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Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3
FEBS Letters xxx (2015) xxx–xxx
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Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3 Yoshifumi Hashimoto, Hiroto Inagaki, Shin-ichi Hoshino ⇑ Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
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Article history: Received 1 May 2015 Revised 26 June 2015 Accepted 27 June 2015 Available online xxxx Edited by Noboru Mizushima Keywords: Translation termination Inhibitor of apoptosis protein eRF3 Calpain Apoptosis
a b s t r a c t The involvement of polypeptide chain-releasing factor eRF3 in translation termination and mRNA decay is well established. Moreover, the finding that the proteolytically processed isoform of eRF3 (p-eRF3) interacts with inhibitors of apoptosis proteins (IAPs) to activate caspase, implies that eRF3 is a cell death regulator. However, the protease(s) responsible for p-eRF3 production and how p-eRF3 regulates apoptosis remain unknown. Here, we show that calpain mediates p-eRF3 production in vitro and in living cells. p-eRF3 is produced in cells treated with ER stressors in a calpain-dependent manner. These findings suggest that p-eRF3 is a novel regulator of calpain-dependent cell death. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction It is generally accepted that eRF3/GSPT works with another releasing factor, eRF1, to execute translation termination. Two subtypes of eRF3, eRF3a/GSPT1 and eRF3b/GSPT2, were identified in mammals [1,2]. Functional domains of eRF3/GSPT are separated into an amino-terminal unstructured domain (N-domain) and a translation elongation factor eEF1A-like GTP-binding domain (C-domain) [2,3]. The C-domain is essential for the interaction with eRF1 to terminate translation, in which eRF3/GSPT hydrolyzes GTP to accelerate the polypeptide chain releasing reaction catalyzed by the ribosome [4,5]. eRF3/GSPT also plays a pivotal role in the initiation of mRNA decay through interaction with the poly(A)-binding protein PABPC1 in its N-domain [6–8]. The interaction between eRF3/GSPT and PABPC1 contributes to translation activation by efficient recycling of the terminating ribosome to the next round of translation initiation [9,10]. These studies define eRF3/GSPT as a multifunctional regulator of gene expression.
Abbreviation: IAP, inhibitor of apoptosis protein Author contributions: YH designed and performed the majority of the experiments and wrote the paper; HI performed the experiments; SH conceived and directed the study and wrote the paper. ⇑ Corresponding author. Fax: +81 52 836 3427. E-mail address: [email protected] (S.-i. Hoshino).
A previous study identified a proteolytically processed isoform of eRF3a/GSPT1 (termed p-eRF3) as a binding partner of inhibitors of apoptosis proteins (IAPs), and showed that p-eRF3 promotes the activation of caspase by the titration of IAPs, which leads to the liberation of caspases from IAP inhibition [11]. p-eRF3 is generated by cleavage at residue 73A of eRF3a/GSPT1, the N-terminus of which exposes the IAP-binding motif (IBM). Since the N-terminally exposed alanine of IBM is essential for the interaction with IAPs, p-eRF3 but not full-length eRF3 interacts with IAPs to activate caspases [12,13]. When p-eRF3 is produced by the cleavage, the nuclear export signal (NES) of eRF3a/GSPT1, which is located immediately upstream of the cleavage site A73, is removed from eRF3a/GSPT1 [14]. This enables p-eRF3 to localize in the nucleus as well as the cytoplasm and to interact efficiently with the nuclear ARF tumor suppressor [14]. These findings reveal that eRF3 becomes a regulator of cell death upon cleavage at 73A. To elucidate the physiological significance of p-eRF3, we have attempted to determine stimuli for p-eRF3 production [15]. To date, we have examined apoptosis-inducing conditions, in which other IBM-containing proteins, Omi/HtrA2 and Smac/DIABLO, function to promote apoptosis, but p-eRF3 was not produced in these conditions [15]. Since Omi/HtrA2 and Smac/DIABLO are mitochondrial proteins and promote mitochondria-dependent apoptosis, p-eRF3 may not function in the mitochondria-dependent apoptosis pathway, consistent with the cytoplasmic localization of eRF3 [16,17]. We also found that the other processed isoform of eRF3
http://dx.doi.org/10.1016/j.febslet.2015.06.041 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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(eRF3-cp), which is cleaved by caspase mainly at 32D, is generally produced during apoptosis [15]. Here, we demonstrate that calpains, calcium-dependent cysteine proteases, are responsible for p-eRF3 production. The calpains are a conserved family of cysteine proteases involved in several physiological processes, including apoptosis and cell survival [18]. We also show that calpain activity is necessary for p-eRF3 production in living cells. These findings characterize p-eRF3 as a novel regulator of calpain-dependent cell death. 2. Materials and methods 2.1. Cell culture and transfection THP-1 cells were cultured in RPMI-1640 (Nissui) supplemented with 5% fetal bovine serum (Sigma) and maintained at 37 °C in 5% CO2. SK-N-SH and HEK293T cells were cultured in DMEM supplemented with 5% fetal bovine serum (Sigma). THP-1 cells were transfected with plasmid using Neon™ Transfection System (Invitrogen). HEK293T cells were transfected with plasmids using polyethylenimine Max (Polysciences, Inc.).
supernatant was incubated with Glutathione Sepharose beads (GE Healthcare) for 1 h in a cold room. The resin was washed 3 times with buffer B. Proteins were eluted in buffer C (buffer B + 10 mM glutathione) for 30 min in a cold room. For the preparation of eRF3, eRF3-FLAG-expressing HEK293T cells were lysed in RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 0.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 2 lg/ml aprotinin, 2 lg/ml leupeptin and 2 lg/ml pepstatin A) and cells were placed on ice for 30 min. The lysate was centrifuged at 15 000 rpm for 20 min at 4 °C and the supernatant was incubated with anti-FLAG IgG agarose (Sigma) for 1 h in a cold room. The resin was washed 3 times with RIPA buffer. Proteins were eluted in buffer D (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 1 mM dithiothreitol, 150 ng/lL 3FLAG peptide (Sigma)) for 30 min in a cold room. 2.6. In vitro calpain-mediated cleavage assay Purified eRF3-FLAG proteins and a combination of m-calpain (Calbiochem) and 2.5 mM CaCl2 were incubated in buffer D (50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol) on ice.
