containing recombinant human caspase-1, 3 or 8 proteins at 37°C for 2 h. Proteolytic cleavage of RIP was analysed by 12% SDS-PAGE and autoradiography.
I-B kinase assay
I-B kinase activity was measured by the method described (Regnier et al., 1997). Vector constructs encoding RIP fragments (4 g) were co-transfected into HEK293 cells (5´106) with 2 g of pRK7-flat-IKK or pRK5-flag-IKK. After incubation for 36 h, cells expressing FLAG-IKK or FLAG-IKK were treated with 20 ng/ml of recombinant human TNF for 20 min. The cells were lysed in a lysis buffer containing 50 mM HEPES (pH 7.6), 100 mM NaCl, 10% glycerol, 1 mM EDTA, 20 mM -glycerophosphate, 20 mM para-niotrophosphate, 1 mM Na3VO4, 1 mM NaF, 1% NP-40, and 1 mM PMSF. FLAG-IKK or FLAG-IKK was immunoprecipitated using anti-FLAG antibody. IKK activity of the immunecomplexes was determined by the incubation at 30°C for 30 min in a reaction buffer containing 20 mM HEPES (pH 7.5), 20 mM -glycerophosphate, 10 mM MgCl2, 100 mM Na3VO4, 2 mM DTT, 20 mM ATP, 100 Ci/ml [32P]ATP (Amersham, USA), and 1 g of GST-I-B. Phosphorylation of GST-I-B was analysed by 10% SDS-PAGE and autoradiography.
Co-immunoprecipitation and Western blotting
Transfected cells were lysed in E1A buffer containing 50 mM HEPES (pH 7.4), 250 mM NaCl, 0.1% NP-40, 5 mM EDTA, 10 g/ml leupeptin and 1 mM PMSF (Hsu et al., 1995). Lysates were incubated with 10 g of anti-TNFR1 antibody at 4°C for 2 h, and the immunecomplexes were incubated with a 1/10 volume of protein G agarose for another 2 h. The beads were washed twice with high salt (500 mM NaCl) E1A buffer and E1A buffer in succession. The precipitates were resolved in SDS-PAGE, and the associated proteins were analysed by immunoblotting using monoclonal antibodies against GFP, Myc, FLAG, HA or TNFR1.
Caspase activity assay
RIP(-) Jurkat cells expressing RIP fragments were lysed in a lysis buffer containing 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.5% Nonidet P-40, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin (Tewari et al., 1995). Caspase activity was measured using colorimetric peptide substrates. Ac-DEVD-pNA, Ac-IETA-pNA, or Ac-LEHD-pNA was employed as a specific substrate for caspase-3, -8, and -9, respectively (Komoriya et al., 2000). Cell lysate proteins (10 g) were incubated with each 100 M of peptide substrate for 2 h at 30°C, and caspase activities were monitored by measuring the absorbance of the substrate solution at 405 nm.
The pEGFP-C1-FADD (1 g), pcDNA3-HA-RIP fragments (2 g) and pEF-crmA (1 g) were co-transfected into HEK293 cells (5´105). After transfection (36 h), cells were treated with 20 ng/ml of TNF for 20 min and washed twice with PBS and fixed in 3.7% formaldehyde in PBS for 30 min. Expression of the GFP-FADD was observed by fluorescence microscope (Nikon, Japan).
We appreciate Dr David Goeddel's (Tularik Inc., USA) provision of various cDNA clones. We thank Dr Brian Seed (Harvard Medical School, USA) for providing the RIP deficient cell line. This work was supported in part by a grant (95-0401-06-3) from the Korea Science and Engineering Foundation (CO Joe) and a grant from the Creative Research Initiatives Program of the Korea Ministry of Science and Technology (E-J Choi).
