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

Oncogene (2002) 21, 3181 ± 3189 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc 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.

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