2.2. Plasmid 3. Results To construct pCMV-MycFLAG-Ub-hGSPT1/eRF3a(A73G), the corresponding region of human GSPT1 cDNA was amplified by inverse PCR using primer pairs YH30 (50 -GTG CCC AAC GTC CAC GCC GCC GAG TTC GTG-30 )/YH31 (50 -GAA GGG CTT GCC GTT GAC GTT GAG TTG CCG-30 ), and pCMV-MycFLAG-Ub-hGSPT1 [15] as a template. Other eRF3-expressing constructs used in this study were as described previously [15]. To construct pCMV-Myc-GST-XIAP, GST cDNA and XIAP cDNA, which was obtained from HeLa cDNA library, were inserted in HindIII and EcoRI/XhoI of pCMV-Myc [7], respectively. 2.3. Preparation of cell extract Cells were lysed in buffer A (20 mM Tris–HCl (pH7.5), 10 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 0.5% NP-40) and placed on ice for 10 min. After centrifugation at 15 000 rpm, the supernatant was used as the cell extract. To produce p-eRF3, cells were lysed in buffer A supplemented with 2.5 mM CaCl2 (CaCl2-containing buffer). The Western blot analysis, antibodies and protease inhibitors used in this study were as described previously [15]. ALLN was purchased from CALBIOCHEM. 2.4. Cell treatment For treatment with stress-inducing reagents (tunicamycin, thapsigargin, A23187 and etoposide), culture medium of SK-N-SH cells was replaced with fresh culture medium and stress-inducing reagents were then added. To examine the effects of protease inhibitors (AEBSF, E-64, z-VAD-fml, MG132, lactacystin (Calbiochem) and calpeptin (Sigma)), cells were pretreated with protease inhibitors for 30 min, and then stress-inducing reagents were added. 2.5. Purification of recombinant proteins For the preparation of XIAP, Myc-GST-XIAP-expressing HEK293T cells were lysed in buffer B (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 2.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 2 lg/ml aprotinin, 2 lg/ml leupeptin and 2 lg/ml pepstatin A) and cells were placed on ice for 30 min. The lysate was centrifuged at 15 000 rpm for 20 min at 4 °C, and the
3.1. p-eRF3 is produced by calcium-dependent protease During the course of investigating proteolytic cleavage of eRF3, we noticed that eRF3 was cleaved in cell extracts that were prepared using EDTA-free buffer. Thus we examined the effects of divalent cations on the cleavage of eRF3 in cell extracts. THP-1 cells were lysed with lysis buffer containing either 2.5 mM MgCl2 or 2.5 mM CaCl2, and the status of eRF3 in each cell extract was analyzed by Western blotting. Fig. 1A clearly shows that eRF3 was cleaved in CaCl2-containing buffer, whereas eRF3 was not affected in MgCl2-, EDTA- or EGTA-containing buffer. To verify that the processed isoform of eRF3 (provisionally named eRF3-Ca2+) is p-eRF3, we attempted to determine whether eRF3 is cleaved at the N-terminal domain or the C-terminal domain. To this end, we prepared cells expressing wild-type eRF3 (eRF3-wt) and 73A-eRF3 (p-eRF3), both of which are N-terminally ubiqutin (Ub)-tagged and C-terminally FLAG-tagged [15]. As described previously, when these eRF3 constructs were expressed in cells, the Ub moiety is removed by multiple ATP-dependent proteases, and eRF3 fragments without Ub can be produced [15]. The eRF3-wt-expressing cells were lysed in CaCl2-containing buffer and C-terminally FLAG-tagged eRF3 was detected by anti-FLAG antibody. Since eRF3-Ca2+, as well as full-length eRF3, was recognized by anti-FLAG antibody, eRF3-Ca2+ is produced by cleavage at the N-terminal region of eRF3 (Fig. 1B). We also confirmed that eRF3-Ca2+ exhibited the same mobility on SDS–PAGE as that of 73A-eRF3 (p-eRF3). We previously reported another form of cleaved eRF3 (eRF3-cp) that is produced by caspase-mediated cleavage of eRF3, mainly at 32D [15]. To distinguish between eRF3-Ca2+ and eRF3-cp, we next prepared cells expressing 33Q-eRF3 (eRF3-cp) and eRF3 (D25A/D29A/D32A/D35A), which is resistant to the cleavage by caspases [15]. As shown in Fig. 1C, both 33Q-eRF3 (eRF3-cp) and eRF3 (D25A/D29A/D32A/D35A) were cleaved in Ca2+-containing buffer. These results show that eRF3-Ca2+ is a distinct isoform from eRF3-cp. Because an important property of p-eRF3 is that it binds to IAPs through its N-terminally exposed IAP-binding motif (IBM) [11,12], we examined the interaction between eRF3-Ca2+ and XIAP. We prepared cells expressing either eRF3 (wt) or eRF3(A73G) mutant, both of which were C-terminally FLAG-tagged. The eRF3
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Fig. 1. p-eRF3 is produced by calcium-dependent protease. (A) Cell extracts were prepared from THP-1 cells using lysis buffer supplemented with EDTA, EGTA, MgCl2 or CaCl2 (each at 2.5 mM). The status of eRF3 was analyzed by Western blotting using anti-eRF3 antibody. GAPDH was also analyzed using anti-GAPDH as a loading control. (B) THP-1 cells were electroporated with pCMV-MycFLAG-Ub-eRF3 and whole cell extracts were prepared using lysis buffer containing either EGTA (CaCl2) or CaCl2 (CaCl2+). Standard sample of p-eRF3 prepared from HEK293T cells transfected with pCMV-MycFLAG-Ub-73A-eRF3 is indicated as 73A-eRF3 (p-eRF3). The status of eRF3 proteins was analyzed by Western blotting using anti-FLAG antibodies. (C) THP-1 cells were electroporated with pCMV-MycFLAG-Ub-eRF3, eRF3 (D25A/D29A/D32A/D35A) or 33Q-eRF3 and wholecell extracts were prepared using lysis buffer containing either EGTA (CaCl2) or CaCl2 (CaCl2+). Western blotting was performed as described in Fig. 1B. (D) Illustration of XIAP binding assay protocol. IP, immunoprecipitation. (E) THP-1 cells expressing FLAG-tagged eRF3 were lysed in EDTA or CaCl2 containing lysis buffer for preparation of fulllength eRF3 (WT (EDTA)) or eRF3-Ca2+ (WT (Ca2+)). The eRF3 proteins were immunoprecipitated using anti-FLAG antibody. As a negative control, FLAG-tagged eRF3 (A73G) (Ca2+) was also prepared as above. eRF3 proteins were purified using anti-FLAG M2 agarose and mixed with Myc-tagged GST-XIAP. Pull down experiment was performed using anti-FLAG antibody. * indicates immunoglobulin. (F) Schematic diagram of the interaction between IAP and eRF3 proteins.
