Interchangeable Binding of Bcl10 to TRAF2 and Ciaps Regulates Apoptosis Signaling

Interchangeable binding of Bcl10 to TRAF2 and cIAPs regulates apoptosis signaling

Daishi Yui*,1,3, Takunari Yoneda1,3, Kayoko Oono1,3, Taiichi Katayama1,3, Kazunori Imaizumi2 and Masaya Tohyama1,3

1Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University. 2-2 Yamadaoka Suita, Osaka 565- 0871, Japan; 2Department of Cellular and Structural Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama Ikoma, Nara 630-0101, Japan; 3CREST of Japan Science and Technology Corporation (JST), 2-2 Yamadaoka Suita, Osaka 565-0871, Japan

Bcl10 was identi®ed as a candidate gene responsible for Recently, Bcl10 has been identi®ed as a mutative gene low grade B cell lymphomas of mucosa-associated with high frequency chromosomal translocation lymphoid tissue. Overexpression of Bcl10 in cultured t(1;14)(p22;q32) (Willis et al., 1999; Zhang et al., cells was reported to promote apoptosis, however, the 1999), suggesting that it is a target gene involved in mechanism of regulation of apoptosis mediated by Bcl10 the pathogenesis of a wide range of human malig- has not been demonstrated. In the present study, we nancies (Bertoni et al., 1999; Du et al., 2000; Simms et analysed the apoptosis signaling pathway mediated by al., 2000; Yamamoto et al., 2000). Bcl10 has a caspase- Bcl10, focusing on phosphorylation of Bcl10 and the recruiting domain (CARD) at its amino-terminal dynamic interaction with its binding partners during region. The CARD domain is suggested as mediating apoptosis. Previously, we have demonstrated that Bcl10 the binding between adapter molecules and caspases. potentially interacts with the other apoptosis regulator, The domain is found in a number of components of TNF receptor associated factor-2 (TRAF2) and inhibitor apoptosis cascades such as pro-apoptotic RAIDD/ of apoptosis proteins (cIAPs). The present results showed CRADD, RIP2/CARDIAK, Ced4/Apaf-1 and anti- that the complex formation of these molecules was apoptotic cIAP1, cIAP2 and ARC (Cecconi, 1999; regulated by phosphorylation of Bcl10, that is, phosphor- Chou et al., 1998; Koseki et al., 1998; McCarthy et al., ylation of Bcl10 resulted in binding of Bcl10 to cIAPs 1998; Rothe et al., 1995). Recently identi®ed protein, and the dissociation of it from TRAF2. Moreover, CARD9, binds to Bcl10 and induces NFkB activation hyperphosphorylation of Bcl10 enhanced apoptosis, (Bertin et al., 2000). These CARD-containing proteins suggesting that changes in the binding partners of interact with each other under a variety of stimuli, Bcl10 were correlated to the promotion of apoptosis as including DNA-damaging agents, growth factor starva- mediated by Bcl10. Indeed, the mutant which was deleted tion and treatment of cell-death ligands such as Fas from the binding site of Bcl10 for cIAPs, could not and TNF. The resultant bindings cause activation of induce apoptosis. These ®ndings indicate that Bcl10 is a downstream signaling, initiating apoptosis (review by mediator of apoptosis signaling, by switching over Homan et al., 1997; Hofmann, 1999). binding to cIAPs from TRAF2 through the events of Previously, we have reported that Bcl10 binds to Bcl10 phosphorylation. Oncogene (2001) 20, 4317 ± both TNF receptor associated factor-2 (TRAF2) and 4323. inhibitors of apoptosis proteins (cIAPs), which form TNF-receptor complexes (Yoneda et al., 2000). It is Keywords: Bcl10; TRAF2; cIAPs; phosphorylation; well known that TRAF2 mediates signaling via TNF- apoptosis receptors and is mainly involved in NF-kB activation (Arch et al., 1998). The activation of NF-kB promotes the expression of genes encoding anti-apoptotic factors Introduction that allow the cell to survive. One of these inducible genes is cIAP2 which can be a potent inhibitor of It is well known that recurrent chromosome 1p22 active caspases, and also activates NF-kB (Rothe et al., abnormalities occur in several