Apoptosis-Linked Gene 2 Binds to the Death Domain of Fas and Dissociates from Fas during Fas-Mediated Apoptosis in Jurkat Cells

Biochemical and Biophysical Research Communications 288, 420 – 426 (2001) doi:10.1006/bbrc.2001.5769, available online at http://www.idealibrary.com on

Apoptosis-Linked Gene 2 Binds to the Death Domain of Fas and Dissociates from Fas during Fas-Mediated Apoptosis in Jurkat Cells Yong-Sam Jung,* Keun-Soo Kim,* Kwang Dong Kim,† Jong-Seok Lim,† Jung-Woo Kim,* and Eunhee Kim* ,1 *Research Center for Biomedicinal Resources and Division of Life Science, PaiChai University, Taejon, Korea 302-735; and †Korea Research Institute of Bioscience and Biotechnology, Taejon, South Korea 305-333

Received September 19, 2001

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca 2ⴙ-binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation. The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward. Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fasmediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein–protein interaction of ALG-2 with Fas was demonstrated using yeast twohybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process. © 2001 Academic Press

Key Words: ALG-2; Fas; Jurkat cell; apoptosis; binding; cleavage; translocation.

Apoptosis is an essential physiological process for normal development and homeostasis in multicellular organisms (1–3). It is well known that improper regulation of this process leads to many pathogenic outcomes. Dysregulation of this process causes autoimmune diseases by allowing persistence of self-reactive 1

To whom correspondence should be addressed. Fax: 82-42-5205463. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

T and B cells and some lymphoproliferative diseases. Cancer cells arise when mutant cells are not eliminated by apoptosis. Neurodegenerative diseases could also result from the disruption of the apoptotic process. Apoptosis-linked gene 2 (ALG-2) has been identified as a protein whose absence causes failure of apoptosis induced by Fas, T cell receptors, and glucocorticoids (4). ALG-2 is expressed in all adult mouse tissues and the most abundant expression of this protein is in the thymus and in the liver. ALG-2 belongs to the calpain small subunit subfamily of Ca 2⫹-binding proteins because ALG-2 contains five EF-hand Ca 2⫹ binding domains (5, 6). Intracellular Ca 2⫹ increases upon Fas signaling and such an intracellular Ca 2⫹ surge is sufficient for caspase activation (7, 8). However the molecule responsible for the link between the intracellular Ca 2⫹ surge and apoptosis had not been discovered. The presence of five EF-hand domains and the dramatic conformational change upon Ca 2⫹ binding suggests that ALG-2 might be one of the relay molecules between the Ca 2⫹ surge and apoptosis (9). It has been proposed that ALG-2 might act downstream of caspase activation because ALG-2 depletion does not affect caspase activation (10). Since ALG-2 depletion blocks apoptosis, it implies that ALG-2 dissociates activation of caspases and the execution of apoptosis. It is certain that ALG-2 is an essential molecule to complete apoptosis. However the mechanism of action and the effector function in the execution of apoptosis of ALG-2 is largely unknown. A new isoform of ALG-2 (ALG-2,1) was detected in mice livers (11). ALG-2,1 is six nucleotides shorter than the previously found type (ALG-2,5) and is missing two internal amino acids (121-Gly and 122-Phe). ALG-2,5 and ALG-2,1 form heterodimers. ALG-2,5 binds to AIP-1 but ALG-2,1 does not. As a result, they proposed that the two isoforms have regulatory functions against each other.

