Inositol 1,4,5-Trisphosphate Receptor Type 1 Is a Substrate for Caspase-3 and Is Cleaved during Apoptosis in a Caspase-3-dependent Manner*

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.

Inositol 1,4,5-trisphosphate (IP 3 ) 1 receptor (IP 3 R), an IP 3gated Ca 2ϩ channel located on intracellular Ca 2ϩ stores, plays a crucial role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals (7,8). Molecular cloning studies revealed that there are three types of IP 3 R: IP 3 R type 1 (IP 3 R1), IP 3 R type 2 (IP 3 R2), and IP 3 R type 3 (IP 3 R3) (9 -12). The involvement of IP 3 Rs during apoptosis has been proposed (13)(14)(15). Khan et al. (13) reported that mRNA and protein of IP 3 R3 increase in B and T lymphocytes in response to anti-IgM antibodies and dexamethasone, respectively. Reduction of IP 3 R3 expression by antisense construct of IP 3 R3 cDNA blocked the dexamethasone-induced apoptosis. Jayaraman and Marks (14) reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of IP 3 R1 is resistant to apoptotic stimuli, including Fas, dexamethasone, and ␥-irradiation, despite the finding that T-cells in IP 3 R1-deficient mice normally develop and respond to proliferative and death signals (16). Sugawara et al. (15) reported that IP 3 /Ca 2ϩ signaling is involved in B-cell antigen receptor-induced apoptosis in a chick B-cell line, DT40 cells. In their experiments, IP 3 R-deficient cells showed a reduction in apoptosis in which the degree of resistance depend on the number of IP 3 Rs depleted, i.e. triple IP 3 R-deficient cells were more resistant than single IP 3 R-deficient cells.
Although it has been demonstrated that IP 3 Rs are involved in the process of apoptosis, less attention has been directed to the relationship between IP 3 Rs and caspases. Among the IP 3 R family, IP 3 R1 is the most widely expressed in tissues and is recognized as an ubiquitous type of IP 3 R. In the primary amino acid sequence of IP 3 R1, there is the DEVD consensus sequence for caspase-3 cleavage at 1889 -1892 amino acids of mouse IP 3 R1, which is conserved among species ( 1888 DEVD rat IP 3 R1 and 1835 DEVD human IP 3 R1). In the present studies, we asked whether IP 3 R1 could serve as a substrate of caspase-3 during apoptosis, and we obtained evidence that IP 3 R1 is a newly identified substrate for caspase-3. Using caspase-3-deficient * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ Present address: Laboratory for Molecular Neurogenesis, Brain Science Inst., Inst. of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan. cells, MCF-7 (17), we found that caspase-3 is essential for the cleavage of IP 3 R1. In addition, effects of the cleavage by caspase-3 on the IP 3 R1 channel function were also given attention.
Cell Lines and Culture Conditions-The human lymphoblastoid Tcell line, Jurkat, was obtained from the ATCC (Manassas, VA), and was maintained in RPMI 1640 medium with 2 mM L-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 mM L-glutamine and 10% fetal calf serum.
Western Blot Analysis-Cells (1 ϫ 10 6 cells) treated with apoptotic stimuli were harvested at the indicated time and washed with phosphate-buffered saline and then were solubilized in 100 l 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 M leupeptin, 10 M pepstatin A, 10 M 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 IP 3 R.
Cleavage of IP 3 R1 by the Recombinant Caspase-3 in Vitro-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 g/ml), in which IP 3 R1 is dominantly expressed was incubated with the purified recombinant caspase-3 (10 or 50 g/ml) at 37°C for 10 min in the presence or absence of 10 M caspase-3 inhibitor, Ac-DEVD-CHO. The reaction mixture were then subjected to Western blot analysis as described above.
Determination of Cleavage Sites of IP 3 R1 by Caspase-3-Cleavage sites of IP 3 R1 by caspase-3 were determined by N-terminal amino acid sequencing, as described, but with some modification (20). Briefly, IP 3 R1 was purified, as described previously (21) 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).
Stable Transfection of Caspase-3 cDNA in MCF-7 Cells-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 g/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 g/ml of G418. IP 3 -induced Ca 2ϩ Release by Caspase-3-digested IP 3 R1-Effect of digestion by caspase-3 on IP 3 R1 channel activity was investigated using mouse cerebellar microsome fractions in the presence or absence of the recombinant caspase-3. Mouse microsome fractions (400 g/ml) were incubated with various concentrations of recombinant caspase-3 (0 -100 g/ml) for 10 min at 30°C with continuous stirring in Ca 2ϩ release assay buffer (110 mM KCl, 10 mM NaCl, 5 mM KH 2 PO 4 , 1 mM 2-mercaptoethanol, 100 M phenylmethylsulfonyl fluoride, 10 M leupeptin, 10 M pepstatin A, 10 M 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 M Ac-DEVD-CHO, and then an equal volume of the Ca 2ϩ release assay buffer supplemented with oligomycin, phosphocreatine, creatine kinase, MgCl 2 , and Fura-2 was added to give final concentrations of 1 g/ml, 10 mM, 10 units/ml, 2 mM, and 2 M, respectively. IP 3 -induced Ca 2ϩ release was measured as described (20,22). In all experiments, IP 3 was added at the same base-line level to minimize effect of deviation of free Ca 2ϩ concentration on IP 3 R channel activity. Free [Ca 2ϩ ] was calculated as described (23), assuming a dissociation constant of 224 nM for Fura-2-Ca 2ϩ . 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).