types of lymphoma, 1995; Shu et al., 1996; Wang et al., 1998). Taken however, the molecular events remain unsettled. together, Bcl10 could play a role in apoptosis by regulating the interaction with TRAF2 or cIAPs. In the present study, we examined the mechanisms responsible for interaction of these three molecules, *Correspondence: D Yui, Department of Anatomy and and demonstrated that the interaction could be Neuroscience, Graduate School of Medicine, Osaka University, 2-2 regulated by alteration of phosphorylated-Bcl10. We Yamadaoka Suita, Osaka 565-0871, Japan; also showed that a combination of interaction with E-mail: [email protected] Received 23 January 2001; revised 9 March 2001; accepted 30 cIAP2 and dissociation from TRAF2 is necessary for April 2001 apoptosis mediated by Bcl10. Phosphorylated Bcl10 enhances apoptosis signaling D Yui et al 4318 Results Bcl10 phosphorylation and apoptosis Regulation of Bcl10 phosphorylation Overexpression of Bcl10 has been known to induce apoptosis in 293T cells. To examine the eects of Western blotting of Bcl10 in cells in which its phosphorylation of Bcl10 on apoptosis signaling, 293T expression plasmids were introduced showed a 32 ± cells, which were beforehand transfected with Bcl10 35 kDa double band (Figure 1), and the higher band of expression plasmids, were treated with PMA and H-7. 35 kDa was demonstrated, by metabolic labeling Treatment with PMA signi®cantly enhanced activities experiments using 32P, to be phosphorylated Bcl10 of caspase-3 like proteases twofold compared with the (data not shown). The ®nding raised the possibility controls, which were not treated with PMA (Figure that the function of Bcl10 may be regulated by its 2a). Morphologically, we con®rmed that cell death phosphorylation. First, we examined the eects of induced by overexpression of Bcl10 was promoted by various agents for protein kinase activators and treatment with PMA (Figure 2b). Conversely, treat- inhibitors on Bcl10 phosphorylation. We treated 293T ment with H-7 inhibited activation of caspase-3 like cells transiently transfected with Bcl10 expression proteases and cell death induced by Bcl10. Neither plasmids with ®ve kinds of agents, 10 nM phorbol 12- PMA nor H-7 aected the viabilities in non-transfected myrisate 13-acetate (PMA) (activator of protein kinase 293T cells, and also did not cause changes in the C(PKC)), 10 mM H-7 (inhibitor of PKC), 10 mM expression levels of Bcl10 (data not shown), suggesting Forskolin (activator of adenylate cyclase and increases that phosphorylated forms of Bcl10 could contribute to cytosolic cyclic-AMP), 10 mM H-89 (inhibitor of activation of Bcl10-mediated cell death. protein kinase A(PKA)) and 1 mM Wortomaninn (inhibitor of PI3 kinase). The levels of phosphorylated Bcl10 were markedly increased by treatment with PMA Phosphorylation of the endogenous Bcl10 and decreased by H-7. Phosphorylation of Bcl10 was Bcl10 is highly expressed in lymphoid tissues (Willis et also promoted by treatment with Forskolin, and al., 1999; Yoneda et al., 2000). From our previous slightly inhibited by Wortomaninn. Treatment with observation of remarkable atrophy of the thymus in H-89 did not aect Bcl10 phosphorylation (Figure 1). Bcl10 overexpressed mice, we used thymocytes as a These results indicated that regulators of PKC model to monitor the endogenous Bcl10 phosphoryla- activities eectively altered the levels of phosphorylated tion followed by cell death. Thymocytes were metabo- Bcl10, however, as some other kinase modulators also lically labeled by 32P for 3 h, and immunoprecipitated aect those, it is still unclear whether Bcl10 is with anti-Bcl10 polyclonal antibody. After treatment phosphorylated by PKC directly or indirectly. From with PMA, the endogenous Bcl10 phosphorylation this data, we used PKC modulators to regulate the level was signi®cantly increased compared with the levels of Bcl10 phosphorylation in the following experiments.

