Proapoptotic Activity of Itm2bs, a BH3-Only Protein Induced Upon IL-2-Deprivation Which Interacts with Bcl-2

Proapoptotic activity of ITM2Bs, a BH3-only protein induced upon IL-2-deprivation which interacts with Bcl-2

Aarne Fleischer1, VeroÂ nica AylloÂ n1, Laure Dumoutier2, Jean-Christophe Renauld2 and Angelita Rebollo*,1

1Centro Nacional de BiotecnologõÂa, Department of Immunology and Oncology, Campus de Cantoblanco, UAM, 28049 Madrid, Spain; 2Ludwig Institute for Cancer Research, Brussels Branch and Experimental Medicine Unit, Christian de Duve Institut of Cellular Pathology, UniversiteÂ de Louvain, Brussels, Belgium

Growth factor deprivation is a physiological mechanism 1998). This is supported by recent discovery of a new to induce apoptosis. We used an IL-2-dependent murine group of cell death members containing only the BH3 T cell line to identify proteins that trigger apoptosis. domain (Fesik, 2000). BH3 domain in these molecules Here we report the identi®cation, the cloning and plays an important role in mediating proapoptotic characterization of ITM2Bs, a protein induced upon function as well as association with the prosurvival IL-2-deprivation. ITM2Bs, which shares the BH3 members of the Bcl-2 family. It has been shown that domain of Bcl-2 family members, is a cytoplasmic and mutagenesis of the critical leucine and aspartic acid mitochondrial protein. Expression of ITM2Bs induces residues within the BH3 domain of Bax is critical for apoptosis in IL-2-stimulated cells, but not in IL-4- dimerization and killing (Wang et al., 1998). stimulated cells, while overexpression of the long form of Most of BH3-only proteins were discovered in the protein is not able to induce apoptosis. In IL-2- protein interaction traps by binding to Bcl-2 or Bcl-x stimulated cells, ITM2Bs interacts with the antiapoptotic or in functional assays. The amphipathic helix formed protein Bcl-2, and does not interact with the proapopto- by the BH3 domain of BH3-only proteins bind to a tic Bad. Mutation of the critical L and D residues within hydrophobic groove on the surface of the antiapoptotic the BH3 domain abolished the ability of ITM2Bs to Bcl-2 family members (Fesik, 2000). Bid can also promote apoptosis. interact with Bax and Bak, resulting in cytochrome c Oncogene (2002) 21, 3181 ± 3189. DOI: 10.1038/sj/ releasing (Wei et al., 2000). This result suggests that onc/1205464 some BH3-only proteins can promote apoptosis by two mechanisms: inactivating prosurvival Bcl-2-like pro- Keywords: apoptosis; BH3; ITM2Bs teins by direct binding or by modi®cation of proapoptotic Bax-like molecules. Many BH3-only proteins are present in healthy cells Introduction in a dormant form and are activated by post- translational modi®cations. Signals from certain Members of the Bcl-2 family proteins are regulators of growth factors results in phosphorylation of Bad at cell death that can be grouped into prosurvival and multiple sites and sequestration of modi®ed Bad by proapoptotic molecules (Adams and Cory, 1998; Gross binding to 14-3-3 protein (Hsu et al., 1997; Zha et al., et al., 1999). They are characterized by the presence of 1996b). In addition to post-translational modi®cations, several conserved motifs, known as the Bcl-2 homology BH3-containing proteins may be regulated at the level (BH) domains, designed BH1, BH2, BH3 and BH4. of expression. Neuronal cell death upon deprivation of Although the BH4 domain is restricted to some nerve growth factor can be blocked by inhibitors of prosurvival members, BH1, BH2 and BH3 can RNA and protein synthesis, suggesting that this death generally be found among members of both, the is dependent on the regulated expression of some prosurvival and the proapoptotic proteins. Mutagenesis apoptotic factor(s) (Lutz, 2000). Finally, an interferon- and structural studies revealed that BH3 domain is induced dual function protein with a BH3 domain can important for dimerization function. In addition to its polymerize ATP and promote apoptosis independently role as a protein ± protein interaction domain, the BH3 (Ghosh et al., 2001). motif of proapoptotic members appears to be Integral membrane protein 2B (ITM2B) is a type II important for mediating their proapoptotic function integral membrane protein that is associated to (Chittenden et al., 1995b; Kelekar and Thompson, chrondrogenic dierentiation (Deleersnijder et al., 1996). ITM2B belongs to a multiple gene family containing, at least, two other members, ITM2A and ITM2C. The three isoforms share 50% identity at the *Correspondence: A Rebollo; E-mail: [email protected] Received 18 October 2001; revised 9 March 2002; accepted 11 amino acid level, with most of the similarity in the March 2002 COOH-terminal domain. An alternative splicing of Apoptotic activity of ITM2Bs A Fleischer et al 3182 ITM2B generates