From the Department of Biochemistry and
§ Department of Cell Biology, Parke-Davis Pharmaceutical
Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, the ¶ Department of Molecular Biology, the
Department of Biochemistry, and the
Department of Immunology, BASF Bioresearch Corporation,
Worcester, Massachusetts 01605, and the ** Department of Pathology,
University of California at San Diego,
La Jolla, California 92093
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ABSTRACT |
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Although the available evidence suggests
that whereas the caspase family plays a major role in apoptosis, they
are not the sole stimulators of death. A random yeast two-hybrid screen
of a lymphocyte cDNA library (using caspase-3 as the bait) found an
interaction between caspase-3 and the regulatory subunit A of
protein phosphatase 2A. This protein was found to be a substrate for
caspase-3, but not caspase-1, and could compete effectively against
either a protein or synthetic peptide substrate.
In Jurkat cells induced to undergo apoptosis with anti-Fas antibody,
protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory A subunit could no longer be
detected in cell lysates. There was no change in the amount of the
catalytic subunit. The effects on PP2A could be prevented by the
caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the
phosphorylated forms of MAP kinase was observed. Again, this loss of
activated MAP kinase could be prevented by the addition of DEVD-cho or
DEVD-fmk. These data are consistent with a pathway whereby induction of
apoptosis activates caspase-3. This enzyme then cleaves the regulatory
A
subunit of PP2A, increasing its activity. These data show that the
activated PP2A will then effect a change in the phosphorylation state
of the cell. These data provide a link between the caspases and signal
transduction pathways.
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INTRODUCTION |
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A role for the caspase family in apoptosis was discovered when a
gene of Caenorhabditis elegans was shown to be required for apoptosis during development in this organism. Additionally, this gene
was found to have a protein sequence that was homologous to caspase-1.
When either ced-3 or the cDNA for caspase-1 was injected into rat-1
fibroblasts, the cells underwent programmed cell death (1). This
finding opened the field of caspase biology to more than just the
processing of pre-interleukin-1. Since then, many more members of
the caspase family have been cloned, and their essential roles in
apoptosis are beginning to be elucidated. Several mechanisms by which
the caspases elicit their effect in apoptosis can be postulated. These
mechanisms include proteolytic cleavage and inactivation of key
substrates involved in maintenance of DNA, the cell cycle, or
structural elements.
After caspase-1, caspase-3 is the most extensively studied of all the caspase family members and may be one of the more downstream apoptotic effector molecules (2). One theory to summarize the role of caspase-3 in apoptosis revolves around the premise that this enzyme can cleave key enzymes involved in DNA repair. These enzymes include poly-(ADP)ribose polymerase and DNA-dependent protein kinase. This cleavage then renders these repair enzymes inactive (3-8). But because PARP1 knock-out mice (9) and severe compromised immunodeficient mice lacking the active DNA-PK catalytic subunit can develop normally, it can be concluded that inactivation of these enzymes is not the only requirement for apoptosis.
Caspase-3 has been demonstrated to cleave additional substrates during cellular apoptosis such as the U1 70-kDa small nuclear ribonuclear protein (which is involved in RNA splicing) (10), structural proteins such as fodrin, non-heme spectrin (11), lamin (12), gas 2 (13), and cell kinases, such as retinoblastoma-associated protein Rb (14). The role of these substrates in apoptosis remains unclear. It may be necessary for each caspase family member, with their different substrate specificities, to cleave many essential substrates in a cooperative fashion for cell death to occur. However, it is apparent that caspase-3 plays an essential role in apoptosis since caspase-3 knock-out mice have a problem with apoptosis and development in the brain (15). Like caspase-1, other family members may have nonapoptotic roles in the cell as well.