Figure 2 Increased activity of caspase-3 and apoptosis by phosphorylated Bcl10. (a) 293T cells transiently transfected with Figure 1 Eects of various agents which activate or inhibit Bcl10 were treated with PMA or H-7 for 6 h, caspase-3 activities protein kinases on phosphorylation of Bcl10. Western blotting were measured in each cell lysate using the ¯uorogenic substrate analyses were performed with anti-Bcl10 antibody using cell Ac-DEVD-AMC. A value of 1.0 corresponds to caspase-3 lysates expressing Bcl10 after treatment with the indicated agents activities of untreated cells transfected with mock vector. (b) for 30 min. Signal intensities for phosphorylated forms of Bcl10 The relative numbers of apoptotic cells were evaluated by Live/ were measured using a densitometer. A value of 1.0 corresponds Dead stainings as described in the Materials and methods. A to the amount of phosphorylated Bcl10 protein in unstimulated value of 1.0 corresponds to the amount of stained apoptotic cells cells. Data from four independent measurements are plotted as in 293T cells transfected with mock vector. Error bars refer to the relative density. P-Bcl10; Phosphorylated Bcl10 standard deviation obtained in three independent experiments

Oncogene Phosphorylated Bcl10 enhances apoptosis signaling D Yui et al 4319 control, which is not treated with PMA (Figure 3a). reverses the eects of PMA on phosphorylation and We also con®rmed that the treatment with PMA binding of Bcl10 to cIAP2 (data not shown). In all caused DNA fragmentation and cell death (Figure these experimental conditions, expression levels of 3b,c) as previously reported (Kizaki et al., 1989). TRAF2 and cIAP2 were not changed (Figure 4a,b). Taken together, endogenous Bcl10 is indeed phosphorylated in thymocytes during apoptosis induced by PMA and the ®ndings lead us to speculate that the phosphorylation of Bcl10 might be playing crucial roles in apoptosis signaling in thymocytes.

Dynamic interaction of Bcl10 with TRAF2 or cIAP2 In our previous paper we reported that Bcl10 binds to both TRAF2 and c-IAPs (Yoneda et al., 2000), suggesting that these proteins could be involved in modulation of Bcl10 functions through direct interaction. We examined whether phosphorylation of Bcl10 altered the complex formation of Bcl10, TRAF2 and c- IAPs. After treatment with 10 nM PMA, or 10 mM H-7 for 6 h, cells were lyzed and immunoprecipitated with anti-Bcl10 polyclonal antibody, followed by immuno- blotting with anti-TRAF2 or anti-cIAP2 polyclonal antibodies. Treatment with PMA decreased amounts of TRAF2 co-immunoprecipitated with Bcl10 (Figure 4a), alternatively, it increased those of cIAP2 bound to Bcl10 (Figure 4b). In contrast, treatment with H-7 Figure 4 Changes in binding partners of Bcl10 by its phosphorylation. 293T cells were co-transfected with 2.0, 1.5, 1.5 (mg) of enhanced both interactions of Bcl10 with TRAF2 and expression plasmids for Bcl10, TRAF2 and cIAP2, respectively. dissociation of Bcl10 from cIAP2 (Figure 4a,b). The Twenty-four hours after transfection, cells were stimulated by same results were obtained in the reverse experiments, PMA or H-7 for 6 h. Each lysate was immunoprecipitated with which were immunoprecipitated with anti-TRAF2 or anti-Bcl10 antibody and detected with antibodies against TRAF2 (a) or cIAP2 (b) by Western blots (lower panel shows Western anti-cIAP2 polyclonal antibodies, followed by immu- blots of TRAF2 (a) and cIAP2 (b) using each cell lysate). (c) The noblotting with anti-Bcl10 antibody. We also tested levels of phosphorylated Bcl10 after treatment with each agents that the co-treatment of cells with H-7 and PMA are shown

Figure 3 Phosphorylation of Bcl10 in thymocytes during apoptosis. (a) Thymocytes were treated with 10 nM PMA for 30 min, and then incubated with 32P for 3 h. Cell lysates were immunoprecipitated with anti-Bcl10 antibody (upper panel). Signal intensities for phosphorylated Bcl10 were measured and presented as Figure 1 (lower panel). (b) After treatment with PMA for 6 h, thymocytes DNA were extracted and subjected to electrophoresis. Lane 1, DNA from thymocytes without PMA treatment; Lane 2, PMA treated thymocytes; M, HindIII digest of lamda phage for DNA marker. (c) Thymocytes were incubated with or without PMA for 48 h. Apoptotic cells were visualized by Live/Dead stainings. Error bars refer to the standard deviation of three independent experiments