Beg AA and Baltimore D. (1996). Science 274, 782-784. Article MEDLINE
Cardone MH, Salvesen GS, Widmann C, Johnson G and Frisch SM. (1997). Cell 90, 315-323. MEDLINE
Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K and Hardwick JM. (1997). Science 278, 1966-1968. Article MEDLINE
Cryns V and Yuan J. (1998). Genes Dev. 12, 1551-1570. MEDLINE
Datta R, Kojima H, Yoshida K and Kufe D. (1997). J. Biol. Chem. 272, 20317-20320. MEDLINE
DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H, Ghosh S and Karin M. (1996). Mol. Cell. Biol. 16, 1295-1304. MEDLINE
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A and Nagata S. (1998). Nature 391, 43-50. Article MEDLINE
Grimm S, Stanger BZ and Leder P. (1996). Proc. Natl. Acad. Sci. USA 93, 10923-10927. Article MEDLINE
Hsu H, Huang J, Shu HB, Baichwal V and Goeddel DV. (1996). Immunity 4, 387-396. MEDLINE
Hsu H, Xiong J and Goeddel DV. (1995). Cell 81, 495-504. MEDLINE
Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi A and Green DR. (1998). Mol. Cell 1, 543-551. MEDLINE
Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ and Leder P. (1998). Immunity 8, 297-303. MEDLINE
Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH and Peter ME. (1995). EMBO J. 14, 5579-5588. MEDLINE
Komoriya A, Packard BZ, Brown MJ, Wu ML and Henkart PA. (2000). J. Exp. Med. 191, 1819-1828. MEDLINE
Li H, Zhu H, Xu CJ and Yuan J. (1998). Cell 94, 491-501. MEDLINE
Lin Y, Devin A, Rodriguez Y and Liu Z. (1999). Genes Dev. 13, 2514-2526. MEDLINE
Liu X, Zou H, Widlak P, Garrard W and Wang X. (1999). J. Biol. Chem. 274, 13836-13840. MEDLINE
McCarthy JV, Ni J and Dixit VM. (1998). J. Biol. Chem. 273, 16968-16975. Article MEDLINE
Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, Young DB, Barbosa M, Mann M, Manning A and Rao A. (1997). Science 278, 860-866. Article MEDLINE
Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME and Dixit VM. (1996). Cell 85, 817-827. MEDLINE
Nagata S. (1997). Cell 88, 355-365. MEDLINE
Regnier CH, Song HY, Gao X, Goeddel DV, Cao Z and Rothe M. (1997). Cell 90, 373-383. MEDLINE
Rothe M, Pan MG, Henzel WJ, Ayres TM and Goeddel DV. (1995). Cell 83, 1243-1252. MEDLINE
Rothe M, Wong SC, Henzel WJ and Goeddel DV. (1994). Cell 78, 681-692. MEDLINE
Rothwarf DM, Zandi E, Natoli G and Karin M. (1998). Nature 395, 297-300. Article MEDLINE
Rudel T and Bokoch GM. (1997). Science 276, 1571-1574. Article MEDLINE
Sanz L, Sanchez P, Lallena MJ, Diaz-Meco MT and Moscat J. (1999). EMBO J. 18, 3044-3053. MEDLINE
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH and Peter ME. (1998). EMBO J. 17, 1675-1687. Article MEDLINE
Shu HB, Takeuchi M and Goeddel DV. (1996). Proc. Natl. Acad. Sci. USA 93, 13973-13978. Article MEDLINE
Stanger BZ, Leder P, Lee TH, Kim E and Seed B. (1995). Cell 81, 513-523. MEDLINE
Sun X, Lee J, Navas T, Baldwin DT, Stewart TA and Dixit VM. (1999). J. Biol. Chem. 274, 16871-16875. Article MEDLINE
Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T and Reed JC. (1998). Cancer Res. 58, 5315-5320. MEDLINE
Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS and Dixit VM. (1995). Cell 81, 801-809. MEDLINE
Ting AT, Pimentel-Muinos FX and Seed B. (1996). EMBO J. 15, 6189-6196. MEDLINE
Varfolomeev EE, Boldin MP, Goncharov TM and Wallach D. (1996). J. Exp. Med. 183, 1271-1275. MEDLINE
Yeh WC, Shahinian A, Speiser D, Kraunus J, Billia F, Wakeham A, de la Pompa JL, Ferrick D, Hum B, Iscove N, Ohashi P, Rothe M, Goeddel DV and Mak TW. (1997). Immunity 7, 715-725. MEDLINE
Zandi E, Chen Y and Karin M. (1998). Science 281, 1360-1363. Article MEDLINE
Zandi E, Rothwarf DM, Delhase M, Hayakawa M and Karin M. (1997). Cell 91, 243-252. MEDLINE
Zhang SQ, Kovalenko A, Cantarella G and Wallach D. (2000). Immunity 12, 301-311. MEDLINE
Figure 1 Proteolytic cleavage of RIP during apoptosis. (a) HEK293 cells were irradiated with 100 J/m2 of UV light and incubated for the indicated time periods. The cellular levels of PARP, RIP, FADD, and TRAF2 were examined by immunoblot analysis using monoclonal antibodies against each respective protein. (b) HEK293 cells were treated with anti-Fas IgM (CH-11) for 16 h in the presence (lower) or absence (upper) of 100 M of zVAD-fmk. (c) HEK293 cells were treated with recombinant human TNF (plus 10 g/ml cycloheximide). Cleavage of RIP was examined by 10% SDS-PAGE and immunoblot analysis using monoclonal antibody against human RIP