(wt)-expressing cells were lysed with EDTA- or Ca2+-containing buffer to obtain full-length eRF3 or eRF3-Ca2+, respectively, and eRF3(A73G)-expressing cells were lysed with Ca2+-containing buffer. The eRF3 proteins were purified using anti-FLAG resin and then mixed with Myc-tagged XIAPs (Fig. 1D). After rotation, pull-down
assay was performed using anti-FLAG resin and interactions between the eRF3 proteins and XIAP were analyzed by Western blotting. As shown in Fig. 1E, only eRF3-Ca2+ but not full-length eRF3 co-precipitated XIAP. The A73G mutation of eRF3 abolished its interaction with XIAP, although the mutation did not affect
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Fig. 2. p-eRF3 is produced in a calpain-dependent manner. (A) THP-1 cells were lysed in CaCl2-containing buffer supplemented with 0.5 mM PMSF and aprotinin, leupeptin or pepstatin A (each at 2 lg/ml). PMSF was added to all samples to prevent unexpected degradation of eRF3. THP-1 cell extracts supplemented with or without all of the proteases were also prepared. The status of eRF3 was analyzed by Western blotting using anti-eRF3 antibody. (B) THP-1 cells were lysed in CaCl2-containing buffer supplemented with either E-64 or ALLN (each at 20 lM) and Western blotting was performed as described in Fig. 2A. (C) Flag-tagged eRF3 protein purified from eRF3-FLAGexpressing HEK293T cells, was incubated with an indicated amounts of recombinant m-calpain on ice for 30 min. Flag-tagged eRF3 incubated with 2 lg/ml of recombinant mcalpain for the indicated time periods were also prepared. (D) Intact eRF3-FLAG and calpain-mediated processed eRF3 (0.2 lg/ml of m-calpain for 30 min and 2 lg/ml mcalpain for 5 min) were loaded with a standard sample of p-eRF3 (73A-eRF3) prepared as in Fig. 1B.
the cleavage of eRF3. Since the N-terminal alanine of IBM is crucial for its binding to IAPs (Fig. 1F), the evidence strongly supports our conclusion that eRF3-Ca2+ is p-eRF3. 3.2. p-eRF3 is produced in a calpain-dependent manner To elucidate the protease(s) responsible for p-eRF3 (eRF3-Ca2+) production, we tested the effects of protease inhibitors including
serine protease inhibitors PMSF and aprotinin, cysteine protease inhibitor leupeptin and aspartyl protease inhibitor pepstatin A on the cleavage of eRF3 in CaCl2-containing buffer (Fig. 2A). Among the proteases tested, only leupeptin inhibited the cleavage of eRF3, indicating that cysteine protease might be responsible for the Ca2+-dependent cleavage of eRF3. Since calpain is a well-known Ca2+-dependent cysteine protease, we next examined another cysteine protease inhibitor, E-64, and also a specific
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Fig. 3. Activation of calpain is necessary for the production of p-eRF3 in living cells. (A) SK-N-SH cells were treated with ER-stress inducers (1 lg/ml tunicamycin (TM), 1 lM thapsigargin (TG) and 1 lg/ml A23187) and cultured for the indicated periods (24 h and 48 h). Cells without incubation were prepared as a control. The status of eRF3 was analyzed by Western blotting using anti-eRF3 antibody. Endogenous GAPDH was detected as a loading control. (B) SK-N-SH cells were treated with either 1 lM thapsigargin (TG) or 1 lg/ml A23187 in the presence of 20 lM z-VAD-fmk. Protease inhibitors (AEBSF (50 lg/ml), E-64 (50 lM), MG132 (10 lM), ALLN (20 lM) and lactacystin (5 lM)) were also added. (C) SK-N-SH cells were treated with etoposide and protease inhibitors (AEBSF (50 lg/ml), E-64 (50 lM), z-VAD-fmk (20 lM), MG132 (10 lM) and lactacystin (5 lM)). Calpain specific inhibitor calpeptin (20 lM) was also tested. (D) Schematic representation of calpain-mediated processing of eRF3 into p-eRF3.