Oncogene Phosphorylated Bcl10 enhances apoptosis signaling D Yui et al 4320 These ®ndings suggest that the changes in phosphor- cIAP2, are essential for activation of apoptosis ylation of Bcl10 may alter its binding partners, that is, signaling by Bcl10. phosphorylated forms of Bcl10 interact with cIAP2, and non-phosphorylated-forms interact with TRAF2. Discussion Functional domains in Bcl10 The mutation of Bcl10 is commonly considered to be As described above (Figure 4), the phosphorylated involved in the pathogenesis of a wide range of human form of Bcl10 interacts with cIAp2 during apoptosis malignancies. Therefore, it has been proposed that induced by overexpression of Bcl10. To examine the Bcl10 is a tumor suppressor gene. Overexpression of binding site of Bcl10 to cIAP2, we engineered ®ve wild-type Bcl10 induces apoptosis in several types of deletion mutants, delta C143 (amino acids 1 ± 143), cells (Thome et al., 1999; Koseki et al., 1999; Yan et delta C191 (1 ± 191), delta N48 (48 ± 233), delta N78 al., 1999). Indeed, our recent report showed that Bcl10 (78 ± 233) and delta N117 (117 ± 233) (Figure 5a). Each transgenic mouse revealed promotion of apoptosis in mutant was tagged with a FLAG epitope sequence at the thymocytes and lymphocytes at postnatal stages. its C-terminal. Immunoprecipitation followed by These aspects raise the possibility that it could play Western blotting was performed after co-transfection crucial roles in regulation of apoptosis in various cells. of each deletion mutant of Bcl10 and full-length cIAP2 In this report, to elucidate the regulatory mechanisms into 293T cells. All constructs except C143 were co- of apoptosis by Bcl10, we analysed biological char- immunoprecipitated with Bcl10 (Figure 5b). This result acteristics of Bcl10, particularly focusing on the indicates that the binding site of Bcl10 to cIAP2 could interchangeable interactions with the other apoptosis be within amino acids 117 ± 143. regulators, TRAF2 and cIAP2, during apoptosis. To understand the mechanisms of Bcl10 induced Although Bcl10 has been known to interact with apoptosis, we tried to determine the essential domains TRAF2 and cIAP2, the binding was demonstrated in of Bcl10 for induction of apoptosis. Morphological this study to be dynamically interchangeable under changes and caspase-3 like activities in 293T cells apoptotic or non-apoptotic conditions. The changes of which were transiently co-transfected with each Bcl10 the binding partners of Bcl10 were closely associated construct and GFP cDNA were examined. The great with the promotion of apoptosis. That is, when majority of GFP-positive cells, which were co-trans- apoptosis is undergone by overexpression of Bcl10, it fected with expression plasmids for Bcl10 and GFP, preferentially interacts with cIAP2 and dissociates from showed morphological characteristics of early stage of TRAF2. Alternatively, when the apoptosis is sup- apoptosis such as shrinking cell bodies (Figure 6a). pressed by agents which decrease levels of phosphory- Activation of caspase-3 like proteases was also lated-Bcl10, it forms a stable complex with TRAF2. observed in these cells (Figure 6b). The same abilities The ®ndings indicate that Bcl10 is a mediator of were observed in cells transfected with C191 and N48. apoptosis signaling by switching binding to cIAP2 The eects of the transfection with N117 on induction from TRAF2. Indeed, the mutant which had the of apoptosis were less than those of full-length, C191 deleted binding site of Bcl10 for cIAP2, could not or N48. Cells transfected with C143 showed no induce apoptosis even if the construct was over- changes in morphology and caspase-3 like activities. expressed. It has been reported that BIR domains of These results indicate the domains of amino acids cIAPs are capable of binding to caspase-3, -7, -9 and 117 ± 143, which predicted to be the binding site for potentially inhibiting the activation of these caspases

Figure 5 The structure and expression of deletion mutants of Bcl10. (a) Each construct of deletion mutants of Bcl10. CARD domains and Flag-epitope tags are shown by gray and black boxes, respectively. (b) Interaction of each mutant with cIAP2. 293T cells were co-transfected with Bcl10 mutants and cIAP2 expression vectors. Cell lysates were immunoprecipitated with anti-Bcl10 antibody, and immune complexes were detected by Western blotting with anti-cIAP2 antibody (upper panel). cIAP2 expression levels were almost equivalent in each cell lysate (lower panel). Note that delta C143 is not co-immunoprecipitated with cIAP2

