DEDD and DEDD2 Associate with Caspase-8/10 and Signal Cell Death

Allison Alcivar, Shimin Hu, Jun Tang and Xiaolu Yang*

Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

An apoptotic signal triggered by cell surface death FADD/MORT via homophilic interaction involving a receptors is disseminated to intracellular compartments conserved motif, termed the death domain, that are through protein–protein interactions mediated by con- present in both proteins. FADD contains another served domains such as the death effector domain (DED). conserved domain known as the death effector domain A unique family of single DED-containing proteins, (DED), which recruits two tandem DED-containing including DEDD and DEDD2, is targeted to the caspases – procaspase-8 and procaspase-10 – to the nucleolus. However, the role of DEDD/DEDD2 in receptors through homophilic DED–DED interactions. apoptosis remains less understood. Here we show that Oligomerization of procaspase-8 (and probably caspase- DEDD and DEDD2 are highly conserved in diverse 10) in this membrane-bound signaling complex leads to species, and that they are potent inducers of apoptosis in their autoproteolytic activation (Martin et al., 1998; various cell types. Deletion analysis indicates that both the Muzio et al., 1998; Yang et al., 1998a). A DED domain N-terminal DED domain and the C-terminal region of is also present in a nucleolus-targeted protein DEDD DEDD2 can induce apoptosis. The cell death activity of (Stegh et al., 1998). Upon its overexpression or during this family appears to be related to their nuclear CD95-mediated apoptosis, DEDD translocates to the localization. DEDD and DEDD2 bind to two tandem nucleus, where it may interfere with the synthesis of DED-containing caspases, caspase -8 and -10, that are rRNA (Stegh et al., 1998; Schickling et al., 2001). engaged by death receptors. Consistent with the nuclear Recently, a human DEDD homologue known as localization of this family, caspase-8 translocates to the DEDD2 or FLAME-3 was identified (Roth et al., nucleus during CD95-induced apoptosis. DEDD and 2002; Zhan et al., 2002). However, to date only weak DEDD2 also readily associate with themselves and with proapoptosis activity has been attributed to this family each other. These results suggest that DEDD and of DED-containing proteins. Whether DEDD and DEDD2 may be important mediators for death receptors DEDD2 play a significant role in apoptosis induction and that they may target caspases to the nucleus. and if so, how they may induce apoptosis remain largely Oncogene (2003) 22, 291–297. doi:10.1038/sj.onc.1206099 unknown. In an effort to study the function of DEDD in Keywords: DEDD; DEDD2; caspase-8/10; apoptosis apoptosis, we isolated a human and a mouse protein that share identity with DEDD throughout their lengths, and were subsequently found to be the same as the recently reported human and mouse DEDD2/ Apoptosis plays critical roles in development, main- FLAME-3, respectively. Interestingly, partial protein tenance of homeostasis, and protection against cancer. sequences remarkably homologous to human DEDD Death receptors in the tumor necrosis factor receptor (with 91% or higher identity) were found in various (TNFR) superfamily – which include TNFR1 (p55/ species including chicken and xenopus (Figure 1a). CD120a), CD95 (Fas/APO-1), death receptor 3 (DR3, Homologues of human DEDD2 also exist in other Apo3/WSL-lATRAMP/LARD), DR4, and DR5 organisms including zebrafish and xenopus (with 44% (Apo2fTRAIL-R2/KILLER/TRICK 2) – are important or higher identity) (Figure 1a). The evolutionary mediators of mammalian cell death (Ashkenazi and conservation of DEDD and DEDD2 suggests that this Dixit, 1998). Over the past several years, the molecular family of proteins may play an important role in cellular events underlying death-receptor-mediated apoptosis functions. have begun to be unraveled (Ashkenazi and Dixit, DEDD2 is expressed in various human tissues as a 1998). Upon binding to their cognate ligands or 2.0-kb major transcript and a 1.8-kb minor transcript agonistic antibodies, death receptors such as CD95 (Roth et al., 2002, Zhan et al., 2002, and Figure 1b). To become associated with a cellular adapter protein determine the expression profile of DEDD2 in various tumor cells, a human multiple cancer cell line Northern *Correspondence: X yang, 421 Curie Blvd., Rm 610 BRBII/III, blot was hybridized with a human DEDD2 cDNA Philadelphia, PA 19104-6160, USA; E-mail: [email protected] probe. The expression level of DEDD2 is relatively high Received 12 June 2002; revised 26 September 2002; accepted 4 in the chronic myologenous leukemia K-562 and the October 2002 colorectal adenocarcinoma SW480 but moderate in the DEDD and DEDD2 are potent inducers of apoptosis A Alcivar et al 292