the long and short forms of ITM2B. upregulated within 3 h of IL-2-deprivation and max- In this manuscript we describe the characterization of imal expression was reached upon 20 h of lymphokine ITM2Bs as a novel regulator of cell death, that deprivation. The upregulation of ITM2Bs expression promotes apoptosis and interacts with Bcl-2. was also con®rmed using a probe of the 5' untranslated region of ITM2Bs, that is absent in ITM2BL (data not shown). A non upregulated 1.4 kb transcript that corresponds to the long form of the Results protein, ITM2BL, was also observed (Figure 1). As internal control of RNA loaded, 28S and 18S signal Identification of ITM2B as an IL-2-deprivation induced s are shown. gene in TS1ab cells The full-length ITM2Bs was cloned by PCR and To identify proteins that could be involved in the encodes a 210 amino acid protein (Figure 2a). Protein induction of apoptosis, we performed a representa- homology searches in the GenBank database with the tional dierence analysis (RDA) of gene expression in Blast program revealed a stretch of six amino acids, IL-2-deprived cells. Cells were IL-2-stimulated or 35 ± 40, with high homology to a BH3 motif (Figure -deprived for 20 h before mRNA extraction. The 2b). This conserved motif is shared by Bcl-2 family isolation of IL-2-deprivation induced transcripts was members such as Bad, Bak, Bax, Bcl-x, Bcl-2, Bid, Bik, performed using the representational dierence analy- Bim, Harakiri, Noxa, Diva, Map1, 2-5(A) synthase and sis technique as previously described (Hubank and Puma (Figure 2b) (Chinttenden et al., 1995a; Zha et Schatz, 1994; Louahed et al., 1999). Oligo (dT)-primed al., 1996a). The mouse genomic database revealed a cDNAs were synthesized, digested with DpnII and clone from chromosome 14 containing the ITM2Bs used to generate the respective amplicons. After three gene. Comparison with the cDNA sequence suggests a rounds of subtractive hybridization, the third product ®ve exon structure (Figure 2c) for ITM2Bs gene. was cloned. Among the dierent IL-2-deprivation To study the eect of IL-2-deprivation on ITM2Bs induced transcripts, one of these clones consisted of protein expression, cells were stimulated or deprived of a 190 nucleotide DpnII fragment showing a 100% IL-2 for dierent periods of time and total ITM2Bs match to a cDNA encoding the integral membrane expression analysed by Western blot. Weak expression protein 2B (ITM2B). The kinetic of ITM2B expression of ITM2B was observed in control IL-2-stimulated was analysed by Northern blot after IL-2-deprivation cells, increasing around threefold upon IL-2-depriva- for dierent periods of time. As shown in Figure 1, a tion (Figure 3a). Reprobing the membrane with pan- transcript of 1.1 kb corresponding to ITM2Bs was Ras showed similar level of Ras expression throughout the kinetic analysed. The speci®city of the generated ITM2Bs antibody was estimated by competition with increasing concentrations of the peptide used for generation of the antibody (Figure 3b). This antibody recognizes both ITM2Bs and ITM2BL, that can be dierentiated by molecular weight. In addition, we also have generated polyclonal antibodies that speci®cally recognize ITM2BL. These results suggest that the dierent induction kinetics at the mRNA and protein level may be attributed to speci®c function acquired after IL-2-deprivation. To determine the subcellular localization of ITM2Bs, we isolated nuclear, mitochondrial, cytosolic and cytoplasmic proteins of IL-2-stimulated or -deprived cells. ITM2Bs was detected by Western blot in cytoplasmic extracts of IL-2-stimulated or -deprived cells and was not observed in nuclear extracts of IL-2-stimulated or -deprived cells (Figure 4). ITM2Bs was detected in the mitochondrial fraction of IL-2-stimulated cells. The amount of ITM2Bs associated to mitochondria increased upon IL-2-deprivation. We were not able to detect ITM2Bs Figure 1 Upregulation of ITM2Bs mRNA expression. Total RNA was isolated from 306106 IL-2-stimulated (C, 5 ng/ml) or in the cytosolic fraction of IL-2-stimulated cells, -deprived cells for dierent periods of time. RNA (15 mg) was being weakly detected in cytosolic fraction of IL-2- electrophoresed in 1% agarose/formaldehyde, blotted and deprived cells (Figure 4). As internal control of 32 hybridized in high stringency conditions with a P-labelled protein fractionation, the blot was probed with pan- ITM2B probe. Two transcripts of 1.1 kb, corresponding to s Ras (cytoplasmic marker), anti-caspase 3 (cytosolic ITM2Bs, and 1.3 kb, corresponding to ITM2BL, are shown. DNA was labelled by random priming. Both 28S and 18S are shown to marker), anti-Mito 2813 (pyruvate dehydrogenase, estimate RNA levels. Data are representative of three independent mitochondria marker) and anti-Histones antibody experiments (nuclear marker).