Oncogene Phosphorylated Bcl10 enhances apoptosis signaling D Yui et al 4321 (Takahashi et al., 1998; Roy et al., 1997). Therefore, we propose a possible mechanism of Bcl10-mediated apoptosis as follows; in cells in which apoptosis is induced by overexpression of Bcl10, or promoted by phosphorylation of Bcl10, Bcl10 causes competitive inhibition of the direct interaction of caspases and cIAPs. The dissociated caspases from cIAPs may result in the cleaving of substrates and lead the cells to apoptosis. Previously, we reported that the transgenic mice expressing Bcl10 exhibited abnormalities in their immune systems; that is, accelerated apoptosis in thymocytes and lymphocytes after the postnatal stage. This phenotype is similar to that of TRAF2-de®cient mice (Yeh et al., 1997), suggesting that these two proteins might play crucial roles in the regulation of survival or cell death in immune systems in response to a number of cytokines. Additionally, as cIAP2 is abundantly expressed in lymphoid tissues, it also may be involved in regulation of apoptosis by co-operating with TRAF2 and Bcl10. The phosphorylation of Bcl10 caused changes in its binding partners, suggesting that the switching of the binding partner is regulated by its phosphorylation. The putative phosphorylation sites serve the motifs that are phosphorylated by PKC and casein kinase 2. As the agents which regulate activities of PKC, such as PMA or H-7, caused alteration of the levels of phosphorylated-Bcl10, Bcl10 could be directly phosphorylated by PKC. A variety of reports have suggested that PKC can act as a potentiator of apoptosis induced by stimuli such as TNF (Laouar et al., 1999; Takada et al., 1999). It is possible that Bcl10 might be a novel target molecule involved in the signaling of PKC-mediated apoptosis. The speculation is supported by the ®ndings that the accelerated phosphorylation of endogenous Bcl10 was observed in thymocytes initiating apoptosis after treatment with PMA. However, some other agents that modulate phosphorylation, dierently from the PKC pathways, also aected the levels of phosphorylated-Bcl10. There- fore, to clarify whether PKC directly phosphorylates Bcl10, further examination is needed. As shown by the present study, Bcl10 interchange- ably binds to TRAF2 and cIAPs through the events of Bcl10 phosphorylation, and the resultant alteration of its binding partner may activate apoptosis signaling. Therefore, regulatory controls of Bcl10 phosphorylation are possible strategies for therapies for some kinds of human cancers in the immune system. Figure 6 Eects of each mutant of Bcl10 on cell death. (a) Apoptotic changes in cells co-transfected with Bcl10 mutants and GFP. 293T cells were co-transfected with expression plasmids for: (A) Mock, (B) Bcl10, (C) delta C143, (D) delta C191, (E) delta N78, (F) delta N117, and GFP as described in Experimental Materials and methods Procedures. Then the morphology was observed by ¯uorescence detection of GFP activity at 48 h after the transfection. The Cell culture and transfections arrowhead shows the early apoptotic cells. (b) Activation of 293T cells were cultured in Dulbecco's Modi®ed Eagle's caspase-3 by each mutant of Bcl10. 293T cells were transfected with each construct of Bcl10 mutant. Twenty-four hours after the medium (DMEM) supplemented with 10% fetal bovine serum, transfection, lysates were measured for caspase-3 activities using penicilin (100 units/ml), streptomycin (100 mg/ml). Transient the ¯uorogenic substrate Ac-DEVD-AMC. Error bars refer to the transfections into 293T cells were undertaken by Lipofect- standard deviation obtained in three independent experiments AMINE reagent (Gibco BRL), according to the manufac-