Figure 2 Proteolytic cleavage of RIP by caspase-8. (a) 35S-labeled human RIP produced by in vitro translation was incubated at 37°C for 2 h with 2 g of E. coli BL21(DE3) lysates containing recombinant caspases. The samples were analysed by 12% SDS-PAGE and autoradiography. Arrow indicates intact or cleavage products of RIP. (b) 35S-labeled RIP fragments were incubated with 2 g of E. coli BL21(DE3) lysates containing recombinant caspase-8 at 37°C for 2 h. Samples were analysed by 15% SDS-PAGE and autoradiography. The asterisks indicate nonspecific bands. (c) Aspartic acid residues at 300, 334, or 380 in RIP were substituted to alanines by site-directed mutagenesis. The 35S-labeled RIP mutants were incubated with 2 g of E. coli lysates containing recombinant caspase-8 at 37°C for 2 h and analysed by 12% SDS-PAGE and autoradiography. (d) HEK293 cells (5´105) were transfected with 2 g of pcDNA3-HA-RIP or pcDNA3-HA-RIPD324A and incubated for 24 h. Cells were treated with specified concentration of recombinant human TNF plus 10 g/ml of cycloheximide for 16 h. Proteolytic cleavage of RIP was examined by 10% SDS-PAGE and immunoblot analysis using monoclonal antibody against HA
Figure 3 Stimulation of apoptosis by the C-terminal cleavage product of RIP. (a) RIP(-) cells (5´105) were transfected with 2 g of pcDNA3-HA-RIP, pcDNA3-HA-NTF, pcDNA3-HA-CTF, or pcDNA3-HA-RIPD324A. After incubation for 36 h, the expression of HA-RIP fragments in cell lysates (100 g) was examined by 10% SDS-PAGE and immunoblot probed with monoclonal antibody against HA. (b) Each vector construct (2 g) was co-transfected into Jurkat or RIP(-) Jurkat cells (5´105) with or without 1 g of pEF-crmA. Apoptosis was analysed by monitoring the percentage of PI stained hypoploid cells at 36 h after transfection. Data represent the results from the three independent experiments
Figure 4 Inhibition of TNF-induced NF-B activation by the C-terminal fragment of RIP. (a) RIP(-) Jurkat cells (5´105) were co-transfected with pcDNA3-3X-B-Luc (0.5 g), pcDNA3--galactosidase (0.5 g), and pcDNA3-HA vector constructs encoding RIP fragments (2 g). After incubation for 24 h, the cells were treated with 20 ng/ml of TNF for 12 h, and the luciferase activities in the transfected cells were examined by Luciferase Assay Kit (Promega, USA). (b) TNF-induced NF-B activation was measured in RIP(-) Jurkat cells expressing crmA. The values were normalized to the level of the activity of co-transfected -galactosidase. Data represent the results of three independent experiments. (c) Cell lysates (100 g) were resolved by 12% SDS-PAGE, and the level of I-B protein was examined by immunoblot analysis using monoclonal antibody against I-B. (d) Vector constructs encoding RIP fragments (4 g) were co-transfected with either pRK7-FLAG-IKK (2 g) or pRK5-FLAG-IKK (2 g) into RIP(-) Jurkat cells (5´106). After incubation for 36 h, cells were treated with 20 ng/ml of TNF for 20 min. FLAG-IKK was immunoprecipitated using anti-FLAG antibody. The immunecomplex was incubated at 30°C with GST-I-B (1 g) in the presence of 100 Ci/ml of [32P]--ATP. Phosphorylation of GST-I-B was analysed by 10% SDS-PAGE and subsequent autoradiography. The cellular levels of FLAG-IKK and FLAG-IKK were examined by immunoblotting using monoclonal antibody against FLAG
Figure 5 The C-terminal fragment of RIP stimulates the association of FADD with TNFR1. (a) The pcDNA3-HA vector constructs (4 g) encoding RIP fragments, pRK5-TNFR1, and pEF-crmA were co-transfected, along with pRK-Myc-TRADD (2 g), pRK7-FLAG-TRAF2 (2 g), or pEGFP-C1-FADD (2 g) into RIP(-) cells (5´106). After incubation for 36 h, the transfected cells were incubated in the presence or absence of TNF (20 ng/ml) for 15 min. Expressed proteins in transfected cells were immunoprecipitated using anti-TNFR1 antibody. The immunoprecipitated proteins were analysed by immunoblotting using monoclonal antibody against GFP, Myc, FLAG, HA or TNFR1. (b) RIP(-) Jurkat cells (5´106) were transfected with the vector constructs encoding RIP fragments (4 g). After incubation for 36 h, cellular caspase activities was measured by using colorimetric peptide substrates. Ac-DEVD-pNA, Ac-IETD-pNA or Ac-LEHD-pNA was employed as a specific substrate for caspase-3, -8, and-9, respectively
Figure 6 Translocation of FADD into cytoplasmic membrane in cells expressing the C-terminal fragment of RIP. HEK293 cells (5´105) were co-transfected with pcDNA3-HA vector constructs encoding RIP fragments (2 g), pEGFP-C1-FADD (1 g), and pEF-crmA (0.5 g). After incubation for 36 h, transfected cells were incubated with or without TNF (20 ng/ml) for 15 min. The subcellular location of GFP-FADD was examined by fluorescent microscope (Nikon, Japan)
|Received 23 February 2000; revised 2 May 2000; accepted 11 July 2000|
|14 September 2000, Volume 19, Number 39, Pages 4491-4499|
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