Oncogene Apoptotic activity of ITM2Bs A Fleischer et al 3183

Figure 2 ITM2Bs is a novel BH3-containing protein. (a) Predicted amino acid sequence of ITM2Bs. The BH3 domain of ITM2Bs is underlined and the conserved BH3 core element is in bold. (b) Alignment of the ITM2Bs BH3 motif with the BH3 domains of Bcl-2 family proteins: antiapoptotic murine Bcl-2 and Bcl-x; proapoptotic murine Bad, Bak, Bax, Bid, Bik, Bim, Harakiri, Diva, Puma, Map1, 2 ± 5 (A) synthetase and Noxa, with two BH3 domains. Conserved leucine (L) and aspartic acid (D) residues are shaded. (c) Exon ± intron organization of the ITM2Bs gene. Murine ITM2Bs contains ®ve exons spanning a 5.6 kb region of murine chromosome 14. Positions of the start and termination codons are indicated, with coding regions in grey blocks. The BH3 domain is located in exon 2

apoptosis, compared with the level of apoptosis Expression of ITM2B induces apoptosis, which is s observed in IL-4-stimulated or -deprived mock trans- repressed by Bcl-2 fected cells (Figure 5b). The level of apoptosis in non The eect of ITM2Bs on cell survival was examined in transfected cells is also shown. Coexpression of TS1ab cells using transient transfection assay. Trans- ITM2Bs and Bcl-2 strongly inhibited the apoptosis- fection of ITM2Bs expression plasmid into IL-2- inducing activity of ITM2Bs in IL-2-stimulated or stimulated cells results in a strong induction of -deprived cells, compared with the high level of apoptosis 20 h after transfection, compared with the apoptosis observed in ITM2Bs transfected cells (Figure level of apoptosis observed in IL-2-stimulated cells 5c). Expression of transiently transfected ITM2Bs or transfected with the empty vector, mock transfectants Bcl-2 in IL-2 or IL-4-stimulated or -deprived cells was (Figure 5a). Transfection of ITM2B into IL-2-deprived con®rmed by direct comparison of ITM2Bs or Bcl-2 cells also results in a dramatic induction of apoptosis protein level in transfected cells and mock controls. compared with the level observed in IL-2-deprived Unaltered Ras expression was shown under all mock transfected cells (Figure 5a). Transfection of transfection conditions as an internal control of protein ITM2BL in IL-2-stimulated or -deprived cells had no loading. eect on apoptosis (data not shown). The level of apoptosis observed in non transfected cells is also The BH3 domain of ITM2B is required for the induction shown. Expression of transfected ITM2B was con- s s of apoptosis ®rmed by Western blot. Ras expression is shown as internal control of protein loading. To determine if the BH3 domain of ITM2Bs is required Interestingly, expression of ITM2Bs in IL-4-stimu- to induce apoptosis, we compared the apoptotic lated or -deprived cells did not have any eect on activity of wild-type ITM2Bs and a BH3 domain