RESULTS
Specific Degradation of IP 3 R1 during Apoptosis-IP 3 R1 has the DEVD consensus sequence for caspase-3 cleavage within its modulatory domain at 1888 DEVD of mouse IP 3 R1, which is conserved among species (rat: 1888 DEVD and human: 1835 DEVD) (Fig. 1A). To determine whether IP 3 R1 is cleaved during apoptosis, Jurkat cells were treated with either 2 M 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 IP 3 R. Fig. 1 (B and C) shows the time course of IP 3 Rs degradation during apoptosis, as induced by either 1 M staurosporine or 500 ng/ml anti-Fas IgM CH-11. During the staurosporine-induced apoptosis, IP 3 R1 was par-FIG. 1. Specific degradation of IP 3 Rs during apoptosis. A, schematic representation of IP 3 R1, which has the DEVD consensus sequence for caspase-3 cleavage. This sequence was conserved among species ( 1888 DEVD 1891 mouse, rat IP 3 R1, and 1835 DEVD 1838 human IP 3 R1). B and C, specific degradation of IP 3 R during apoptosis induced by 2 M staurosporine (B) and 500 ng/ml anti-Fas IgM CH-11 (C). IP 3 Rs were detected with type-specific mAbs against the C terminus of IP 3 Rs, KM1112 (anti-IP 3 R1), KM1083 (anti-IP 3 R2), and KM1082 (anti-IP 3 R3).
tially cleaved, whereas IP 3 R2 and IP 3 R3 were resistant to specific degradation. All three types of IP 3 R, 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 IP 3 R1 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 IP 3 R1, KM1112.
To determine whether degradation of IP 3 R1 during apoptosis is mediated by caspase-3 or caspase-3-like proteases, we examined effects of caspase inhibitors on IP 3 R1 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-CH 2 DCB) inhibited the degradation of IP 3 R1 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 and N-acetyl-Leu-Leu-norleucinal for calpain, did not block IP 3 R1 degradation during apoptosis (data not shown).
Specific Digestion of IP 3 R1 by Recombinant Caspase-3-To confirm that IP 3 R1 is cleaved by caspase-3, digestion of IP 3 R1 was tested by treatment with the purified recombinant caspase-3 in vitro. Fig. 3 shows cleavage of IP 3 R1 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).
Cleavage Site of IP 3 R1 by Caspase-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 with 1892 RDAPSR of mouse IP 3 R1, indicating that IP 3 R1 is cleaved at just after DEVD consensus sequence for caspase-3 (Fig. 1A). 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.
Caspase-3 Is Essential for the Specific Degradation of IP 3 R1-To determine whether IP 3 R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the degradation of IP 3 R1 during apoptosis in the caspase-3 deficient cell line, MCF-7 cells (breast carcinoma cell) (17). As noted by other investigators (17), 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-␣ or staurosporine induced apoptosis in MCF-7 cells, caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP 3 R1 (Fig. 4). To confirm that caspase-3 is essential for IP 3 R1 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-␣/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 IP 3 R1 degradation upon apoptotic stimuli, and the fragmentation patterns were the same as seen in Jurkat cells and in vitro cleavage.

Caspase-3 Digestion of IP 3 R1 Resulted in Inhibition of IP 3induced Ca 2ϩ
Release Activity-Effects of the cleavage of IP 3 R1 by caspase-3 on channel activity were then investigated using mouse cerebellar microsome fractions, in which IP 3 R1 is dominantly expressed. Fig. 5A shows typical profiles of ATP-induced Ca 2ϩ uptake and IP 3 -induced Ca 2ϩ release (IICR) in microsome fractions treated with various concentrations of caspase-3. Control microsomes or caspase-3-digested microsomes were loaded with Ca 2ϩ by adding 2 mM of ATP, and then 1 M of IP 3 was added to induce Ca 2ϩ release. Although the ATP-induced Ca 2ϩ 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 IP 3 R1 were measured by densitometric analysis as described under "Experimental Procedures." The percentage of digested IP 3 R1 and the channel activity are summarized in Fig. 5B. Increasing caspase-3 concentrations (0, 10, 20, 50, and 100 g/ml) resulted in increase in the percentage of . IICR activities were not significantly inhibited by caspase-3 until less than 50% of IP 3 R1 was digested, yet when over half of the IP 3 R1 was digested, the channel activities decreased.