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Two-hybrid screening in order to understand the function of ALG-2 found an ALG-2 interacting protein, AIP1/Alix, whose yeast homolog is a component of the Pkc1p-MAP kinase pathway (12, 13). This suggests ALG-2 is a link molecule between apoptosis and the MAP kinase pathway. Chen et al. (2000) reported the interaction of SETA (SH3 domain-containing protein expressed in tumorigenic astrocytes) with ALG-2/AIP complex (14). The interaction between SETA and ALG2/AIP complex is essential for astrocyte’s apoptosis. In addition, AIP was strongly upregulated by E2F1, a key cellular regulator not only for proliferation but also for apoptosis (15). Based upon these data, it was hypothesized that ALG-2/AIP complex is a modulator at the interface between proliferation and cell death (16). Recently ALG-2 was reported to interact with perflin, another member of PEF family whose function is largely unknown, in the absence of Ca 2⫹ (17). However, there has been no report of ALG-2’s interaction with well-known members of the apoptotic machinery. Many apoptotic components are regulated at the posttranslational level. Bcl-2 family members are good examples of such regulation. Bid, Bax and Bad are translocated to the mitochondria during apoptosis and induce the release of apoptogenic proteins into the cytosol (18 –21). For instance, cell death stimuli trigger a conformational change of Bax exposing its N- and C-termini, that appears to be required for its insertion into mitochondria and for the cleavage at the N-terminus (22, 23). Caspase-8 is another example in which the removal of its N-terminal death effector domain is necessary for its proteolytic activation. The proteolytically activated caspase-8 translocates from the cytoplasmic tail of Fas to the cytoplasm, and subsequently initiates the caspase-cascade (24, 25). The translocation of FKHRL1, a member of the forkhead transcription factor family, is regulated by phosphorylation at its Akt sites (26). Here we investigated direct molecular interaction with apoptotic machinery and molecular changes of ALG-2 during Fas-mediated apoptosis in Jurkat cells. MATERIALS AND METHODS Materials. Jurkat cells were purchased from ATCC. Mouse monoclonal anti-ALG-2 antibody (Ab) was purchased from Transduction Laboratories. Two kinds of anti-Fas Abs were used in this study. Abs from Upstate Biotechnology, CH11, and from Dr. Peter H. Krammer, anti-APO-1 (30), were used for Fas signaling and for immunoprecipitation, respectively. Monoclonal antibody against caspase-8 was purchased from Cell Signaling technology. Antihuman poly-ADP-ribose polymerase (PARP) Ab, protein A/G PLUSagarose and anti-mouse alkaline phosphatase conjugated secondary Ab were obtained from Santa Cruz Biotechnology. Anti-mouse IgGTRITC secondary Ab was purchased from Jackson ImmunoResearch Laboratories. Cell culture supplements containing FBS (fetal bovine serum) were obtained from GIBCO-BRL. [ 35S]Methionine and PVDF (polyvinylidene difluoride) membrane were from NEN and Millipore, respectively. The pcDNA3 vector was from Invitrogen.

Cells and cell culture. Jurkat cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. Cells were maintained in 5% CO 2 at 37°C. Cells were washed once in phosphate-buffered saline (PBS) and then placed in fresh serum-free medium for at least 12 h prior to the administration of CH11 Ab for Fas signaling. Western blot analysis. For Western blot analysis, cells were washed twice with ice-cold PBS and collected by centrifugation. Cells were then lysed in mammalian cell lysis buffer (50 mM Tris–Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.4 mM PMSF). After centrifugation, the supernatant was stored at ⫺80°C. Protein concentrations were determined by Bradford’s method using a kit from Bio-Rad. Protein samples were separated by SDS–PAGE using 15% polyacrylamide gels, and then transferred to PVDF membranes using a semidry blotter in the transfer buffer (20 mM Tris–Cl, pH 8.3, 150 mM glycine, 20% methanol). After blocking in blocking buffer (5% skim milk, 50 mM Tris–Cl, pH 8.0, 2 mM CaCl 2, 0.01% antifoam A, 0.05% Tween 20, 0.02% sodium azide) at 4°C for 2 h, the membranes were incubated with the primary Abs in blocking buffer for 4 h at 4°C. Primary Abs were used at a dilution of 1:1000, except for the monoclonal ALG-2 Ab, which was diluted at 1:5000. The membranes were washed three times with the blocking buffer and then incubated with secondary Abs, which were conjugated with alkaline phosphatase at a final dilution of 1:1000. Following three washings in 1⫻ TBST (100 mM Tris–Cl, pH 8.0, 1.5 M NaCl, 0.5% Tween 20), immunosignals were detected by the method of color development using a kit from Bio-Rad. In the case of caspase-3, blot was incubated with HRP-conjugated secondary antibody diluted 1:1000. After washing with TBST, the blot was developed with the chemiluminescence method following the manufacturer’s protocol (Boehringer Mannheim). Yeast two-hybrid assay. The two-hybrid assay was conducted using the MATCHMAKER LexA system from CLONTECH according to the manufacturer’s instructions. Yeast strain EGY48 was co-transformed with a possible pair of pLexA and pJG4-5 constructs by lithium acetate/polyethylene glycol 4000 procedure and selected on synthetic dropout plates lacking uracil (Ura), histidine (His) and tryptophan (Trp) and grown for 3 days at 30°C. Colonies positive for growth on selective media were blotted on synthetic dropout/agar plates lacking SD medium plate lacking Ura, His and Trp in the presence of 40 ␮g/ml 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). Blue colors on the plates containing X-gal were analyzed after 2–3 days. In vitro binding assay. For the detection of binding of ALG-2 and Fas protein in vitro, ALG-2, ALG-2-N (a.a. 1–95), ALG-2-C (a.a. 96 –191) and ALG-2(-23N) (a.a. 24 –191) were cloned in frame into pGEX4T-1 (Amersham Pharmacia Bioptech) and glutathione S-transferase (GST) fusion proteins were expressed in E. coli BL21(DE3) and purified on glutathione–Sepharose 4B according to the manufacturer’s protocols (Amersham Pharmacia Biotech). Full size Fas protein (45 kDa) was made with TNT Reticulocyte Lysate System (TNT system; Promega). 35S-labeled Fas was incubated with 10 ␮g of each GST fusion protein in 0.2 ml of binding buffer (50 mM Hepes, pH 7.6, 50 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 10% glycerol), washed three times, and separated on an SDS–polyacrylamide gel and exposed to X-ray film. Immunoprecipitation. To test for an association between ALG-2 and Fas in Jurkat cells, cells were activated with 200 ng/ml of CH11 anti-human Fas Ab in medium at 37°C. Cells were washed with phosphate-buffered saline, lysed in RIPA buffer (1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 10 mM sodium phosphate, pH 7.2) and incubated with 10 ␮g of anti-APO-1 Ab for 16 h at 4°C. Immunoprecipitates were washed four times with RIPA buffer and resuspended in 2⫻ SDS sample buffer (100 mM Tris–Cl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) and boiled for 3 min at 100°C. Western blot analysis of immune complexes was performed as described above.