Oncogene Apoptotic activity of ITM2Bs A Fleischer et al 3184

Figure 4 ITM2Bs is a non-nuclear intracellular protein. Nuclear, mitochondrial, cytosolic and cytoplasmic proteins were isolated from IL-2-stimulated or -deprived cells, separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted sequentially with anti-ITM2B, pan-Ras (cytoplasmic marker), anti-caspase 3 (cytosolic marker), anti-Mito 2813 (pyruvate dehydrogenase, mitochondria marker) and anti-histones (nuclear marker). Similar results were obtained in two independent experiments

Figure 3 Upregulation of ITM2Bs expression at the protein level. (a) TS1ab cells were IL-2-stimulated (c) or -deprived for the times indicated and lysed. Proteins were separated by SDS ± protein level in transfected cells and mock controls. PAGE, transferred to nitrocellulose and probed with anti-ITM2B This result suggests that integrity of ITM2Bs BH3 antibody. Protein bands were detected using the ECL system. The domain is necessary for induction of apoptosis. blot was reprobed with pan-Ras antibody as internal control of To determine the ability of ITM2B to associate with protein loading. Similar results were obtained in three indepen- s dent experiments. Molecular weights of the corresponding antiapoptotic molecules, we performed immunoprecipitation experiments using ITM2B and Bcl-2 speci®c proteins are shown. Densitometric analysis of ITM2Bs Western s blot is shown. (b) TS1ab cells were lysed, proteins separated by antibodies. ITM2Bs was detected by Western blot in SDS ± PAGE and transferred to nitrocellulose. Anti-ITM2B anti-Bcl-2 immunoprecipitates of IL-2-stimulated cells antibody was pre-incubated with increasing concentrations of and was not observed in anti-Bad immunoprecipitates the peptide used for the immunization and then incubated with the blot (Figure 7a). ITM2Bs was not observed in anti-Bcl-2 or anti-Bad immunoprecipitates of IL-2-deprived cells. Reprobing the membrane with anti-Bcl-2 and anti-Bad antibodies showed both proteins in IL-2-stimulated mutant of ITM2Bs protein (ITM2Bs*), in which critical cells and only Bad expression in IL-2-deprived cells. To leucine (L) and aspartic acid (D) residues were mutated con®rm the interaction, we performed reciprocal to glutamic acid (E) and alanine (A) respectively. experiment, Bcl-2 was detected in anti-ITM2Bs im- Transient transfection shows that mutation of BH3 munoprecipitates of IL-2-stimulated cells (Figure 7a) domain inhibits apoptosis when compared with the and was not observed in anti-ITM2B immunoprecipi- ability of wild-type ITM2Bs to induce apoptosis in IL- tates of IL-2-deprived cells. We were not able to detect 2-stimulated or -deprived cells (Figure 6a). Expression association of Bad to ITM2Bs upon ITM2B immuno- of ITM2Bs* had no eect on apoptosis in IL-4- precipitation (Figure 7a). Reprobing the membrane stimulated or -deprived transfected cells compared to with anti-ITM2B showed the presence of the protein. mock transfectants (Figure 6b). Expression of transi- The speci®city of the described interaction was ently transfected proteins in IL-2 or IL-4-stimulated or con®rmed by immunoprecipitation of cytoplasmic -deprived cells was con®rmed by direct comparison of lysates with an irrelevant antibody, anti-IL-2.