calpain inhibitor, ALLN. As shown in Fig. 2B, E-64 and ALLN completely blocked the cleavage of eRF3. These results strongly suggest that the Ca2+-dependent cleavage of eRF3 is mediated by calpain. Essentially the same results were obtained using other cell types, although the extent of the cleavage of eRF3 varied among cell types
(data not shown). To confirm that the Ca2+-dependent cleavage of eRF3 is mediated by calpain, we examined whether recombinant calpain cleaves eRF3. Purified eRF3 proteins were incubated with recombinant m-calpain. A single cleaved isoform of eRF3 was preferentially produced by the recombinant m-calpain (Fig. 2C),
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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though increased amount of calpain and prolonged incubation cause minor cleavages or degradation of eRF3. As expected, the principal cleaved product of eRF3 exhibited the same mobility as p-eRF3 (eRF3-Ca2+) on SDS–PAGE (Fig. 2D). From these results, we conclude that calpain is responsible for the Ca2+-dependent cleavage of eRF3. 3.3. Activation of calpain is necessary for the production of p-eRF3 in living cells We next examined whether p-eRF3 is produced in living cells when calpain is activated. Because challenge with ER stress was reported as one of the calpain-activating conditions [19,20], we tested the status of eRF3 when cells were treated with tunicamycin, thapsigargin and A23187, which are commonly used as ER-stress-inducing agents. Fig. 3 shows representative results of p-eRF3 production under ER-stress conditions. When human neuroblastoma SK-N-SH cells were treated with the ER stressors, two cleavage products corresponding to p-eRF3 and eRF3-cp were detected, although only a modest effect was observed for tunicamycin (Fig. 3A). As reported previously, the upper band corresponds to eRF3-cp, since the band was abrogated by the caspase inhibitor zVAD.fmk (Fig. 3B). To ensure that the lower band corresponds to p-eRF3, we examined the effect of protease inhibitors on the production of p-eRF3 under condition that inhibits caspases by zVAD.fmk (Fig. 3B). Calpain inhibitors E-64 and ALLN completely abrogated the appearance of the lower band. The results are consistent with the idea that the lower band corresponds to p-eRF3 and the production of p-eRF3 depends on calpain. Proteasome inhibitor MG132, which is known to inhibit calpain [21], also blocked the production of p-eRF3, whereas another proteasome inhibitor, lactacystin, had no obvious effect. These results also support the idea that the production of p-eRF3 is mediated by calpain [22]. Although the extent of p-eRF3 production varied depending on the cell lines, p-eRF3 was reproducibly detected by the treatment with ER stressors in several cell lines (data not shown). Especially by the treatment with calcium ionophore A23187, which is directly linked to calpain activation, p-eRF3 was observed in almost all cell lines tested. p-eRF3 was also produced in a calpain-dependent manner when SK-N-SH cells were treated with the non-ER stressor etoposide (Fig. 3C). Thus, conditions leading to p-eRF3 production are not limited to the challenge with ER stress. Overall, these results indicate that the activation of calpain is necessary for the production of p-eRF3. 4. Discussion Previous studies suggested that eRF3 may function in the regulation of cell death; processed eRF3 (p-eRF3) interacts with IAPs through its IBM and is able to promote caspase activation, IAP ubiquitination and apoptosis. However, lack of knowledge regarding the conditions to produce p-eRF3 has hampered progress in investigating the role of eRF3 in cell death. In this study, we have demonstrated that calpain is responsible for the processing of eRF3 into p-eRF3 (Fig. 3D), based on the following five features; (i) Ca2+ dependence, (ii) sensitivity to cysteine protease inhibitors, leupeptin and E-64, (iii) sensitivity to calpain-specific inhibitor ALLN, (iv) the ability of the processed isoform of eRF3 to bind to XIAP, and (v) the ability of recombinant m-calpain to process eRF3 into p-eRF3. The finding that calpain is responsible for the production of p-eRF3 provides a basis for understanding the role of p-eRF3 in apoptosis (Fig. 3D). Consistent with the apoptotic function of p-eRF3, the pro-apoptotic functions of calpain are well established. Although calpain is involved in mitochondria-dependent apoptosis, it was also
reported to activate mouse caspase-12 and human caspase-4, which are crucial caspases for ‘mitochondria-independent ER-stressinduced apoptosis’ [19,23]. ER-stress-induced apoptosis usually crosstalks with mitochondria-dependent and mitochondriaindependent pathways [24,25], but in some cases, the contribution of the mitochondrial pathway is not observed [26,27]. In the latter cases, IAP-binding proteins including Smac/DIABLO and Omi/HtrA2, which are located in the mitochondrial inner membrane, cannot promote apoptosis. These observations are consistent with the idea that p-eRF3, instead of Smac/DIABLO, Omi/HtrA2, is a cytoplasmic IAP-binding protein that promotes mitochondria-independent ER-stress-induced apoptosis (Fig. 3D). Calpain is also known to regulate oncogenic stress-induced apoptosis and senescence [28–30]. These functions of calpain may provide a clue to understand the second physiological role of p-eRF3 in apoptosis, since p-eRF3, which is devoid of an NES, interacts with ARF tumor suppressor in the nucleus (Fig. 3D) [14]. It is thus tempting to speculate that p-eRF3 mediates calpain-activating signal to the nucleus, in which p-eRF3 might regulate the function of ARF, a crucial regulator of oncogene-related apoptosis and senescence [31,32]. Thus far, we have reported two types of cleaved isoforms of eRF3, p-eRF3 [11,14] and eRF3-cp [15] (Fig. 3D). These cleaved eRF3 isoforms are produced upstream or downstream of caspase activation; p-eRF3 is produced by calpain to activate caspase whereas eRF3-cp is produced as a result of caspase activation. A relationship between the two isoforms of eRF3 is not clear at present. However, our results show eRF3-cp might be a poor target of calpain (Fig. 1C), suggesting that N-terminal residues of eRF3, which are removed by caspase-mediated cleavage, support calpain-mediated cleavage. Noteworthy, polyglycine expansions included in this N-terminal region of eRF3 associates with cancer susceptibility [33,34]. Thus it is tempting to speculate that polyglycine expansions affect calpain-mediated cleavage and apoptosis, and therefore cancer susceptibility. These possibilities are now under investigation in our laboratory. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 25291004) from Japan Society for the Promotion of Science, and a Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan (to S H). References [1] Hoshino, S., Miyazawa, H., Enomoto, T., Hanaoka, F., Kikuchi, Y., Kikuchi, A. and Ui, M. (1989) A human homologue of the yeast GST1 gene codes for a GTPbinding protein and is expressed in a proliferation-dependent manner in mammalian cells. EMBO J. 8, 3807–3814. [2] Hoshino, S., Imai, M., Mizutani, M., Kikuchi, Y., Hanaoka, F., Ui, M. and Katada, T. (1998) Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1. J. Biol. Chem. 273, 22254–22259. [3] Hoshino, S., Hosoda, N., Araki, Y., Kobayashi, T., Uchida, N., Funakoshi, Y. and Katada, T. (1999) Novel function of the eukaryotic polypeptide-chain releasing factor 3 (eRF3/GSPT) in the mRNA degradation pathway. Biochemistry (Moscow) 64, 1367–1372. [4] Alkalaeva, E.Z., Pisarev, A.V., Frolova, L.Y., Kisselev, L.L. and Pestova, T.V. (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell 125, 1125–1136. [5] Cheng, Z. et al. (2009) Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev. 23, 1106–1118. [6] Hosoda, N., Kobayashi, T., Uchida, N., Funakoshi, Y., Kikuchi, Y., Hoshino, S. and Katada, T. (2003) Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation. J. Biol. Chem. 278, 38287–38291.