Oncogene DEDD and DEDD2 are potent inducers of apoptosis A Alcivar et al 293 cervical carcinoma HeLa, the Burkitt’s lymphoma Raji, region homologous to certain DNA-binding proteins the lung carcinoma A549, and the melonoma G361. In such as histones (Stegh et al., 1998; Roth et al., 2002). contrast, two leukemia cell lines, HL-60 (promyelocytic To delineate the effector domain of DEDD2, GFP leukemia) and MOLT-4 (lymphoblastic leukemia), show fusions of each of these regions (GFP-DEDD2-N1 and relatively low levels of DEDD2 expression (Figure 1b). -C) were introduced into HeLa cells and 3T3 cells. The human DEDD2 gene was mapped to chromosome Interestingly, both regions induced high levels of 19ql3.2, and abnormalities in this region are frequently apoptosis (Figure 2b,c). DEDD2 also contains three found in various tumors. Mouse DEDD2, on the other putative nuclear localization signals (Roth et al., 2002; hand, is expressed as a 2.0-transcript (Figure 1b). The Zhan et al., 2002). To study a possible correlation expression level of mouse DEDD2 is high in liver, heart, between the cellular localization of DEDD2 and its kidney, and testis but low in brain, spleen, lung, and proapoptosis activity, we examined the localization of skeleton muscle, and this pattern of expression does not the full-length and the deletion mutants of DEDD2. mirror that of human DEDD2. Consistent with previous reports and with the localiza- Overexpression of certain DED-containing proteins tion of DEDD, GFP-DEDD2 was found predominantly such as FADD and caspase-8 leads to robust apoptosis in the nucleolus colocalizing with UBF (upstream in mammalian cells. The proapoptotic activity of binding factor), a nucleolus-specific protein (Figure 3a). DEDD/DEDD2 was previously examined by using a Similarly, GFP-DEDD2-C was also found in the cotransfected reporter gene to label the DEDD/ nucleolus. GFP-DEDD2-N1, on the other hand, ap- DEDD2-expressing cells (Stegh et al., 1998; Roth et al., peared to be both cytosolic and nuclear, and was 2002; Zhan et al., 2002). Using a similar approach, we notably excluded from the nucleolus (Figure 3a), also only detected minimal apoptosis activity of DEDD/ suggesting that the COOH- but not the NH2-terminal DEDD2 (data not shown). However, because both region targets DEDD2 to the nucleolus. To assess DEDD and DEDD2 translocate to the nucleolus upon whether the putative nuclear localization signal in overexpression and are likely to inhibit protein synthesis DEDD2-N1 is required for its cytotoxicity, the signal (Stegh et al., 1998; Roth et al., 2002; Zhan et al., 2002, was deleted and the resulting fusion, GFP-DEDD2-N2, and see below), high levels of the reporter gene was able to kill cells with slightly reduced efficiency expression in transfected cells may very well indicate compared with GFP-DEDD-N1 (Figure 2b, c). This weak expression of DEDD/DEDD2 and vice versa. region, however, still resided in the nucleus (Figure 1a), Thus, using a fusion protein instead of a cotransfected which was possibly mediated by a cellular factor(s). To reporter was deemed important to accurately reflect the further determine whether nuclear localization is critical expression levels of DEDD2/DEDD. Green fluores- for DEDD/DEDD2-mediated apoptosis, we mutated all cence protein (GFP) fusions of DEDD and DEDD2 previously identified nuclear localization signals on each were transiently transfected into HeLa cells, and cell of these proteins. The resulting DEDD mutant (GFP- death was scored among GFP positive cells based on the DEDDDNLS) was almost exclusively cytosolic, consis- morphological changes typical of adherent cells under- tent with previous reports (Schickling et al., 2001), going apoptosis such as rounded cell shape, membrane whereas the GFP-DEDD2DNLS mutant was present in blebbing, detachment from plates, and refractory both the cytosolic and the nuclear compartments appearance. Both proteins induced specific apoptosis (Figure 3b). In the apoptosis assay, the former failed in over 60% of the transfected cells (Figure 2b). These to kill cells while the latter retained substantial cell death levels of apoptosis were comparable to that stimulated activity (Figure 2f). Taken together, these results suggest by FADD under the same conditions (Figure 2b), that the DEDD/DEDD2’s proapoptotic activity is suggesting that both DEDD and DEDD2 are potent related to their nuclear localization. apoptosis inducers. To ensure that DEDD/DEDD2- To examine whether the proapoptotic effect of induced apoptosis was not peculiar to HeLa cells, we DEDD/DEDD2 is mediated by caspases, we cotrans- also examined the effects of DEDD and DEDD2 fected them with crmA, a serpin caspase inhibitor that overexpression on human 293, green monkey COS-1, directly targets the active sites of caspases, particularly and mouse fibroblast 3T3 cells. As was seen in HeLa those of caspase-1 and -8 (Chang and Yang, 2000). As cells, DEDD and DEDD2 showed similar proapoptotic shown in Figure 2g, DEDD- and DEDD2-induced activities as FADD in these cells (Figure 1c–e). apoptosis was inhibited by crmA. Additionally, it was DEDD2, like DEDD, contains an NH2-terminal noted that in the presence of crmA, both the number of region with the DED domain and a COOH-terminal cells expressing the GFP-DEDD/DEDD2 protein and