Oncogene Apoptotic activity of ITM2Bs A Fleischer et al 3185

Figure 5 Expression of ITM2Bs induces apoptosis, which is repressed by Bcl-2. (a) Cells were transfected with or without ITM2Bs using the DEAE-dextran method and maintained after transfection with or without IL-2 for 20 h. Cells were washed, permeabilized and stained with propidium iodide. Samples were analysed by ¯ow cytometry. Non transfected cells maintained in the presence or absence of IL-2 were also used as control. Standard deviation (s.d.) is shown were n=3. IL-2-stimulation, white bars; IL-2- deprivation, shaded bars. Expression of the transiently transfected ITM2Bs was con®rmed by direct comparison of protein level in mock and ITM2Bs transfected cells. The blot was probed with pan-Ras as internal control of protein loading. IL-2-stimulation, white bars; IL-2-deprivation, shaded bars. (b) Cells were transfected with or without ITM2Bs as in a and maintained after transfection with or without IL-4 for 24 h. Samples were analysed by ¯ow cytometry. Non transfected cells maintained in the presence or absence of IL-4 were also used as control. Standard deviation (s.d.) is shown were n=3. IL-4-stimulation, white bars; IL-4-deprivation, shaded bars. Expression of the transiently transfected ITM2Bs was con®rmed by Western blot. The blot was proved with pan-Ras as internal control of protein loading. IL-4-stimulation, white bars; IL-4-deprivation, shaded bars. (c) Cells were cotransfected with or without ITM2Bs or ITM2Bs and Bcl-2 as in a and maintained after transfection in the presence or absence of IL-2 for 24 h. Samples were analysed by ¯ow cytometry. Expression of the transfected ITM2Bs was con®rmed by Western blot. The blot was probed with pan-Ras as internal control of protein loading. IL-2-stimulation, white bars; IL-2- deprivation, shaded bars. Similar results were obtained in three independent experiments

To study the eect of IL-2-deprivation in Bad and Bcl-2 Discussion expression, TS1ab cells were IL-2-stimulated or -deprived for dierent periods of time and total protein expression We have identi®ed ITM2Bs as a novel regulator of cell was analysed by Western blot. Expression of Bad was death that induces apoptosis in T cells. ITM2Bs shares observed in IL-2-stimulated cells and not modi®ed homology with the BH3 domain of Bcl-2 family throughout the starvation period (Figure 7b). Bcl-2 members, but lacks conserved BH1, BH2 and BH4 expression was observed in IL-2-stimulated cells, decreas- domain. BH3-only proteins may be regulated by ing throughout lymphokine deprivation (Figure 7b). distinct signals and they may also have dierent