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Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3 Yoshifumi Hashimoto, Hiroto Inagaki, Shin-ichi Hoshino ⇑ Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
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Article history: Received 1 May 2015 Revised 26 June 2015 Accepted 27 June 2015 Available online xxxx Edited by Noboru Mizushima Keywords: Translation termination Inhibitor of apoptosis protein eRF3 Calpain Apoptosis
a b s t r a c t The involvement of polypeptide chain-releasing factor eRF3 in translation termination and mRNA decay is well established. Moreover, the finding that the proteolytically processed isoform of eRF3 (p-eRF3) interacts with inhibitors of apoptosis proteins (IAPs) to activate caspase, implies that eRF3 is a cell death regulator. However, the protease(s) responsible for p-eRF3 production and how p-eRF3 regulates apoptosis remain unknown. Here, we show that calpain mediates p-eRF3 production in vitro and in living cells. p-eRF3 is produced in cells treated with ER stressors in a calpain-dependent manner. These findings suggest that p-eRF3 is a novel regulator of calpain-dependent cell death. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction It is generally accepted that eRF3/GSPT works with another releasing factor, eRF1, to execute translation termination. Two subtypes of eRF3, eRF3a/GSPT1 and eRF3b/GSPT2, were identified in mammals [1,2]. Functional domains of eRF3/GSPT are separated into an amino-terminal unstructured domain (N-domain) and a translation elongation factor eEF1A-like GTP-binding domain (C-domain) [2,3]. The C-domain is essential for the interaction with eRF1 to terminate translation, in which eRF3/GSPT hydrolyzes GTP to accelerate the polypeptide chain releasing reaction catalyzed by the ribosome [4,5]. eRF3/GSPT also plays a pivotal role in the initiation of mRNA decay through interaction with the poly(A)-binding protein PABPC1 in its N-domain [6–8]. The interaction between eRF3/GSPT and PABPC1 contributes to translation activation by efficient recycling of the terminating ribosome to the next round of translation initiation [9,10]. These studies define eRF3/GSPT as a multifunctional regulator of gene expression.
Abbreviation: IAP, inhibitor of apoptosis protein Author contributions: YH designed and performed the majority of the experiments and wrote the paper; HI performed the experiments; SH conceived and directed the study and wrote the paper. ⇑ Corresponding author. Fax: +81 52 836 3427. E-mail address: [email protected] (S.-i. Hoshino).
A previous study identified a proteolytically processed isoform of eRF3a/GSPT1 (termed p-eRF3) as a binding partner of inhibitors of apoptosis proteins (IAPs), and showed that p-eRF3 promotes the activation of caspase by the titration of IAPs, which leads to the liberation of caspases from IAP inhibition [11]. p-eRF3 is generated by cleavage at residue 73A of eRF3a/GSPT1, the N-terminus of which exposes the IAP-binding motif (IBM). Since the N-terminally exposed alanine of IBM is essential for the interaction with IAPs, p-eRF3 but not full-length eRF3 interacts with IAPs to activate caspases [12,13]. When p-eRF3 is produced by the cleavage, the nuclear export signal (NES) of eRF3a/GSPT1, which is located immediately upstream of the cleavage site A73, is removed from eRF3a/GSPT1 [14]. This enables p-eRF3 to localize in the nucleus as well as the cytoplasm and to interact efficiently with the nuclear ARF tumor suppressor [14]. These findings reveal that eRF3 becomes a regulator of cell death upon cleavage at 73A. To elucidate the physiological significance of p-eRF3, we have attempted to determine stimuli for p-eRF3 production [15]. To date, we have examined apoptosis-inducing conditions, in which other IBM-containing proteins, Omi/HtrA2 and Smac/DIABLO, function to promote apoptosis, but p-eRF3 was not produced in these conditions [15]. Since Omi/HtrA2 and Smac/DIABLO are mitochondrial proteins and promote mitochondria-dependent apoptosis, p-eRF3 may not function in the mitochondria-dependent apoptosis pathway, consistent with the cytoplasmic localization of eRF3 [16,17]. We also found that the other processed isoform of eRF3
http://dx.doi.org/10.1016/j.febslet.2015.06.041 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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(eRF3-cp), which is cleaved by caspase mainly at 32D, is generally produced during apoptosis [15]. Here, we demonstrate that calpains, calcium-dependent cysteine proteases, are responsible for p-eRF3 production. The calpains are a conserved family of cysteine proteases involved in several physiological processes, including apoptosis and cell survival [18]. We also show that calpain activity is necessary for p-eRF3 production in living cells. These findings characterize p-eRF3 as a novel regulator of calpain-dependent cell death. 2. Materials and methods 2.1. Cell culture and transfection THP-1 cells were cultured in RPMI-1640 (Nissui) supplemented with 5% fetal bovine serum (Sigma) and maintained at 37 °C in 5% CO2. SK-N-SH and HEK293T cells were cultured in DMEM supplemented with 5% fetal bovine serum (Sigma). THP-1 cells were transfected with plasmid using Neon™ Transfection System (Invitrogen). HEK293T cells were transfected with plasmids using polyethylenimine Max (Polysciences, Inc.).
supernatant was incubated with Glutathione Sepharose beads (GE Healthcare) for 1 h in a cold room. The resin was washed 3 times with buffer B. Proteins were eluted in buffer C (buffer B + 10 mM glutathione) for 30 min in a cold room. For the preparation of eRF3, eRF3-FLAG-expressing HEK293T cells were lysed in RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 0.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 2 lg/ml aprotinin, 2 lg/ml leupeptin and 2 lg/ml pepstatin A) and cells were placed on ice for 30 min. The lysate was centrifuged at 15 000 rpm for 20 min at 4 °C and the supernatant was incubated with anti-FLAG IgG agarose (Sigma) for 1 h in a cold room. The resin was washed 3 times with RIPA buffer. Proteins were eluted in buffer D (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 1 mM dithiothreitol, 150 ng/lL 3FLAG peptide (Sigma)) for 30 min in a cold room. 2.6. In vitro calpain-mediated cleavage assay Purified eRF3-FLAG proteins and a combination of m-calpain (Calbiochem) and 2.5 mM CaCl2 were incubated in buffer D (50 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol) on ice.