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polypeptide fragment (a.a. 162–181) of ALG-2. The fact that 19-kDa ALG-2 protein bound to this antibody suggests that the 19-kDa protein contains that fragment. Thus it seems logical to assume that 22-kDa ALG-2 is cleaved at its N-terminus and the 19-kDa protein does not contain the N-terminal amino acids.

FIG. 1. Cleavage of ALG-2 during Fas-mediated apoptosis in Jurkat cells. ALG-2 cleavage in Jurkat cells was monitored by time course with 200 ng/ml of CH11 anti-Fas Ab stimulation. PARP cleavage was also monitored as a marker for caspase 3 activation with anti-PARP Ab which detects the C-terminus of PARP. Numbers on the left represent molecular weights in kDa. Immunofluorescence and subcellular distribution. For Fas signaling, Jurkat cells were treated with CH11 anti-Fas Ab for 6 h. The treated cells were then washed and stained with FITC-conjugated annexin V for 15 min. After washing with annexin V binding buffer, they were plated on poly-L-lysine-treated coverslips and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. After washing with PBS three times, cells were incubated for 15 min with 0.2% Triton X-100 in PBS, and then for 1 h with 3% skim milk as a blocking solution. Coverslips were stained with anti-ALG-2 (1:1000) Ab in blocking solution for 1 h, followed by staining with goat antimouse IgG-TRITC. Coverslips containing stained cells were mounted on glass slides and subjected to a confocal laser scanning microscope (Carl Zeiss LSM 410). Cell fractionation. After washing in PBS, Jurkat cells were subfractionated as follows. To prepare the nucleus, 2 ⫻ 10 8 cells were incubated in 500 ␮l of Buffer A (20 mM Hepes–KOH, pH 7.5, 10 mM KCl, 1 mM sodium EGTA, 1 mM DTT) for 15 min at 4°C to swell the cell. The cells were passed through a 21-gauge needle 10 –12 times with microscopic evaluation. Cell suspension was centrifuged at 1000g for 10 min at 4°C. The pellet was solubilized with 200 ␮l of Buffer A and used as the nucleus. The supernatant was centrifuged at 100,000g for 1 h to separate the cytoplasm from the membrane fraction. The pellet was solubilized with 100 ␮l of Buffer A and used as the membrane. The supernatant was used as the cytoplasm and subjected immediately to Western blot analysis.