Oncogene Apoptotic activity of ITM2Bs A Fleischer et al 3186

Figure 7 ITM2Bs interacts with Bcl-2 in vivo.(a) Cytoplasmic lysates from IL-2-stimulated or -deprived (20 h) cells were immunoprecipitated with anti-ITM2B antibody, transferred to nitrocellulose and immunoblotted with anti-Bcl-2, anti-Bad and anti-ITM2B, the latter as internal control. Molecular weights of the corresponding proteins are shown. Similar results were obtained in three independent experiments. Alternatively, cytoplasmic lysates from IL-2-stimulated or -deprived cells were immunoprecipitated with anti-Bcl-2 or anti-Bad antibodies, Figure 6 The BH3 domain of ITM2Bs is required for induction transferred to nitrocellulose and immunoblotted with anti- of apoptosis. (a) Cells were transfected with or without ITM2Bs ITM2B, anti-Bcl-2 or anti-Bad, the two latter as internal control. or ITM2Bs containing a mutated BH3 domain (ITM2Bs*) in Similar results were obtained in three independent experiments. which critical L and D residues were mutated to E and A As a negative control, cytoplasmic lysates from IL-2-stimulated or respectively. Cells were maintained after transfection in the -deprived cells were immunoprecipitated with anti-IL-2 antibody presence or the absence of IL-2 for 24 h. Samples were analysed and blotted with anti-ITM2B. (b) Cells were IL-2-stimulated or by ¯ow cytometry. Expression of the transfected ITM2Bs was -deprived for dierent periods of time, then lysed. Protein extracts con®rmed by Western blot. Hybridization with pan-Ras was used were separated by SDS ± PAGE, transferred to nitrocellulose and as internal control of protein loading. IL-2-stimulation, white probed with anti-Bad, anti-Bcl-2 and pan-Ras, the latter as bars; IL-2-deprivation, shaded bars. Similar results were obtained internal control of protein loading. Similar results were obtained in two independent experiments. (b) Cells were transfected with or in three independent experiments without ITM2Bs or ITM2Bs containing a mutated BH3 domain (ITM2Bs*) as in a and maintained after transfection in the presence or absence of IL-4 for 24 h. Samples were analysed as in a. Expression of the transfected proteins was analysed by Western blot. IL-4 stimulation, white bars; IL-4-deprivation, shaded bars con®rm previous results showing that mutation of the critical amino acids leucine (L) and aspartic acid (D) within ITM2Bs BH3 domain inhibits apoptotic activity. Thus, BH3 domain of ITM2Bs appears to be critical eector functions. It has been described that BH3 for induction of apoptosis. Interestingly, Diva and domain of Bax and Bak is required for association to Map1 are the only described proapoptotic members Bcl-2 and Bcl-x (Chinttenden et al., 1995a; Han et al., that lack one of the conserved residues of the BH3 1996; Simonain et al., 1996; Zha et al., 1996a). In domain. addition, deletion of BH3 domain blocks apoptotic ITM2Bs interacts with Bcl-2, but not with Bad. Low activity of Bak, Bad and Noxa (Chittenden et al., level of Bcl-2/ITM2Bs association was observed in IL- 1995b; Oda et al., 2000). Our results demonstrate and 2-deprived cells because Bcl-2 was the limiting molecule

