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• W1994447752 abstract "The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, modulates intracellular Ca2+signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP3R type 1 (IP3R1) was found to be cleaved. IP3R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH2DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP3R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP3R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888DEVD*1892R (mouse IP3R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP3R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-α- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP3R1 degradation upon apoptotic stimuli. Therefore IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP3R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP3R1 was digested, indicating that caspase-3 inactivates IP3R1 channel functions. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, modulates intracellular Ca2+signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP3R type 1 (IP3R1) was found to be cleaved. IP3R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH2DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP3R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP3R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888DEVD*1892R (mouse IP3R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP3R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-α- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP3R1 degradation upon apoptotic stimuli. Therefore IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP3R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP3R1 was digested, indicating that caspase-3 inactivates IP3R1 channel functions. d-myo-inositol 1,4,5-trisphosphate IP3 receptor IP3R type 1 IP3R type 2 IP3R type 3, mAb, monoclonal antibody IP3-induced Ca2+ release tumor necrosis factor-α Apoptosis is an evolutionary conserved form of cell death by which normal cellular development and homeostasis are maintained. This programmed cell death is regulated by a series of biochemical events, namely activation of a family of cysteine proteases, caspases, which in turn cleave specific intracellular proteins resulting in an irreversible commitment to cell death. Among the caspase family, caspase-3 plays a crucial role in execution of apoptosis. Known substrates for caspase-3 (1Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar) involve poly(ADP-ribose) polymerase, p21-activated kinase 2 (1Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar, 2Porter A.G. Ng P. Janicke R.U. Bioessays. 1997; 19: 501-507Crossref PubMed Scopus (166) Google Scholar), gelsolin (3Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1042) Google Scholar), DNA-dependent protein kinase catalytic subunit, DNA fragmentation factor 45 kDa subunit (4Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar), and α-fodrin (5Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 6Martin S.J. O'Brien G.A. Nishioka W.K. McGahon A.J. Mahboubi A. Saido T.C. Green D.R. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). By analogy of the cleavage site of these substrates, amino acid sequence of DEXD is considered to be a recognition motif of caspase-3. Inositol 1,4,5-trisphosphate (IP3)1 receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, plays a crucial role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals (7Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar, 8Mikoshiba K. Curr. Opin. Neurobiol. 1997; 7: 339-345Crossref PubMed Scopus (161) Google Scholar). Molecular cloning studies revealed that there are three types of IP3R: IP3R type 1 (IP3R1), IP3R type 2 (IP3R2), and IP3R type 3 (IP3R3) (9Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (826) Google Scholar, 10Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (320) Google Scholar, 11Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 12Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Receptors Channels. 1994; 2: 9-22PubMed Google Scholar). The involvement of IP3Rs during apoptosis has been proposed (13Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S.H. Ross C.A. Snyder S.H. Science. 1996; 273: 503-507Crossref PubMed Scopus (245) Google Scholar, 14Jayaraman T. Marks A.R. Mol. Cell. Biol. 1997; 17: 3005-3012Crossref PubMed Scopus (235) Google Scholar, 15Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar). Khanet al. (13Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S.H. Ross C.A. Snyder S.H. Science. 