2.2. Plasmid 3. Results To construct pCMV-MycFLAG-Ub-hGSPT1/eRF3a(A73G), the corresponding region of human GSPT1 cDNA was amplified by inverse PCR using primer pairs YH30 (50 -GTG CCC AAC GTC CAC GCC GCC GAG TTC GTG-30 )/YH31 (50 -GAA GGG CTT GCC GTT GAC GTT GAG TTG CCG-30 ), and pCMV-MycFLAG-Ub-hGSPT1 [15] as a template. Other eRF3-expressing constructs used in this study were as described previously [15]. To construct pCMV-Myc-GST-XIAP, GST cDNA and XIAP cDNA, which was obtained from HeLa cDNA library, were inserted in HindIII and EcoRI/XhoI of pCMV-Myc [7], respectively. 2.3. Preparation of cell extract Cells were lysed in buffer A (20 mM Tris–HCl (pH7.5), 10 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 0.5% NP-40) and placed on ice for 10 min. After centrifugation at 15 000 rpm, the supernatant was used as the cell extract. To produce p-eRF3, cells were lysed in buffer A supplemented with 2.5 mM CaCl2 (CaCl2-containing buffer). The Western blot analysis, antibodies and protease inhibitors used in this study were as described previously [15]. ALLN was purchased from CALBIOCHEM. 2.4. Cell treatment For treatment with stress-inducing reagents (tunicamycin, thapsigargin, A23187 and etoposide), culture medium of SK-N-SH cells was replaced with fresh culture medium and stress-inducing reagents were then added. To examine the effects of protease inhibitors (AEBSF, E-64, z-VAD-fml, MG132, lactacystin (Calbiochem) and calpeptin (Sigma)), cells were pretreated with protease inhibitors for 30 min, and then stress-inducing reagents were added. 2.5. Purification of recombinant proteins For the preparation of XIAP, Myc-GST-XIAP-expressing HEK293T cells were lysed in buffer B (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 2.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 2 lg/ml aprotinin, 2 lg/ml leupeptin and 2 lg/ml pepstatin A) and cells were placed on ice for 30 min. The lysate was centrifuged at 15 000 rpm for 20 min at 4 °C, and the
3.1. p-eRF3 is produced by calcium-dependent protease During the course of investigating proteolytic cleavage of eRF3, we noticed that eRF3 was cleaved in cell extracts that were prepared using EDTA-free buffer. Thus we examined the effects of divalent cations on the cleavage of eRF3 in cell extracts. THP-1 cells were lysed with lysis buffer containing either 2.5 mM MgCl2 or 2.5 mM CaCl2, and the status of eRF3 in each cell extract was analyzed by Western blotting. Fig. 1A clearly shows that eRF3 was cleaved in CaCl2-containing buffer, whereas eRF3 was not affected in MgCl2-, EDTA- or EGTA-containing buffer. To verify that the processed isoform of eRF3 (provisionally named eRF3-Ca2+) is p-eRF3, we attempted to determine whether eRF3 is cleaved at the N-terminal domain or the C-terminal domain. To this end, we prepared cells expressing wild-type eRF3 (eRF3-wt) and 73A-eRF3 (p-eRF3), both of which are N-terminally ubiqutin (Ub)-tagged and C-terminally FLAG-tagged [15]. As described previously, when these eRF3 constructs were expressed in cells, the Ub moiety is removed by multiple ATP-dependent proteases, and eRF3 fragments without Ub can be produced [15]. The eRF3-wt-expressing cells were lysed in CaCl2-containing buffer and C-terminally FLAG-tagged eRF3 was detected by anti-FLAG antibody. Since eRF3-Ca2+, as well as full-length eRF3, was recognized by anti-FLAG antibody, eRF3-Ca2+ is produced by cleavage at the N-terminal region of eRF3 (Fig. 1B). We also confirmed that eRF3-Ca2+ exhibited the same mobility on SDS–PAGE as that of 73A-eRF3 (p-eRF3). We previously reported another form of cleaved eRF3 (eRF3-cp) that is produced by caspase-mediated cleavage of eRF3, mainly at 32D [15]. To distinguish between eRF3-Ca2+ and eRF3-cp, we next prepared cells expressing 33Q-eRF3 (eRF3-cp) and eRF3 (D25A/D29A/D32A/D35A), which is resistant to the cleavage by caspases [15]. As shown in Fig. 1C, both 33Q-eRF3 (eRF3-cp) and eRF3 (D25A/D29A/D32A/D35A) were cleaved in Ca2+-containing buffer. These results show that eRF3-Ca2+ is a distinct isoform from eRF3-cp. Because an important property of p-eRF3 is that it binds to IAPs through its N-terminally exposed IAP-binding motif (IBM) [11,12], we examined the interaction between eRF3-Ca2+ and XIAP. We prepared cells expressing either eRF3 (wt) or eRF3(A73G) mutant, both of which were C-terminally FLAG-tagged. The eRF3
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Fig. 1. p-eRF3 is produced by calcium-dependent protease. (A) Cell extracts were prepared from THP-1 cells using lysis buffer supplemented with EDTA, EGTA, MgCl2 or CaCl2 (each at 2.5 mM). The status of eRF3 was analyzed by Western blotting using anti-eRF3 antibody. GAPDH was also analyzed using anti-GAPDH as a loading control. (B) THP-1 cells were electroporated with pCMV-MycFLAG-Ub-eRF3 and whole cell extracts were prepared using lysis buffer containing either EGTA (CaCl2) or CaCl2 (CaCl2+). Standard sample of p-eRF3 prepared from HEK293T cells transfected with pCMV-MycFLAG-Ub-73A-eRF3 is indicated as 73A-eRF3 (p-eRF3). The status of eRF3 proteins was analyzed by Western blotting using anti-FLAG antibodies. (C) THP-1 cells were electroporated with pCMV-MycFLAG-Ub-eRF3, eRF3 (D25A/D29A/D32A/D35A) or 33Q-eRF3 and wholecell extracts were prepared using lysis buffer containing either EGTA (CaCl2) or CaCl2 (CaCl2+). Western blotting was performed as described in Fig. 1B. (D) Illustration of XIAP binding assay protocol. IP, immunoprecipitation. (E) THP-1 cells expressing FLAG-tagged eRF3 were lysed in EDTA or CaCl2 containing lysis buffer for preparation of fulllength eRF3 (WT (EDTA)) or eRF3-Ca2+ (WT (Ca2+)). The eRF3 proteins were immunoprecipitated using anti-FLAG antibody. As a negative control, FLAG-tagged eRF3 (A73G) (Ca2+) was also prepared as above. eRF3 proteins were purified using anti-FLAG M2 agarose and mixed with Myc-tagged GST-XIAP. Pull down experiment was performed using anti-FLAG antibody. * indicates immunoglobulin. (F) Schematic diagram of the interaction between IAP and eRF3 proteins.