DISCUSSION
Specific Degradation of IP 3 R1 during Apoptosis-In the IP 3 R1 primary amino acid sequence, there is the DEVD consensus sequence for caspase-3 cleavage at 1888 -1891 amino acids of mouse IP 3 R1 (Fig. 1A), which is conserved among mice, rats, and humans. In Jurkat cells, IP 3 R1 was cleaved during apoptosis. One major fragment of 95,000 and two minor fragments of 215,000 and 160,000 of IP 3 R1 were detected using an mAb against the C terminus of IP 3 R1, indicating that IP 3 R1 is cleaved at three sites during apoptosis (Fig. 1, B and C). The different degrees of degradation of IP 3 R1 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 IP 3 R1 is cleaved at DEVD consensus sequence for caspase-3. Therefore, IP 3 R1 is probably degraded by caspase-3 or caspase-3-like proteases. IP 3 R2 and IP 3 R3 were resistant to specific degradation. In IP 3 R2 and IP 3 R3, 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. (13) reported down-regulation of IP 3 R1 and upregulation IP 3 R3 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 IP 3 R3 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). IP 3 R1 Is Cleaved by Caspase-3 (-like Protease)-Caspase-3 (-like protease) inhibitor or a caspase inhibitor of broad specificity but not caspase-1 inhibitor inhibited the degradation of IP 3 R1 during apoptosis, thereby indicating that IP 3 R1 is cleaved by caspase-3 or caspase-3-like protease (Fig. 2). It was reported that IP 3 R1 was down-regulated in response to chronic activation of cell surface receptors, an event that was caused by IP 3 R1 degradation by the cysteine protease, calpain (24). In the case of apoptosis, cysteine protease inhibitors, E-64d for cathepsin B/H/L, and calpain and N-acetyl-Leu-Leu-norleucinal for calpain did not block IP 3 R1 degradation, thus supporting our observation that IP 3 R1 is cleaved by caspase-3 (-like protease) but not by calpain. In addition, in the case of the recombinant caspase-3 digested IP 3 R1 in vitro, the fragmentation pattern is consistent with that seen in Jurkat cells. This means that IP 3 R1 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 IP 3 R1 is a newly identified caspase-3 substrate and that one of the cleavage sites contains the DEVD consensus sequence for caspase-3 cleavage.

FIG. 5. Effects of digestion by caspase-3 on IP 3 -induced Ca 2؉ release.
A, typical profile of IICR by mouse cerebellar microsome fractions treated with caspase-3 at the indicated concentrations. Control microsome or caspase-3-digested microsomes were loaded with Ca 2ϩ by adding 2 mM of ATP, and then 1 M of IP 3 was added to induce Ca 2ϩ release, as indicated by arrows. Treatment with caspase-3 dosedependently inhibited IP 3 R channel activity. B, relation between IP 3induced Ca 2ϩ release activity and percentage of digestion. Percentages of digestion were measured by densitometric analysis of Western blot of intact IP 3 R1, as described under "Experimental Procedures." IICR activity and the percentages of digestion were normalized against control ([caspase-3] ϭ 0; values are means Ϯ S.D., n ϭ 3). 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-␣ (25). 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 ␣-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 (25). 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 (3,4,26,27), despite the finding that they were cleaved in the absence of caspase-3, possibly by other caspases.
In the case of IP 3 R1, caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP 3 R1 (Fig. 4), indicating that IP 3 R1 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, IP 3 R1 was cleaved when MCF-7 cells were stably transfected with caspase-3, indicating that caspase-3 is essential for the cleavage of IP 3 R1. Thus, our data show that IP 3 R1 is a specific substrate for caspase-3 and that this cleavage cannot occur with other caspases in MCF-7 cells.
Caspase-3 Inactivates IP 3 -induced Ca 2ϩ Release Activity-Effects of the cleavage of IP 3 R1 by caspase-3 on channel activity were also investigated using mouse cerebellar microsome fractions, in which IP 3 R1 is dominantly expressed (Fig. 5). The time course of Ca 2ϩ uptake was not affected by caspase-3 treatment, indicating that the Ca 2ϩ -ATPase function is resistant to cleavage by caspase-3 (Fig. 5A). On the contrary, the IICR was inhibited by caspase-3 in a dose-dependent manner (Fig. 5A).
Digestion of up to 50% did not significantly inhibit the channel activity, suggesting that even partially digested IP 3 R1 can function as a Ca 2ϩ channel (Fig. 5B), as was observed in trypsinized IP 3 R1 (20). Therefore 90% of the digested IP 3 R1 has 25% IICR activity. Alternatively, IICR is not highly cooperative, because if the Hill coefficient of IICR is 4, the digested IP 3 R subunit could have a dominant negative effect on IP 3 R channel activity. Moreover, inhibitory effects on the IICR were observed in over 50% of the digested IP 3 R1, suggesting that cleavage of at least two subunits of IP 3 R is needed to inactivate the IP 3 R channel. These results are in accord with our previous studies on kinetics of the purified IP 3 R1, in which the Hill coefficient of IICR was 2 (28).
In conclusion, IP 3 R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for cleavage of IP 3 R1 during apoptosis. One of the cleavage sites of IP 3 R1 is the DEVD consensus sequence for caspase-3. Cleavage of IP 3 R1 by caspase-3 resulted in inhibition of IP 3 -induced Ca 2ϩ release activity, in a digestion-dependent manner, an event that may possibly interfere with the IP 3 /Ca 2ϩ signaling pathway and intracellular Ca 2ϩ homeostasis within cells undergoing apoptosis.