1996; 273: 503-507Crossref PubMed Scopus (245) Google Scholar) reported that mRNA and protein of IP3R3 increase in B and T lymphocytes in response to anti-IgM antibodies and dexamethasone, respectively. Reduction of IP3R3 expression by antisense construct of IP3R3 cDNA blocked the dexamethasone-induced apoptosis. Jayaraman and Marks (14Jayaraman T. Marks A.R. Mol. Cell. Biol. 1997; 17: 3005-3012Crossref PubMed Scopus (235) Google Scholar) reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of IP3R1 is resistant to apoptotic stimuli, including Fas, dexamethasone, and γ-irradiation, despite the finding that T-cells in IP3R1-deficient mice normally develop and respond to proliferative and death signals (16Hirota J. Baba M. Matsumoto M. Furuichi T. Takatsu K. Mikoshiba K. Biochem. J. 1998; 333: 615-619Crossref PubMed Scopus (29) Google Scholar). Sugawara et al. (15Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar) reported that IP3/Ca2+ signaling is involved in B-cell antigen receptor-induced apoptosis in a chick B-cell line, DT40 cells. In their experiments, IP3R-deficient cells showed a reduction in apoptosis in which the degree of resistance depend on the number of IP3Rs depleted, i.e. triple IP3R-deficient cells were more resistant than single IP3R-deficient cells. Although it has been demonstrated that IP3Rs are involved in the process of apoptosis, less attention has been directed to the relationship between IP3Rs and caspases. Among the IP3R family, IP3R1 is the most widely expressed in tissues and is recognized as an ubiquitous type of IP3R. In the primary amino acid sequence of IP3R1, there is the DEVD consensus sequence for caspase-3 cleavage at 1889–1892 amino acids of mouse IP3R1, which is conserved among species (1888DEVD rat IP3R1 and 1835DEVD human IP3R1). In the present studies, we asked whether IP3R1 could serve as a substrate of caspase-3 during apoptosis, and we obtained evidence that IP3R1 is a newly identified substrate for caspase-3. Using caspase-3-deficient cells, MCF-7 (17Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar), we found that caspase-3 is essential for the cleavage of IP3R1. In addition, effects of the cleavage by caspase-3 on the IP3R1 channel function were also given attention. Monoclonal antibodies (mAbs) KM1112, KM1083, and KM1082 against the C terminus of IP3R1, IP3R2, and IP3R3, respectively, were prepared as described elsewhere (18Sugiyama T. Furuya A. Monkawa T. Yamamoto-Hino M. Satoh S. Ohmori K. Miyawaki A. Hanai N. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 354: 149-154Crossref PubMed Scopus (82) Google Scholar, 19Sugiyama T. Yamamoto-Hino M. Miyawaki A. Furuichi T. Mikoshiba K. Hasegawa M. FEBS Lett. 1994; 349: 191-196Crossref PubMed Scopus (58) Google Scholar). A monoclonal antibody against pro-caspase-3 was obtained from Transduction Laboratories (Lexington, KY). Anti-Fas IgM CH-11 was purchased from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). Recombinant human tumor necrosis factor-α (TNF-α) was obtained from Pepro Tech EC, Ltd. (London, UK). Staurosporine and cycloheximide were purchased from Sigma and Wako Chemical (Tokyo, Japan), respectively. Ac-YVAD-CHO (inhibitor for caspase-1-like protease) and Ac-DEVD-CHO (inhibitor for caspase-3-like protease) were purchased from Peptide Institute (Osaka, Japan). z-VAD-CH2DCB (inhibitor for caspases with broad specificity) was obtained from Phoenix Pharmaceutical, Inc. (Mountain View, CA). The human lymphoblastoid T-cell line, Jurkat, was obtained from the ATCC (Manassas, VA), and was maintained in RPMI 1640 medium with 2 mml-glutamine and 10% of fetal calf serum. The human breast carcinoma cell line, MCF-7, was obtained from Dainippon-Seiyaku, Co., Ltd. (Osaka, Japan) and was maintained in Dulbecco's modified Eagle's medium containing 2 mml-glutamine and 10% fetal calf serum. To induce apoptosis, Jurkat cells were treated with staurosporine (1 or 2 μm) or with anti-Fas IgM CH-11 (500 μg/ml). MCF-7 cells or MCF-7/Casp-3 cells were treated with staurosporine (1 μm) or a combination of TNF-α (30 ng/ml) and cycloheximide (10 μg/ml). Cells (1 × 106cells) treated with apoptotic stimuli were harvested at the indicated time and washed with phosphate-buffered saline and then were solubilized in 100 μl of the lysis buffer (150 mm NaCl, 5 mm EDTA, 1 mm 2-mercaptoethanol, 