Oncogene Apoptotic activity of ITM2Bs A Fleischer et al 3187 in the formation of the complex. Western blot of total apoptosis in IL-2-stimulated cells. In addition, ITM2Bs extracts from IL-2-deprived cells con®rmed the down- is not able to induce apoptosis in IL-4-stimulated cells, regulation of Bcl-2, but not ITM2Bs, expression. Some indicating that the observed apoptotic eect of ITM2Bs BH3-only proteins can promote apoptosis by two in IL-2-stimulated or -deprived cells is physiologically mechanisms: inactivating prosurvival Bcl-2 proteins or relevant and that the mechanisms that control modifying proapoptotic molecules. ITM2Bs could apoptosis via IL-2R and IL-4R are dierent, as it induce apoptosis by inhibiting the antiapoptotic has been previously shown (Cerezo et al., 1998, 1999; activity of Bcl-2. Alternatively, ITM2Bs may be a Rebollo et al., 2000). The results presented here show a protein with apoptotic activity and Bcl-2 may serve as new role for ITM2Bs as an apoptotic-associated gene inhibitor. As IL-2-deprived cells downregulate Bcl-2 with a BH3 domain that appears upon IL-2-depriva- expression, there is no inhibitor of ITM2Bs activity. In tion, a stimulus that trigger apoptosis. agreement, overexpression of Bcl-2 blocks apoptotic activity of ITM2Bs. The function of apoptotic Bcl-2 members may be regulated by binding to antiapoptotic members, inhibiting activation of caspases. Besides Materials and methods interaction with sequestering proteins, the activity of apoptotic Bcl-2 family proteins can be suppressed by Cells, lymphokines, antibodies and reagents other mechanisms, including post-translational mod- The murine T cell line TS1ab can be propagated indepen- i®cations. For example, Bad protein is inactivated by dently in IL-2, IL-4 or IL-9 (Pitton et al., 1993). Cells were phosphorylation (Datta et al., 1997; Del Peso et al., cultured in RPMI-1640 supplemented with 5% heat-inacti- 1997). Other post-translational modi®cation that vated foetal calf serum, 2 mM glutamine, 10 mM HEPES, activates proapoptotic proteins is proteolysis. Bid is 0.55 mM arginine, 0.24 mM asparagine, 50 mM 2-ME and N-myristoylated when cleaved by caspase 8 and this 5 ng/ml of IL-2 or 60 U/ml of IL-4. Murine rIL-4 or postproteolytic N-myristoylation enhances apoptosis supernatant of a HeLa subline transfected with pKCRIL- and release of cytochrome c (Zha et al., 2000). 4.neo was used as a source of murine IL-4. The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, ITM2B resides on mouse chromosome 14. It is CA, USA). Trizol reagent and MessageMaker were from Life interesting to note that other proapoptotic molecules Technologies (Gaitersburg, MD, USA). also localize in chromosome 14, some of them in the same locus; BNIP32 (Bcl-2 adenovirus E1B), which contains a BH3-domain and induces apoptosis; CIDEB Representational difference analysis (RDA) (cell death inducing eector), that induces apoptosis by Total RNA was prepared from IL-2-stimulated or -deprived DNA damage; PTK2B, which induces apoptosis in TS1ab cells using TRIZOL Reagent. Polyadenylated RNA ®broblast; the tumour suppressor Fhit; UBE1C was puri®ed from total RNA with oligo (dT) cellulose columns (GIBCO ± BRL). Double-stranded cDNA was (ubiquitin-activating enzyme E1C); VDAC2 (voltage + dependent anion channel), which is involved in calcium generated from 5 mg poly (A) RNA using an oligo (dT) primer and the SuperScript Choice System for cDNA release and DNAse 1L3, that is involved in cleavage of synthesis, according to the manufacturer's recommendations DNA. It is interesting to notice that the mRNA and (Life Technologies). Representational dierence analysis was protein level of ITM2Bs are upregulated in TS1ab cells performed as described (Hubank and Schatz, 1994; Louahed induced to undergo apoptosis by IL-2 deprivation. et al., 1999). After three rounds of subtraction, ®nal Low quantity of mRNA and protein are synthesized in dierence products were digested with DpnII and cloned into IL-2-stimulated cells. This quantity of ITM2Bs is the BamHI site of pTZ19R. Double-stranded plasmid DNA presumably not enough to induce apoptosis. The was prepared and sequenced on both strands with an mRNA level of ITM2B is not upregulated upon IL- automatic sequencer. Sequence comparisons with the Gen- L Bank Database number were performed with the Blast search 2 deprivation. Moreover, expression of ITM2BL is not able to induce apoptosis. It is interesting to note that program. Quanti®cation of Western blot was made using the program Quantity One version 4.2.0 (BioRad). the N-terminal 57 amino acid of ITM2BL contains a binding motif for IAPs (inhibitory apoptosis protein). It is tempting to speculate that IAPs are associated to RNA isolation the N-terminal region of ITM2BL, avoiding its Total RNA was isolated using Trizol reagent according to proapoptotic role. the manufacturer's recommendations. For Northern blot Some BH3-only regulators of apoptosis are consti- analysis, 15 mg of RNA sample were electrophoresed in a tutively expressed in cells and their apoptotic function 1% agarose gel in the presence of formaldehyde and is usually triggered by other events. ITM2B synthesis transferred to nitrocellulose ®lter. After hybridization with s 32 is regulated by alternative splicing. Alternative splicing the P labelled probe, the ®lter was washed and exposed to aecting the properties of apoptotic proteins is well X-ray ®lm at 7708C with intensifying screen. documented in the case of Bcl-x protein. Bcl-xL and Cloning of ITM2B Bcl-xs are encoded by two alternatively spliced s mRNAs. Bcl-x is antiapoptotic and Bcl-x is proa- L s According to the published sequence (AB030204), ITM2Bs poptotic (Boise et al., 1993). Alternative splicing aects was cloned by PCR using the following primers: 5'-TTT TGT the apoptotic properties of ITM2B; ITM2Bs acts as a CGA CGC CGC CAT GTG TTT CGG ACT GGC CTT proapoptotic protein whereas ITM2BL does not induce CAT G and 3'-TTT TGC GGC CGC TCA AGA ACA AAT