(wt)-expressing cells were lysed with EDTA- or Ca2+-containing buffer to obtain full-length eRF3 or eRF3-Ca2+, respectively, and eRF3(A73G)-expressing cells were lysed with Ca2+-containing buffer. The eRF3 proteins were purified using anti-FLAG resin and then mixed with Myc-tagged XIAPs (Fig. 1D). After rotation, pull-down
assay was performed using anti-FLAG resin and interactions between the eRF3 proteins and XIAP were analyzed by Western blotting. As shown in Fig. 1E, only eRF3-Ca2+ but not full-length eRF3 co-precipitated XIAP. The A73G mutation of eRF3 abolished its interaction with XIAP, although the mutation did not affect
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Fig. 2. p-eRF3 is produced in a calpain-dependent manner. (A) THP-1 cells were lysed in CaCl2-containing buffer supplemented with 0.5 mM PMSF and aprotinin, leupeptin or pepstatin A (each at 2 lg/ml). PMSF was added to all samples to prevent unexpected degradation of eRF3. THP-1 cell extracts supplemented with or without all of the proteases were also prepared. The status of eRF3 was analyzed by Western blotting using anti-eRF3 antibody. (B) THP-1 cells were lysed in CaCl2-containing buffer supplemented with either E-64 or ALLN (each at 20 lM) and Western blotting was performed as described in Fig. 2A. (C) Flag-tagged eRF3 protein purified from eRF3-FLAGexpressing HEK293T cells, was incubated with an indicated amounts of recombinant m-calpain on ice for 30 min. Flag-tagged eRF3 incubated with 2 lg/ml of recombinant mcalpain for the indicated time periods were also prepared. (D) Intact eRF3-FLAG and calpain-mediated processed eRF3 (0.2 lg/ml of m-calpain for 30 min and 2 lg/ml mcalpain for 5 min) were loaded with a standard sample of p-eRF3 (73A-eRF3) prepared as in Fig. 1B.
the cleavage of eRF3. Since the N-terminal alanine of IBM is crucial for its binding to IAPs (Fig. 1F), the evidence strongly supports our conclusion that eRF3-Ca2+ is p-eRF3. 3.2. p-eRF3 is produced in a calpain-dependent manner To elucidate the protease(s) responsible for p-eRF3 (eRF3-Ca2+) production, we tested the effects of protease inhibitors including
serine protease inhibitors PMSF and aprotinin, cysteine protease inhibitor leupeptin and aspartyl protease inhibitor pepstatin A on the cleavage of eRF3 in CaCl2-containing buffer (Fig. 2A). Among the proteases tested, only leupeptin inhibited the cleavage of eRF3, indicating that cysteine protease might be responsible for the Ca2+-dependent cleavage of eRF3. Since calpain is a well-known Ca2+-dependent cysteine protease, we next examined another cysteine protease inhibitor, E-64, and also a specific
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Fig. 3. Activation of calpain is necessary for the production of p-eRF3 in living cells. (A) SK-N-SH cells were treated with ER-stress inducers (1 lg/ml tunicamycin (TM), 1 lM thapsigargin (TG) and 1 lg/ml A23187) and cultured for the indicated periods (24 h and 48 h). Cells without incubation were prepared as a control. The status of eRF3 was analyzed by Western blotting using anti-eRF3 antibody. Endogenous GAPDH was detected as a loading control. (B) SK-N-SH cells were treated with either 1 lM thapsigargin (TG) or 1 lg/ml A23187 in the presence of 20 lM z-VAD-fmk. Protease inhibitors (AEBSF (50 lg/ml), E-64 (50 lM), MG132 (10 lM), ALLN (20 lM) and lactacystin (5 lM)) were also added. (C) SK-N-SH cells were treated with etoposide and protease inhibitors (AEBSF (50 lg/ml), E-64 (50 lM), z-VAD-fmk (20 lM), MG132 (10 lM) and lactacystin (5 lM)). Calpain specific inhibitor calpeptin (20 lM) was also tested. (D) Schematic representation of calpain-mediated processing of eRF3 into p-eRF3.