10% glycerol, 1% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm pepstatin A, 10 μm E-64, and 20 mm Tris-HCl, pH 7.5) on ice for 15 min. The insoluble fraction was removed by centrifugation at 15,000 rpm for 15 min at 4 °C. The resultant supernatant was subjected to SDS-5% polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunodetected using mAbs specific for each type of IP3R. The recombinant histidine-tagged human caspase-3 (CPP32) was kindly provided by Dr. M. Miura (Osaka University, Osaka, Japan). The recombinant caspase-3 was purified using a nickel column according to the protocol of the manufacturer (Amersham Pharmacia Biotech). The cerebellar microsome fraction (200 μg/ml), in which IP3R1 is dominantly expressed was incubated with the purified recombinant caspase-3 (10 or 50 μg/ml) at 37 °C for 10 min in the presence or absence of 10 μm caspase-3 inhibitor, Ac-DEVD-CHO. The reaction mixture were then subjected to Western blot analysis as described above. Cleavage sites of IP3R1 by caspase-3 were determined by N-terminal amino acid sequencing, as described, but with some modification (20Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Briefly, IP3R1 was purified, as described previously (21Nakade S. Rhee S.K. Hamanaka H. Mikoshiba K. J. Biol. Chem. 1994; 269: 6735-6742Abstract Full Text PDF PubMed Google Scholar) then was treated with the recombinant caspase-3. The reaction mixture was applied to SDS-10% polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After staining the membrane with Coomassie Brilliant Blue R-250, three fragments of 215,000, 160,000, and 95,000 were cut and applied to a gas-phase protein sequencer (Applied Biosystem). The expression vector for FLAG-tagged human caspase-3, pM136, was a kindly provided by Dr. M. Miura (Osaka University). pM136 was used to transfect MCF-7 cells using LipofectAMINE (Life Technologies, Inc.). After 2 weeks of selection in growth medium containing 700 μg/ml of G418, about 40 resistant colonies were isolated and examined for caspase-3 expression by Western blot analysis, and then the positive clones were reseeded, isolated, and maintained in culture medium with 100 μg/ml of G418. Effect of digestion by caspase-3 on IP3R1 channel activity was investigated using mouse cerebellar microsome fractions in the presence or absence of the recombinant caspase-3. Mouse microsome fractions (400 μg/ml) were incubated with various concentrations of recombinant caspase-3 (0–100 μg/ml) for 10 min at 30 °C with continuous stirring in Ca2+ release assay buffer (110 mm KCl, 10 mm NaCl, 5 mm KH2PO4, 1 mm 2-mercaptoethanol, 100 μmphenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm pepstatin A, 10 μm E-64, and 50 mm HEPES/KOH, pH 7.2) to produce different degrees of digestion. The reaction of digestion was stopped by adding 100 μm Ac-DEVD-CHO, and then an equal volume of the Ca2+ release assay buffer supplemented with oligomycin, phosphocreatine, creatine kinase, MgCl2, and Fura-2 was added to give final concentrations of 1 μg/ml, 10 mm, 10 units/ml, 2 mm, and 2 μm, respectively. IP3-induced Ca2+ release was measured as described (20Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 22Nakade S. Maeda N. Mikoshiba K. Biochem. J. 1991; 277: 125-131Crossref PubMed Scopus (83) Google Scholar). In all experiments, IP3 was added at the same base-line level to minimize effect of deviation of free Ca2+ concentration on IP3R channel activity. Free [Ca2+] was calculated as described (23Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar), assuming a dissociation constant of 224 nm for Fura-2-Ca2+. Following these measurements, the reaction mixture was subjected to Western blots, and the extent of digestion was determined by densitometric analysis using NIH Image 1.58 (National Institutes of Health, Bethesda, MD). IP3R1 has the DEVD consensus sequence for caspase-3 cleavage within its modulatory domain at 1888DEVD of mouse IP3R1, which is conserved among species (rat:1888DEVD and human: 1835DEVD) (Fig.1 A). To determine whether IP3R1 is cleaved during apoptosis, Jurkat cells were treated with either 2 μm staurosporine or 500 ng/ml anti-Fas IgM CH-11 and then were subjected to Western blot analysis with specific mAbs against the C terminus of each type of IP3R. Fig. 1 (B and C) shows the time course of IP3Rs degradation during apoptosis, as induced by either 1 μm staurosporine or 500 ng/ml anti-Fas IgM