Oncogene Apoptotic activity of ITM2Bs A Fleischer et al 3188 TAA AGT CTC CAC AGC. Ampli®ed DNA of the correct pH 8, 0.5 M sucrose, 1 mM EDTA, 0.5 mM spermidine, size was con®rmed by sequencing and then cloned in the 0.15 mM spermine and 0.2% Triton X-100). Lysates were pCIneo expression vector. centrifuged (4500 g, 23 min, 48C). Nuclei were resuspended in 1 ml buer B (50 mM NaCl, 10 mM HEPES, pH 8, 25% glycerol, 0.1 m EDTA, 0.5 mM spermidine and 0.15 mM Peptide synthesis and antibody production M spermine) and centrifuged (4500 g, 3 min, 48C). Nuclear A peptide of ITM2Bs spanning amino acid sequence 39 ± 55 proteins were extracted at 48C for 30 min in 60 ml of buer C was synthesized on an automated multiple peptide synthesizer (350 mM NaCl, 10 mM HEPES, pH 8, 25% glycerol, 0.1 mM using the solid phase procedure and standard Fmoc EDTA, 0.5 mM spermidine and 0.15 mM spermine). Super- chemistry. Purity and peptide composition were con®rmed natants were cleared by centrifugation and stored at 7808C. in reverse-phase HPLC and by amino acid analysis. The All buers were supplemented with protease inhibitors. peptide, glutaraldehyde-coupled to KLH via N-terminal was injected into outbred New Zealand rabbits. Sera were Isolation of mitochondria and S100 fraction collected 7 ± 10 days after the last injection. This antibody recognizes both ITM2Bs and ITM2BL that can be dier- Mitochondria were isolated using a modi®cation of Yang et entiated by molecular weight. We also have generated al. (1997). Brie¯y, 206106 cells were IL-2-stimulated or polyclonal antibodies that speci®cally recognize ITM2BL. -deprived, harvested and washed with ice-cold PBS. Cell pellet was suspended in 5 volumes of ice-cold buer A (20 m HEPES KOH pH 7.5, 10 mM KCl, 1.5 mM MgCl , Cell cycle analysis, transient transfection, immunoprecipitation M 2 1m EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF and and Western blot M 250 mM sucrose) supplemented with protease inhibitors. Cells Transient transfections, immunoprecipitations and Western were disrupted in a Dounce homogenizer, the nuclei blot were made as previously described in Romero et al., centrifuged (1000 g, 10 min, 48C) and the supernatant further 1999. centrifuged (10 000 g, 15 min, 48C). The resulting mitochondrial pellet was resuspended in buer A and stored at 7808C. The supernatant was centrifuged (100 000 g,1h, Site-directed mutagenesis 48C) and the resulting S100 fraction stored at 7808C. Site-directed mutagenesis was performed using the Quik- Change mutagenesis kit. The primers used for mutation were: ITM2Bs*5': GTG TAC TAC TGT GGA GAA AAG TAC ATC AAA GCT GAC ATC CTG; ITM2Bs*3': CAG GAT GAC GTC AGC TTT GAT GTA CTT TTC TCC ACA GTA GTA CAC. The critical leucine (L) and aspartic acid Acknowledgments (D) from the BH3 domain were mutated to glutamic acid (E) This work was partially supported by grants from Associa- and alanine (A) respectively. Point mutations are in bold and tion pour la Recherche sur le Cancer (ARC), DireccioÂ n underlined. Mutated ITM2Bs sequence was con®rmed by General de InvestigacioÂ n y Ciencia (DGIyC), Belgian sequencing. Federal Service for Scienti®c, Technical and Cultural Aairs and Actions de Recherche ConcerteÂ es, Direction de la Recherche Scienti®c. The Department of Immunology Nuclear extracts and Oncology was founded and is supported by the IL-2-stimulated or -deprived cells (36107) were resuspended Spanish Research Council (CSIC) and Pharmacia Corpora- for 2 min in 1 ml of buer A (50 mM NaCl, 10 mM HEPES tion.

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Oncogene