RESULTS ALG-2 is cleaved in Jurkat cells upon Fas signaling. Many apoptotic molecules undergo post-translational regulation (27–29). Since ALG-2 is required for Fasmediated apoptosis (4), we examined the fate of ALG-2 protein during the death process. Prior to Fas activation, 22-kDa ALG-2 protein was detected in Jurkat cells (Fig. 1). However, after the treatment of anti-Fas Ab capable of Fas activation (CH11), 19-kDa ALG-2 protein appeared in Jurkat cells. The 19-kDa ALG-2 protein increased as time proceeded over a period of 4 hours after anti-Fas Ab treatment. PARP cleavage was observed under the same experimental conditions, which indicates that the caspase cascade was properly activated. The mouse monoclonal anti-ALG-2 Ab used for Western analysis was raised against the C-terminal

ALG-2 translocates from the cytoplasmic membrane to the cytosol during Fas-induced apoptosis. Confocal microscopic analysis was performed with anti-ALG-2 Ab using goat anti-mouse IgG-TRITC as a secondary antibody in Jurkat cells. ALG-2 was present predominantly in the cytoplasmic membrane without anti-Fas Ab treatment (Fig. 2A). Most of the cells were stained with annexin V, an early marker for apoptosis, at 6 h after anti-Fas Ab treatment. At that time, the majority of ALG-2 was detected in the cytoplasm in the same cells stained with annexin V. ALG-2 remained in the cytoplasmic membrane in annexin-negative cells (see arrow) in which the apoptotic process has not been started. The fractionation experiment showed the enrichment of 19-kDa ALG-2 in the cytosol and the decrease of 19-kDa protein in the membrane after 6 h of 200 ng/ml of CH11 anti-Fas Ab treatment (Fig. 2B). According to the fractionation data, ALG-2 was also detected in the nucleus. PARP was stained as the nuclear marker. ALG-2 interacts with Fas. Based on the confocal microscopy data, i.e., ALG-2’s presence in the plasma membrane, we questioned if ALG-2 is bound to Fas, an apoptotic component in the plasma membrane. Twohybrid assay was performed with the death domain of human Fas fused to the DNA-binding protein, LexA, and ALG-2 fused with B42 activation domain (Fig. 3). ALG-2 interacted with Fas in yeast and the interaction was specific because the single amino acid point mutant of Fas, Fas lpr, failed to interact with ALG-2. Since ALG-2 has demonstrated differential binding characteristics depending on the presence of calcium ions (13, 17), we investigated the influence of calcium ions upon ALG-2 and Fas binding. GST-ALG-2 fusion protein pulled down in vitro translated Fas with 2 nM, 2 ␮M and 2 mM of Ca 2⫹ but not without added Ca 2⫹ (Fig. 4A). To learn if the PEF domain is sufficient to allow the binding, GST-ALG-2(-23N) which has deleted N-terminal 23 amino acids was tested (Fig. 4B). GST-ALG-2(-23N) showed similar intensity of binding with Fas implying that PEF is sufficient for the binding. Both the N-terminal half of ALG-2 which contains two proximal EF domains (ALG-2-N) and the C-terminal half of ALG-2 with three distal EF domains (ALG-2-C) were able to interact with Fas even though with markedly reduced intensity. These data show that the interaction of ALG-2 and Fas is Ca 2⫹-dependent and the PEF domain is sufficient to allow the binding.

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FIG. 2. Subcellular localization of ALG-2 in Jurkat cells. Jurkat cells were treated with or without 200 ng/ml of CH11 anti-Fas Ab and analyzed by confocal microscopy (A) and subcellular fractionation (B) for 6 h. (A) Confocal images of Jurkat cells stained with anti-ALG-2 Ab followed by goat anti-mouse IgG-TRITC. For the detection of annexin V, cells were stained with FITC-conjugated annexin V, and subjected to confocal laser scanning microscope. The arrow indicates annexin-negative cell. Red indicates ALG-2, green indicates annexin V. (B) Each fraction from Jurkat cells were prepared as described under Materials and Methods. Subcellular fractions were noninduced (⫺) or induced (⫹) by CH11 anti-Fas Ab. M, membrane; C, cytosol; N, nucleus. Numbers on the left represent molecular weights in kDa.