CH-11. During the staurosporine-induced apoptosis, IP3R1 was partially cleaved, whereas IP3R2 and IP3R3 were resistant to specific degradation. All three types of IP3R, however, disappeared 10 h after stimulation, a time when almost all the Jurkat cells had died, as determined by trypan blue exclusion. In the case of anti-Fas antigen, specific degradation of IP3R1 was also observed during apoptosis. In both cases, one major fragment with an estimated molecular weight of 95,000 and two minor fragments of 215,000 and 160,000 were detected, using a mAb against the C terminus of IP3R1, KM1112. To determine whether degradation of IP3R1 during apoptosis is mediated by caspase-3 or caspase-3-like proteases, we examined effects of caspase inhibitors on IP3R1 degradation. Jurkat cells were pretreated with caspase inhibitors 1 h prior to apoptotic stimuli and then were incubated together during the stimuli. Fig. 2 shows that pretreatment of Jurkat cells with either a caspase-3-like protease inhibitor (Ac-DEVD-CHO) or a caspase inhibitor of broad specificity (z-VAD-CH2DCB) inhibited the degradation of IP3R1 during the apoptosis induced by staurosporine, whereas caspase-1 inhibitor (Ac-YVAD-CHO) showed no such inhibition (Fig. 2). The same results were obtained in the case of anti-Fas stimulation (data not shown). Other cysteine protease inhibitors tested, E-64d for cathepsin B/H/L and calpain andN-acetyl-Leu-Leu-norleucinal for calpain, did not block IP3R1 degradation during apoptosis (data not shown). To confirm that IP3R1 is cleaved by caspase-3, digestion of IP3R1 was tested by treatment with the purified recombinant caspase-3 in vitro. Fig.3 shows cleavage of IP3R1 by recombinant caspase-3, in a concentration-dependent manner, and the fragmentation pattern was consistent with that seen in Jurkat cells undergoing apoptosis. In the presence of the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, this specific cleavage was inhibited (Fig. 3). To determine the cleavage sites, the purified fragments of 215,000, 160,000, and 95,000 were subjected to N-terminal amino acid sequencing. The N-terminal amino acid sequence of the major fragment of 95,000 is RDAPXR (X is not determined), consistent with1892RDAPSR of mouse IP3R1, indicating that IP3R1 is cleaved at just after DEVD consensus sequence for caspase-3 (Fig. 1 A). N-terminal amino acid sequences of two minor fragments of 215,000 and 160,000, however, could not be determined, because they were too faint to examine the sequence or N termini blocking. To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the degradation of IP3R1 during apoptosis in the caspase-3 deficient cell line, MCF-7 cells (breast carcinoma cell) (17Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar). As noted by other investigators (17Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar), pro-caspase-3 was not detected in MCF-7 cells, where we used an anti-caspase-3 antibody that recognizes pro-caspase-3 but not the active form of caspase-3 (Fig. 4). Although TNF-α or staurosporine induced apoptosis in MCF-7 cells, caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1 (Fig. 4). To confirm that caspase-3 is essential for IP3R1 degradation during apoptosis, we established MCF/Casp-3 cells that were stably transfected to express caspase-3. Fig. 4 shows that two independent stable transformants of MCF/Casp-3 cells, 2B1 and 2B5 (two representative clones out of seven tested), express pro-caspase-3. In these cell lines, no spontaneous activation of caspase-3 and no morphological changes were observed (data not shown). Treatment of MCF/Casp-3 cells, 2B1 and 2B5, with TNF-α/cycloheximide or staurosporine resulted in decreases in pro-caspase-3 because of processing into an active form. In contrast to the caspase-3-deficient MCF-7 cells, MCF-7/Casp-3 2B1 and 2B5 cells showed IP3R1 degradation upon apoptotic stimuli, and the fragmentation patterns were the same as seen in Jurkat cells andin vitro cleavage. Effects of the cleavage of IP3R1 by caspase-3 on channel activity were then investigated using mouse cerebellar microsome fractions, in which IP3R1 is dominantly expressed. Fig.5 A shows typical profiles of ATP-induced Ca2+ uptake and IP3-induced Ca2+ release (IICR) in microsome fractions treated with various concentrations of caspase-3. Control microsomes or caspase-3-digested microsomes were loaded with Ca2+ by adding 2 mm of ATP, and then 1 μm of IP3 was