3 Figure 1 Interspecies conservation of DEDD and DEDD2. (a) Comparison of DEDD and DEDD2 proteins from various species. Sequence alignment and homology evaluation were performed using the Mac Vector software (CLUSTAL). The zebrafish DEDD2 shown is the full-length protein sequence while the xenopus and chicken DEDD and the xenopus DEDD2 are partial sequences. Amino- acid residue numbers are given to the right of the sequence. Dotted lines indicate unidentified sequences and dots are inserted gaps for optimal alignment. Identical residues are boxed. (b) Expression profile of human and mouse DEDD2 mRNA. A human multiple tumor cell line mRNA blot (left) and a human and a mouse multiple tissue mRNA blots (middle and right, respectively) were hybridized with a radioisotope-labeled DEDD2 cDNA probe from the same species according to the manufacturer’s instruction (Clontech). The sources of the mRNA are indicated above. Expression of b-actin is shown below as a loading control and size markers are indicated. SI: small intestine. BL: blood leukocyte

Oncogene DEDD and DEDD2 are potent inducers of apoptosis A Alcivar et al 294

a DEDD2 326

DEDD2-N1 115

DEDD2-N2 103

DEDD2-C 101 DEDD2∆NLS

DEDD 318 DEDD∆NLS ///

HeLa c NIH 3T3 293 b 100 100 d 50 75 75 40

30 50 50

% Cell Death 20 % Cell Death

25 25 % Cell Death 10

2 C 2 C 2 -N1 N2 DD DD -N1 -N2 2- DD DD DD GFP 2 2- GFP DD 2 2 GFP DEDD DD2- FA DE FA DE FA DEDDDD DD DE DD DD DEDD DE DEDD DE DE DE DE

e COS-1 f 75 g 100 Vector 50 CrmA 75 40 50 30 50 20 % Cell Death % Cell Death 25 % Cell Death 10 25

2 2 D DD DD DD D 2 GFP GFP DD NLS NLS DD DD DE FA ∆ DE ∆ FA GFP DD DEDD DE DD DE FA DD2 DE DE DE

Figure 2 Apoptosis induced by DEDD2 and its deletion mutants. (a) Schematic diagram of the full-length and mutant DEDD/ DEDD2 proteins. The HA and FLAG tags and the GFP protein were placed at the N-terminus of each indicated protein in mammalian expression plasmids pRK5 and pEGFP-N1, respectively. Filled boxes: the DED domain. Shaded boxes: putative nuclear localization signals. The NLS sequences in DEDD2 were deleted while those in DEDD were mutated (the arginine and lysine residues were changed to leucines). (b–e) Apoptosis induction in various mammalian cells by DEDD, DEDD2, and the DEDD2 mutants. HeLa (b), NIH 3T3 (c), HEK 293 (d), and COS-1 (e) cells seeded in 12-well plates were transfected with 1 mg of the indicated GFP fusion constructs. GFP-expressing cells were scored for apoptosis 48 h later as previously described (Yang et al., 1998b). Data (mean 7 s.d.) from three independent experiments are shown. The expression level of GFP-FADD was consistently higher than those of GFP- DEDD and -DEDD2 in transfected cells (data not shown). (f) Cell killing by DEDDDNLS and DEDD2DNLS. HeLa cells were transfected with 1 mg indicated GFP fusion plasmids and apoptosis was determined as in (b)–(c). (g) Inhibition of DEDD2-induced cell death by crmA. HeLa cells were transfected with 0.25 mg of the indicated GFP fusion construct together with 1.5 mg pRK5 or pRK5- crmA. The percentages of apoptosis were determined as above