Oncogene Phosphorylated Bcl10 enhances apoptosis signaling D Yui et al 4322 turer's procedures. To alter phosphorylation of Bcl10, 293T Cleavage of Ac-DEVD-AMC was then measured using a cells were treated with 10 nM PMA (SIGMA), 1 mM H-7 and luminescence spectrometer. H-89 (Seikagaku Kogyo, Tokyo, Japan), 10 mM Forskolin (SIGMA) and 1 m Wortomaninn (SIGMA). Thymocytes M Detection of DNA fragmentation were obtained from Wistar rat purchased from Nippon Charles River (Yokohama, Japan) and were suspended in Thymocytes were cultured with or without PMA in serum RPMI supplemented with 10% fetal bovine serum. free RPMI for 12 h at 378C. Then thymocyte DNA were puri®ed using an Apoptotic DNA-Ladder Kit (Roche, USA). Aliquots of 2 mg DNA were electrophoresed in 1.5% agarose Antibodies, immunoprecipitation and Western blotting gel. Rabbit antisera were raised against a synthetic peptide corresponding to residues 220 ± 233 of Bcl10. Anti-TRAF2 Construction of Bcl10 mutants H-249 (Santa Cruz Biotechnology) and cIAP2 (Santa Cruz Biotechnology) polyclonal antibodies and Anti-Flag mono- Wild-type Bcl10 expression vector has been described clonal antibody (Eastman Kodak) were purchased. For previously (Yoneda et al., 2000). Deletion mutants were immunoprecipitation, cells were lysed in Nonidet P-40 buer ampli®ed by PCR using the following oligos; For the carboxi- (0.05 M Tris, pH 7.6, 0.15 M NaCl, 1 mM PMSF, 0.2% terminal deletion mutants C143 (1 ± 143 amino acids), 5'- Nonidet P-40). Samples were incubated for 1 h at 48C with TCACTGATGTTCCTGCTGCTACTGTTCGAAGTTGCT- 3 mg antibody. Recombinant protein-G-agarose (GIBCO) CTCATCAGAATT-3'; C191 (1 ± 191 amino acids), 5'- was added to each of the samples, which were then incubated TCACTGATGTTCCTGCTGCTACTGTTCAAGCGAGG- further for 1 h at 48C. The beads were washed ®ve times in CTGAAGAGAAGCT-3' as a sense and for the antisense, 5'- lysis buer then boiled in SDS sample buer and loaded in CCACCATGGAGGCTCCCGCGCCGTCT-3'. For the ami- 12% SDS-polyacrilamide gel. After electrophoresis, proteins no-terminal deletion mutant; N48 (48 ± 233 amino acids), 5'- were transferred to Immobilion-P membrane and immuno- CCACCATGAGTAGAGAAGACACAGAA-3'; N78 (78 ± detection was done using an ECL Western blotting detection 233 amino acids), 5'-CCACCATGCTGGACACCCTTGTG- system (Amersham). GAA-3'; N117 (117 ± 233 amino acids), 5'-CCACCATGCT- GAAGTGCAGTAGCTGTGAG-3' as an antisense and for the sense, 5'-TCACTGATGTTCCTGCTGCTACTGTTC- 32P-Orthophosphate labeling CTGCCGTGAAAGAGCCC-3'. The sequence coding Flag- For in vivo metabolic labeling assay, 293T cells were epitope was tagged with each construct at its 3'-site and was incubated in phosphate-free DMEM for 30 min before subcloned into the EcoRI site of pcDNA3.1 vector. labeling. 150 mCi/ml 32P-orthophosphate (NEN Life Science Products) was added to medium and incubated for Morphological evaluation of apoptosis approximately 3 h at 378C. Cells were then washed two times with Dulbecco's phosphate-buer saline and lysed in For morphological evaluation of cell death, a Live/Dead Nonidet P-40 lysing buer (0.05 M Tris, pH 7.6, 0.15 M staining kit (Molecular Probes) was used. For apoptosis NaCl, 1 mM PMSF, 0.2% Nonidet P-40). Radiolabeled lysate assay of deletion mutants, 293T cells were transfected with from each sample was immunoprecipitated with anti-Bcl10 pairs of expression plasmids encoding Bcl10 or its mutants antibody. The immunoprecipitated complexes were washed and GFP at a 1 : 10 ratio of GFP plasmid to Bcl10 ®ve times in wash buer (same as lysis buer) then boiled in plasmid. Fluorescence activity of GFP was viewed 48 h SDS sample buer and electrophoresed in 12% SDS- after transfection in a Nikon Diaphoto microscope polyacrilamide gel. For detection of 32P, the gel was dried equipped with epi¯uorescence. Fluorescence experiments and then exposed to X-ray ®lm. were repeated on six occasions. Representative photomicro- graphs were shown. Caspase-3 assay 293T cells transfected with Bcl10 or its mutant expression vectors were lysed in a lysis buer (50 mM Tris-HCl, pH 7.4, Acknowledgments 1mM EDTA, 10 mM EGTA, 10 mM digitonin, 10 mM We would like to thank Akemi Arakawa for technical dithiothreitol). Caspase-3 activities in cell lysates were assistance, and Dr Ryosuke Takahashi (Brain Science assayed with ¯uorogenic substrate Ac-DEVD-AMC (Peptide Research Center, RIKEN) for providing the cIAPs cDNA Institute). Aliquots of 40 mg total proteins were incubated for vectors. This work was supported by grants from CREST 30 min at 378C in the presence of 0.1 mg of substrate. of Japan Science and Technology Corporation (JST).

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