added to induce Ca2+ release. Although the ATP-induced Ca2+ uptake was not affected by caspase-3, IICR was inhibited by caspase-3 in a dose-dependent manner. To quantify the degree of digestion, control microsomes and caspase-3-treated microsomes were subjected to Western blots, and amounts of intact IP3R1 were measured by densitometric analysis as described under “Experimental Procedures.” The percentage of digested IP3R1 and the channel activity are summarized in Fig. 5 B. Increasing caspase-3 concentrations (0, 10, 20, 50, and 100 μg/ml) resulted in increase in the percentage of the digested IP3R1 (0, 25.8 ± 8.2, 46.4 ± 15, 81.1 ± 6.7, and 89.2 ± 4.7, respectively;n = 3). IICR activities were not significantly inhibited by caspase-3 until less than 50% of IP3R1 was digested, yet when over half of the IP3R1 was digested, the channel activities decreased. In the IP3R1 primary amino acid sequence, there is the DEVD consensus sequence for caspase-3 cleavage at 1888–1891 amino acids of mouse IP3R1 (Fig. 1 A), which is conserved among mice, rats, and humans. In Jurkat cells, IP3R1 was cleaved during apoptosis. One major fragment of 95,000 and two minor fragments of 215,000 and 160,000 of IP3R1 were detected using an mAb against the C terminus of IP3R1, indicating that IP3R1 is cleaved at three sites during apoptosis (Fig. 1,B and C). The different degrees of degradation of IP3R1 seen with use of staurosporine and anti-Fas IgM would be attributed to different caspases activated or to different activities of these proteases. In both cases, the main fragment of 95,000 was observed, and the molecular size was consistent with that of the expected fragment if IP3R1 is cleaved at DEVD consensus sequence for caspase-3. Therefore, IP3R1 is probably degraded by caspase-3 or caspase-3-like proteases. IP3R2 and IP3R3 were resistant to specific degradation. In IP3R2 and IP3R3, there is no DEXD sequence and no potential cleavage site, which resembles the tetrapeptide sequences identified in various substrates for caspase-3. Khan et al. (13Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S.H. Ross C.A. Snyder S.H. Science. 1996; 273: 503-507Crossref PubMed Scopus (245) Google Scholar) reported down-regulation of IP3R1 and up-regulation IP3R3 during apoptosis in WEHI-B cells induced by IgM, thymocytes, and S49 cells, as induced by dexamethasone. In the present study, there was no apparent increase in IP3R3 expression. These differences may relate to different cells used and to the stimuli used to induce apoptosis. We found the time course of cell death induced by staurosporine or anti-Fas IgM to be more rapid (10–20 h) than that seen with dexamethasone (24–96 h). Caspase-3 (-like protease) inhibitor or a caspase inhibitor of broad specificity but not caspase-1 inhibitor inhibited the degradation of IP3R1 during apoptosis, thereby indicating that IP3R1 is cleaved by caspase-3 or caspase-3-like protease (Fig. 2). It was reported that IP3R1 was down-regulated in response to chronic activation of cell surface receptors, an event that was caused by IP3R1 degradation by the cysteine protease, calpain (24Wojcikiewicz R.J.H. Oberdorf J.A. J. Biol. Chem. 1996; 271: 16652-16655Crossref PubMed Scopus (57) Google Scholar). In the case of apoptosis, cysteine protease inhibitors, E-64d for cathepsin B/H/L, and calpain andN-acetyl-Leu-Leu-norleucinal for calpain did not block IP3R1 degradation, thus supporting our observation that IP3R1 is cleaved by caspase-3 (-like protease) but not by calpain. In addition, in the case of the recombinant caspase-3 digested IP3R1 in vitro, the fragmentation pattern is consistent with that seen in Jurkat cells. This means that IP3R1 serves as a substrate for caspase-3 (-like protease). The N-terminal amino acid sequence revealed that the major fragment of 95,000 is produced by cleavage at caspase-3 recognition motif, the DEVD tetrapeptide. We propose that IP3R1 is a newly identified caspase-3 substrate and that one of the cleavage sites contains the DEVD consensus sequence for caspase-3 cleavage. Janicke et al. (17Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1727) Google Scholar) demonstrated that MCF-7 breast carcinoma cells have genetically lost caspase-3 and that caspase-3 is essential for DNA fragmentation and for blebbing. However, MCF-7 cells do respond to certain apoptotic stimuli, suggesting that caspase-3 is not essential for apoptosis and that other