ALG-2 dissociates from Fas upon Fas activation in Jurkat cells. An immunoprecipitation experiment was performed with anti-APO-1 Ab (30) in Jurkat cells with or without Fas activation in order to confirm the interaction in Jurkat cells. Endogenous ALG-2 was present in the immunoprecipitates of resting Jurkat

cells (Fig. 5A). Time courses of caspase-8 and PARP processing are shown (Fig. 5B). Endogenous ALG-2 was present in the immunoprecipitates of Jurkat cells which received up to 1 h of CH11 anti-Fas Ab treatment. However, endogenous ALG-2 was not detected in the immunoprecipitates of Jurkat cells treated with CH11 anti-Fas Ab more than 2 h. This result agrees well with the confocal microscopy data in which ALG-2 moves out of the plasma membrane after Fas activation and with subcellular fractionation experiment data in which ALG-2 in the membrane fraction decreased significantly. DISCUSSION

FIG. 3. Interaction between ALG-2 and Fas in yeast. The yeast two-hybrid system was used to test the interaction between ALG-2 and Fas. ALG-2 was fused to the B42 activation domain. The death domain of human Fas and Fas lpr were fused Lex A DNA-binding domain. The cotransformants with pJG4-5/TRADD and pLexA/ FADD in EGY48 as positive control and those of pJG4-5/Daxx and pLexA/FADD as negative control were streaked on SD medium containing X-gal.

Failure in executing apoptosis upon transfection of antisense ALG-2 implies that ALG-2 is essential for apoptosis (4). Several groups searched for the function of ALG-2 during the execution of apoptosis. However, no data revealed the effector function of ALG-2 during apoptosis, if any, and presented present direct molecular link of ALG-2 to any well-known apoptotic component. This study, for the first time, presents data on a

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FIG. 4. Ca 2⫹-dependent interaction between ALG-2 and Fas in vitro. (A) Effect of calcium ions on the interaction in vitro between ALG-2 and Fas. GST-fusion recombinant proteins were purified and immobilized on glutathione Sepharose 4B beads. The beads were incubated at 4°C overnight with 35S-Fas protein in binding buffer containing no added Ca 2⫹ or different concentrations of Ca 2⫹. After extensive washing, the mixtures were resolved on a 15% SDS– polyacrylamide gel. (B) Binding of full-length and truncated GST fusion recombinant proteins of ALG-2 to 35S-Fas. GST-ALG-2-N and GST-ALG-2-C contain N-terminal 95 amino acids (a.a. 1–95) and C-terminal 96 amino acids of ALG-2 (a.a. 96 –191), respectively. GST-ALG-2(-23N) contains a.a. 24 –191. Numbers on the left represent molecular weights in kDa. (C) Schematic diagram of ALG-2 deletion constructs used in this experiment. The shaded boxes represent the N-terminal hydrophobic domains and the black boxes indicate EF-hand motifs.

direct molecular link of ALG-2 to Fas, an important member of apoptotic machinery. ALG-2 interacts with Fas prior to Fas activation and dissociates from Fas during Fas-mediated apoptotic process in Jurkat cells. In addition to the dissociation from Fas, disappearance from the cytoplasmic membrane and the cleavage of ALG-2 occurs after Fas activation.

ALG-2 binds to Fas in vitro in the presence of nanomolar concentration of Ca 2⫹, which is equivalent to the intracellular Ca 2⫹ concentration in resting cells. However ALG-2 binds to Fas at a micromolar to millimolar concentration of Ca 2⫹. This leads us to think that the change of Ca 2⫹ concentration during apoptosis might not be the cause for the dissociation of ALG-2 from Fas. The fact that deletion of N-terminal 23 amino acids of ALG-2 did not weaken Fas binding implies that PEF is sufficient for Fas binding. However, Fas binding was significantly weakened when the N- and C-terminal halves of ALG-2 were used. This result suggests that N-terminal and C-terminal EF domains act synergistically for the binding. The D’Adamio group has suggested that ALG-2 might act downstream of caspase 3 based on the fact that apoptotic execution failed even though caspase 3 was properly activated in antisense ALG-2 transfected cells (10). Cleavage of PARP and ALG-2 occurs at similar times during Fas-mediated apoptosis. Our preliminary data shows ALG-2 cleavage does not occur when caspase 3 inhibitor was administered (data not shown). Thus ALG-2 cleavage seems to be a downstream event of caspase 3 activation. If ALG-2 must be cleaved in order to execute apoptosis, our data would agree with the suggestion by the D’Adamio group. At present, we do not know if ALG-2 is a direct substrate of caspase 3 or is a substrate of another protease activated by caspase 3. Experiments to answer this question are underway. Our data suggests that the ALG-2 cleavage might occur at its N-terminus and would generate protein product downsized by 3 kDa. The consequence of this cleavage would remove the cluster of 16 hydrophobic amino acids stretch in the N-terminus (a.a. 8 –23; PGPGAGPGPAAGAALP). The N-terminal regions of the other members of the PEF-domain family, such as calpain small subunit, grancalcin, sorcin and peflin, are rich in hydrophobic residues as well (6, 31). In the case of the calpain small subunit, it has been suggested that these hydrophobic amino acids might serve as a transmembrane domain or as an anchor to cytoplasmic membrane (32, 33). If the hydrophobic residues of ALG-2 accomplish a similar role, it is possible that cleavage releases ALG-2 from the cytoplasmic membrane and result in its dissociation from Fas which is present in the membrane. Confocal microscopic analysis demonstrated that the majority of ALG-2 is present in the cytoplasmic membrane of Jurkat cells prior to Fas activation. Fas activation caused intracellular translocation of ALG-2 from the cytoplasmic membrane to the cytoplasm. The confocal data corresponds well with subcellular fractionation data. However, a discrepancy exists in the subcellular localization of ALG-2. There has been a report that ALG-2 is present essentially in the cytosolic fraction of L66 cells (13). Kitaura et al. (2001) showed