the expression levels of GFP-DEDD/DEDD2 were with an anti-FLAG antibody. This assay revealed that greatly enhanced (data not shown). Therefore, DEDD/ DEDD2 interacted speciﬁcally with both caspase-8 and - DEDD2-induced apoptosis is dependent on caspase 10 (Figure 4a, lanes 2, 3). The interaction was mediated activation. by the N-terminal region of DEDD2 that encompassed Given that the DED domain mediates homophilic mainly the DED domain (Figure 4a, lane 5). Similarly, interactions and the possible involvement of caspase in DEDD also associated with both caspases, but no DEDD/DEDD2-mediated apoptosis, we examined interaction between DEDD/DEDD2 and FADD was whether DEDD2 interacts with the two tandem DED- detected (data not shown). Thus, DEDD and DEDD2 containing caspases, caspase-8 and -10, using a mam- may engage both caspase-8 and -10 to deliver the lethal malian cell immunoprecipitation assay. Hemagglutinin stimulus to the nucleus. (HA)-tagged DEDD2 together with FLAG-tagged We also found that DEDD and DEDD2 can readily caspase-8 or -10 were transiently expressed in human associate with themselves and with each other 293 cells and the cell lysates were immunoprecipitated (Figure 4b, lane 2, 3), and both the N-terminal DED

Oncogene DEDD and DEDD2 are potent inducers of apoptosis A Alcivar et al 295

Figure 3 Cellular localization of the full-length DEDD2 and mutant DEDD/DEDD2 proteins. (a) HeLa cells were transfected with 0.5 mg of the indicated GFP-DEDD2 constructs. At 24 h after transfection, cells were ﬁxed and immunostained with an anti-hUBF antibody (Santa Cruz) following by Texas red-conjugated anti-rabbit IgG antibody (Vector). The expression of GFP (left) and hUBF (middle) and the merged images (right) are shown. (b) Localization of GFP-DEDDDNLS and -DEDD2DNLS. HeLa cells transfected with the indicated GFP constructs were counterstained with DAPI (blue) for the nucleus. GFP images alone (left) and the GFP and DAPI-merged images (right) are shown

domain and the C-terminal domain contribute to the were found in the nucleus. The former contains the interactions (Figure 4b, lanes 4, 5). The latter was DED domains and should retain the ability to interact confirmed by the GST pull-down assay using recombi- with DEDD/DEDD2. The translocation of caspase-8 nant GST-DEDD2-N1 and -C and in vitro translated, occurred before detectable apoptosis and caspase-3 [35S]methionine-labeled DEDD and DEDD2 (Figure 4c). activation. The specificity of the fractionation proce- Activation of initiator caspases such as caspase-8 is dures was confirmed by the detection of b-tubulin, a triggered by adapter-mediated oligomerization (Chang cytosolic protein, and PML, a protein found mainly in and Yang, 2000). Thus, it is possible that DEDD and the nucleus, in the expected fractions. Additionally, the DEDD2 may promote activation of caspase-8/10 translocation was dependent on caspase activation through their ability to bind to caspase-8/10 as well as because it was inhibited by a caspase inhibitor zVAD- their ability to self-oligomerize. fmk. Caspase-8 is a cytosolic protein in living cells. If In summary, we show that DEDD and DEDD2 are DEDD/DEDD2 is to target caspase-8 to the nucleus, conserved through evolution and they are strong then caspase-8 is expected to be found in the nucleus at inducers of apoptosis. The function of DEDD/DEDD2 the early stage of CD95-mediated apoptosis. To test this in apoptosis is unique in that they are targeted to the possibility, HeLa cells were treated with agonistic anti- nucleolus, a subnuclear structure critical for the regula- CD95 antibody. The cell lysates were fractionated into tion of protein synthesis. Inhibition of protein synthesis the cytosolic and nuclear fractions. Interestingly, in the was shown to be an early event upon Fas engagement presence but not in the absence of CD95 stimulation, (Zhou et al., 1998). Given that DEDD/DEDD2 inter- both partially processed and mature caspase-8 molecules acts with both caspase-8 and -10, they may target these