caspases may be activated. Actually, caspase-2, -5, -7, -8, -9, and -10 were detected in MCF-7 cells, and caspase-8 is activated in MCF-7 during the apoptosis induced by TNF-α (25Janicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. Chem. 1998; 273: 15540-15545Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). These same authors reported that most substrates of caspase-3 were cleaved during apoptosis in caspase-3-deficient MCF-7 cells, and they stated that caspase-3 is essential for cleavage of α-fodrin but dispensable for the cleavage of poly(ADP-ribose) polymerase, Rb, p21-activated kinase 2, DNA-dependent protein kinase catalytic subunit, gelsolin, and DNA fragmentation factor 45-kDa subunit, which suggested that caspases other than caspase-3 are activated and cleaved these substrates in MCF-7 cells (25Janicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. Chem. 1998; 273: 15540-15545Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). It is, however, uncertain whether cleavage of these substrates by other caspases in caspase-3-deficient cells is functional for apoptosis, because other groups reported that DNA fragmentation factor 45-kDa subunit and gelsolin require caspase-3 for proper cleavage and its function (3Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1042) Google Scholar, 4Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 26Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1427) Google Scholar, 27Tang D. Kidd V.J. J. Biol. Chem. 1998; 273: 28549-28552Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), despite the finding that they were cleaved in the absence of caspase-3, possibly by other caspases. In the case of IP3R1, caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1 (Fig. 4), indicating that IP3R1 was not cleaved by remaining caspases, such as caspase-8. In MCF/Casp3 cells, pro-caspase-3 became the active form, as induced by apoptotic stimuli (Fig. 4), thereby indicating that the caspase-3 activation pathway was functional. Upon apoptotic stimuli, IP3R1 was cleaved when MCF-7 cells were stably transfected with caspase-3, indicating that caspase-3 is essential for the cleavage of IP3R1. Thus, our data show that IP3R1 is a specific substrate for caspase-3 and that this cleavage cannot occur with other caspases in MCF-7 cells. Effects of the cleavage of IP3R1 by caspase-3 on channel activity were also investigated using mouse cerebellar microsome fractions, in which IP3R1 is dominantly expressed (Fig. 5). The time course of Ca2+uptake was not affected by caspase-3 treatment, indicating that the Ca2+-ATPase function is resistant to cleavage by caspase-3 (Fig. 5 A). On the contrary, the IICR was inhibited by caspase-3 in a dose-dependent manner (Fig.5 A). Digestion of up to 50% did not significantly inhibit the channel activity, suggesting that even partially digested IP3R1 can function as a Ca2+ channel (Fig. 5 B), as was observed in trypsinized IP3R1 (20Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Therefore 90% of the digested IP3R1 has 25% IICR activity. Alternatively, IICR is not highly cooperative, because if the Hill coefficient of IICR is 4, the digested IP3R subunit could have a dominant negative effect on IP3R channel activity. Moreover, inhibitory effects on the IICR were observed in over 50% of the digested IP3R1, suggesting that cleavage of at least two subunits of IP3R is needed to inactivate the IP3R channel. These results are in accord with our previous studies on kinetics of the purified IP3R1, in which the Hill coefficient of IICR was 2 (28Hirota J. Michikawa T. Miyawaki A. Furuichi T. Okura I. Mikoshiba K. J. Biol. Chem. 1995; 270: 19046-19051Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In conclusion, IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for cleavage of IP3R1 during apoptosis. One of the cleavage sites of IP3R1 is the DEVD consensus sequence for caspase-3. Cleavage of IP3R1 by caspase-3 resulted in inhibition of IP3-induced Ca2+ release activity, in a digestion-dependent manner, an event that may possibly interfere with the IP3/Ca2+ signaling pathway and intracellular Ca2+ homeostasis within cells undergoing apoptosis. We thank Dr. M. Miura for the human recombinant CPP32/caspase-3 and for the FLAG-tagged caspase-3 expression construct, pM136, and M. Ohara for helpful comments and language assistance." @default.
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