calpain inhibitor, ALLN. As shown in Fig. 2B, E-64 and ALLN completely blocked the cleavage of eRF3. These results strongly suggest that the Ca2+-dependent cleavage of eRF3 is mediated by calpain. Essentially the same results were obtained using other cell types, although the extent of the cleavage of eRF3 varied among cell types
(data not shown). To confirm that the Ca2+-dependent cleavage of eRF3 is mediated by calpain, we examined whether recombinant calpain cleaves eRF3. Purified eRF3 proteins were incubated with recombinant m-calpain. A single cleaved isoform of eRF3 was preferentially produced by the recombinant m-calpain (Fig. 2C),
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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though increased amount of calpain and prolonged incubation cause minor cleavages or degradation of eRF3. As expected, the principal cleaved product of eRF3 exhibited the same mobility as p-eRF3 (eRF3-Ca2+) on SDS–PAGE (Fig. 2D). From these results, we conclude that calpain is responsible for the Ca2+-dependent cleavage of eRF3. 3.3. Activation of calpain is necessary for the production of p-eRF3 in living cells We next examined whether p-eRF3 is produced in living cells when calpain is activated. Because challenge with ER stress was reported as one of the calpain-activating conditions [19,20], we tested the status of eRF3 when cells were treated with tunicamycin, thapsigargin and A23187, which are commonly used as ER-stress-inducing agents. Fig. 3 shows representative results of p-eRF3 production under ER-stress conditions. When human neuroblastoma SK-N-SH cells were treated with the ER stressors, two cleavage products corresponding to p-eRF3 and eRF3-cp were detected, although only a modest effect was observed for tunicamycin (Fig. 3A). As reported previously, the upper band corresponds to eRF3-cp, since the band was abrogated by the caspase inhibitor zVAD.fmk (Fig. 3B). To ensure that the lower band corresponds to p-eRF3, we examined the effect of protease inhibitors on the production of p-eRF3 under condition that inhibits caspases by zVAD.fmk (Fig. 3B). Calpain inhibitors E-64 and ALLN completely abrogated the appearance of the lower band. The results are consistent with the idea that the lower band corresponds to p-eRF3 and the production of p-eRF3 depends on calpain. Proteasome inhibitor MG132, which is known to inhibit calpain [21], also blocked the production of p-eRF3, whereas another proteasome inhibitor, lactacystin, had no obvious effect. These results also support the idea that the production of p-eRF3 is mediated by calpain [22]. Although the extent of p-eRF3 production varied depending on the cell lines, p-eRF3 was reproducibly detected by the treatment with ER stressors in several cell lines (data not shown). Especially by the treatment with calcium ionophore A23187, which is directly linked to calpain activation, p-eRF3 was observed in almost all cell lines tested. p-eRF3 was also produced in a calpain-dependent manner when SK-N-SH cells were treated with the non-ER stressor etoposide (Fig. 3C). Thus, conditions leading to p-eRF3 production are not limited to the challenge with ER stress. Overall, these results indicate that the activation of calpain is necessary for the production of p-eRF3. 4. Discussion Previous studies suggested that eRF3 may function in the regulation of cell death; processed eRF3 (p-eRF3) interacts with IAPs through its IBM and is able to promote caspase activation, IAP ubiquitination and apoptosis. However, lack of knowledge regarding the conditions to produce p-eRF3 has hampered progress in investigating the role of eRF3 in cell death. In this study, we have demonstrated that calpain is responsible for the processing of eRF3 into p-eRF3 (Fig. 3D), based on the following five features; (i) Ca2+ dependence, (ii) sensitivity to cysteine protease inhibitors, leupeptin and E-64, (iii) sensitivity to calpain-specific inhibitor ALLN, (iv) the ability of the processed isoform of eRF3 to bind to XIAP, and (v) the ability of recombinant m-calpain to process eRF3 into p-eRF3. The finding that calpain is responsible for the production of p-eRF3 provides a basis for understanding the role of p-eRF3 in apoptosis (Fig. 3D). Consistent with the apoptotic function of p-eRF3, the pro-apoptotic functions of calpain are well established. Although calpain is involved in mitochondria-dependent apoptosis, it was also
reported to activate mouse caspase-12 and human caspase-4, which are crucial caspases for ‘mitochondria-independent ER-stressinduced apoptosis’ [19,23]. ER-stress-induced apoptosis usually crosstalks with mitochondria-dependent and mitochondriaindependent pathways [24,25], but in some cases, the contribution of the mitochondrial pathway is not observed [26,27]. In the latter cases, IAP-binding proteins including Smac/DIABLO and Omi/HtrA2, which are located in the mitochondrial inner membrane, cannot promote apoptosis. These observations are consistent with the idea that p-eRF3, instead of Smac/DIABLO, Omi/HtrA2, is a cytoplasmic IAP-binding protein that promotes mitochondria-independent ER-stress-induced apoptosis (Fig. 3D). Calpain is also known to regulate oncogenic stress-induced apoptosis and senescence [28–30]. These functions of calpain may provide a clue to understand the second physiological role of p-eRF3 in apoptosis, since p-eRF3, which is devoid of an NES, interacts with ARF tumor suppressor in the nucleus (Fig. 3D) [14]. It is thus tempting to speculate that p-eRF3 mediates calpain-activating signal to the nucleus, in which p-eRF3 might regulate the function of ARF, a crucial regulator of oncogene-related apoptosis and senescence [31,32]. Thus far, we have reported two types of cleaved isoforms of eRF3, p-eRF3 [11,14] and eRF3-cp [15] (Fig. 3D). These cleaved eRF3 isoforms are produced upstream or downstream of caspase activation; p-eRF3 is produced by calpain to activate caspase whereas eRF3-cp is produced as a result of caspase activation. A relationship between the two isoforms of eRF3 is not clear at present. However, our results show eRF3-cp might be a poor target of calpain (Fig. 1C), suggesting that N-terminal residues of eRF3, which are removed by caspase-mediated cleavage, support calpain-mediated cleavage. Noteworthy, polyglycine expansions included in this N-terminal region of eRF3 associates with cancer susceptibility [33,34]. Thus it is tempting to speculate that polyglycine expansions affect calpain-mediated cleavage and apoptosis, and therefore cancer susceptibility. These possibilities are now under investigation in our laboratory. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 25291004) from Japan Society for the Promotion of Science, and a Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan (to S H). References [1] Hoshino, S., Miyazawa, H., Enomoto, T., Hanaoka, F., Kikuchi, Y., Kikuchi, A. and Ui, M. (1989) A human homologue of the yeast GST1 gene codes for a GTPbinding protein and is expressed in a proliferation-dependent manner in mammalian cells. EMBO J. 8, 3807–3814. [2] Hoshino, S., Imai, M., Mizutani, M., Kikuchi, Y., Hanaoka, F., Ui, M. and Katada, T. (1998) Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1. J. Biol. Chem. 273, 22254–22259. [3] Hoshino, S., Hosoda, N., Araki, Y., Kobayashi, T., Uchida, N., Funakoshi, Y. and Katada, T. (1999) Novel function of the eukaryotic polypeptide-chain releasing factor 3 (eRF3/GSPT) in the mRNA degradation pathway. Biochemistry (Moscow) 64, 1367–1372. [4] Alkalaeva, E.Z., Pisarev, A.V., Frolova, L.Y., Kisselev, L.L. and Pestova, T.V. (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell 125, 1125–1136. [5] Cheng, Z. et al. (2009) Structural insights into eRF3 and stop codon recognition by eRF1. Genes Dev. 23, 1106–1118. [6] Hosoda, N., Kobayashi, T., Uchida, N., Funakoshi, Y., Kikuchi, Y., Hoshino, S. and Katada, T. (2003) Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation. J. Biol. Chem. 278, 38287–38291.
Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041
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Please cite this article in press as: Hashimoto, Y., et al. Calpain mediates processing of the translation termination factor eRF3 into the IAP-binding isoform p-eRF3. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.06.041