Oncogene DEDD and DEDD2 are potent inducers of apoptosis A Alcivar et al 296

Figure 4 Interaction of DEDD2 with caspase-8 and -10 and the homo- and heteroassociation of DEDD2 and DEDD. (a) DEDD2 interacts with caspase-8 and -10. Expression plasmids for HA-DEDD2 and HA-DEDD2-N1 were transfected into 293 cells together with the vector control, FLAG-caspase-8, or FLAG-caspase-10 as indicated. Cell lysates were made in IP-lysis buffer (Yang et al., 1998b) and incubated with anti-FLAG M2 beads conjugated on agarose beads (Sigma). The anti-FLAG immunoprecipitates (IP) and extracts were subjected to Western blotting analysis (W) with rabbit anti-FLAG or anti-HA antibodies (Santa Cruz Biotech). Similar results were obtained for DEDD. Caspase-8/10 did not interact with the C-terminal region of DEDD2 (data not shown). (b) Homo- and heteroassociation of DEDD2 and DEDD 293 cells were transfected with HA-DEDD2 (top two panels) or HA-DEDD (middle two panels) together with expression plasmids for the indicated FLAG-tagged proteins. Anti-FLAG IP and extracts were analysed by Western blotting using indicated antibodies. The bottom panel shows the representative relative levels of FLAG-tagged proteins in the anti-FLAGIP for both HA-DEDD2- and HA-DEDD- transfected cells. The data shown in (a) and (b) are representative of three independent experiments. (c) In vitro GST pull-down assay. DEDD2-N1 and DEDD2-C were cloned into pGEX-1lT (Amersham Pharmacia). DH5a transformants expressing the desired GST fusions were lysed and the insoluble pellet was dissolved in 1/50 volume 8m urea in 1 Â PBS, which was subsequently dialyzed stepwise to 1 Â PBS with decreasing concentration of urea to allow for protein renaturation. The resulting solution was subsequently incubated with glutathione-conjugated beads (Amersham Pharmacia). For in vitro binding experiments, 10 mg of each GST fusion immobilized on beads was incubated for 2 h with in vitro translated, [35S]methionine proteins. The binding proteins were analysed by SDS–PAGE and autoradiography. The inputs represent 5% of the samples used for binding. (d) Translocation of caspase-8 during CD95-mediated apoptosis. HeLa cells were treated for 24 h with anti- CD95 mAb CH11 (MBL, 50 ng/ml) plus IFN-g (Roche, 5 ng/ml), in the presence or absence of caspase inhibitor z-VAD (ESP), or treated with IFN-g alone (cell death is o5%). The cell pellets were suspended in buffer A (10 mm Tris-HCl pH 8.0, 3 mm CaCl2,2mm MgCl2, and 0.32 m sucrose) plus 0.5% Nonidet P-40, and homogenized in douncer (Wheaton, loose pestle), followed by centrifugation at l000 g for 5 min. The supernatant was designated as the cytosolic fraction. The pellets were re-suspended in buffer A, and mixed with equal volume of buffer B (10 mm Tris-HCl, pH 8.0, 2 m sucrose, 5 mm Mg(Ac)2, and 0.1 mm EDTA). The mixture was layered onto the buffer B and centrifuged at 30 000 g for 45 min. The pellet was designated as the nuclear fraction. The cytosolic (C) and the nuclear (N) fractions were resolved on SDS–PAGE and analysed by immunoblotting using antibodies against indicated proteins. *A nonspeciﬁc band

Oncogene DEDD and DEDD2 are potent inducers of apoptosis A Alcivar et al 297 two caspases to the nucleolus to inhibit protein that DEDD and DEDD2 may play a critical role in translation apparatus. Consistent with this possibility, death receptor-induced apoptosis. caspase-8 was found to translocate to the nucleus during CD95-mediated apoptosis and the cell death activity of DEDD/DEDD2 is related to their nuclear localization. Acknowledgments Furthermore, DEDD and DEDD2 form homo- and We thank Vanessa Capacio for excellent technical assistance. heterodimers and they could facilitate activation of Supported by an NIH Cell and Molecular training grant (to caspase-8/10 through aggregation. These results suggest AA), a cystic ﬁbrosis foundation fellowship (to JT), and an NIH Grant CA88868.

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