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FIG. 5. Dissociation of ALG- 2 from Fas upon Fas activation in Jurkat cells. (A) Total cell lysate of Jurkat cells which received 10 min to 6 h treatment of CH11 anti-Fas Ab were immunoprecipitated with another anti-Fas Ab (anti-APO-1) which is capable of immunoprecipitation. After electrophoresis on a 15% polyacrylamide gel, proteins were transferred on PVDF membrane and analyzed by staining with anti-ALG-2 monoclonal Ab. Mouse immunoglobulin chains of anti-APO-1 Ab in the immunoprecipitates were also detected by alkaline phosphatase-conjugated anti-mouse IgG used as a secondary Ab. Numbers on the left represent molecular weights in kDa. (B) Time course of caspase-8 and PARP processing. Kinetics of caspase processing was followed using Western blot immunoblotted with anti-caspase-8 and PARP antibodies, respectively. The migration position of full-length caspase-8 which exists as two isoforms, the cleavage products p43 and p41, full-length PARP and the cleavage product p85 is indicated.

nuclear as well as cytoplasmic presence of ALG-2 in resting Jurkat cells (17). Krebs and Klemenz (2000) showed nuclear localization of ALG-2 in breast cancer cells and observed a significant concentration of ALG-2 in cells prior to cell division and subsequent disappearance of the nuclear localization at the onset of mitosis (16). Venn and Conway observed predominant neuronal distribution of ALG-2 in the brain (34). These data suggest that the subcellular localization of ALG-2 might vary depending on the cell types and their physiological status. However, the disagreement between our data and that of Kitaura et al. (2001) on the subcellular localization of ALG-2 in Jurkat cells is worth mentioning. One possible explanation might come from the specificity differences between ALG-2 antibodies. Kitaura et al. mentioned in their paper the possibility that their antibody might recognize only a fraction of ALG-2 that retains a specific conformation favoring interaction with peflin and poorly recognizes ALG-2 monomers and homodimers under undenatured conditions (17). The antibody used for Western analysis in this study was monoclonal Ab whose immunogen was C-terminal amino acids (a.a. 162–181) of ALG-2. Our antibody might recognize a different subset of ALG-2 whose conformation is different from those interacting

with peflin. It is possible that there exist heterogeneous populations of ALG-2 with different conformations which have different binding characteristics and therefore different functions. Considering the fact that Fas bound ALG-2 dissociates from Fas upon Fas activation, two possibilities arise. First, the release of ALG-2 from Fas might free Fas so that it facilitates Fas interacting with other signal transducers and to modulates the apoptotic signal. Second, ALG-2 can exert its effector function by dissociating from Fas. Both possibilities are not mutually exclusive. The increase of intracellular calcium is directly coupled to apoptosis. ALG-2 has been suggested as the first example of a Ca 2⫹ regulated molecule upon apoptotic execution (35). The identification of a Fas/ALG-2 connection would accelerate the understanding of a molecular aspect of Fas signaling as well as the function of ALG-2. ACKNOWLEDGMENTS The authors thank Dr. Peter H. Krammer at the German Cancer Research Center who generously provided the Fas Ab, anti-APO-1. This work was supported by the Korea Research Foundation (Grant 1999-015-D10077).

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