Although the available evidence suggests that the caspase-1 homologs play a major role in apoptosis, they are not the sole stimulators of death. Fraser et al. (16) concluded that almost everything, including oncoproteins, tumor suppressor proteins, cytokines, and signaling proteins, seems to both induce and suppress apoptosis. However, little is known about how all of these pathways are connected. Protein phosphorylation provides the cell with a basis for the control of growth, metabolism, differentiation, and possibly programmed cell death. The phosphorylation state of cells relies on a very complex but carefully orchestrated set of kinases and phosphatases. The phosphatases are broadly classified into two groups, those preferring phosphorylated tyrosine and those preferring phosphorylated serine or threonine. The serine/threonine phosphatases are grouped into classes based on their substrates and sensitivity to inhibitors. The functions of these protein phosphatases in the cell are extensive. It has been reported that the Ser/Thr phosphatases play roles in metabolism, meiosis, mitosis, and the cell cycle (17). It has been suggested that these phosphatases also play a role in apoptosis (18).
Song and Lavin (19) demonstrated that the inhibitors of protein phosphatases 1 and 2, calyculin A and okadaic acid, could inhibit apoptosis in the irradiated Burkitt's lymphoma cell line BM13674. Recently, Morana et al. (20) have demonstrated that regulation of protein phosphatase 1 activity is essential in regulating apoptosis via activation of a caspase-1/CED-3 protease, intracellular acidification, and DNA digestion. With regard to the caspase-phosphatase interaction, they demonstrated that incubation of ML-1 cells with 1 µM okadaic acid inhibited DNA fragmentation and caspase-induced cleavage of PARP in cells treated with etoposide. Thus, a plausible link between cell cycle stimulators of death and the caspases may be found in signaling pathways via the serine/threonine phosphatases.
Protein phosphatase 2A (PP2A), the most abundant
serine/threonine-specific phosphatase in mammals, plays a role in many
fundamental cellular processes, including cell division, signal
transduction, gene expression, and development. PP2A consists of three
subunits, the catalytic C subunit and the 65-kDa regulatory A subunit,
which together form the core enzyme, and the regulatory B subunit,
which binds to the core enzyme yielding the holoenzyme. The A and C subunits both exist as two isoforms ( and
) and the B subunit as
multiple isoforms, which are subdivided into three families, B, B', and
B", unrelated to each other by primary sequence. The A subunit
polypeptide consists of 15 nonidentical repeats that form a rod-shaped
molecule. The B subunit binds to repeats 1-10 and the C subunit to
repeats 11-15 of the A subunit. Binding of the C subunit to the A
subunit occurs in the absence of the B subunit, whereas binding of the
B subunit requires the presence of both the A and C subunit for
stability (17). The purpose of the studies reported here was to
demonstrate an interaction between caspase-3 and the Ser/Thr
phosphatases.
The yeast two-hybrid system (21) has been used to find unknown protein ligands that bind with known receptors. The data presented here will demonstrate that the yeast two-hybrid system can also be used to detect an interaction between an enzyme and a substrate and thus was used to find a new putative substrate for caspase-3. This interaction provides a link between the cell cycle, metabolic control, and the tumor necrosis factor/FAS-derived death pathways.
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MATERIALS AND METHODS |
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Yeast Two-hybrid Screen-- A yeast two-hybrid screen was performed according to the method of Field and Song (22) utilizing the Matchmaker Two-hybrid System (CLONTECH). The yeast strains used in the screen were HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::(GAL 17-mers)3-CYCI-lacZ) as the primary strain and Y190 (MATa, ura3-52, his3-D200, ade2-101, trp1-901, leu2-3, 112, gal4Dgal80D, LYS2::GAL1-HIS3, URA3::(GAL-lacZ, cyhr2, LYS2::GAL-HIS3CYCI-lacZ) as the secondary screening strain. Yeast were grown in YPD media until transformation. After transformation the yeast were maintained in the appropriate selection media.
The p29 form of caspase-3Expression of Caspase-3 and Caspase-1-- The His-tagged, 29-kDa form of caspase-3 was expressed from the cDNA cloned into the E. coli expression vector pMCH-1 as described previously (24). The vector was maintained in the host strain MM294A and propagated at 28-30 °C to avoid induction of the protein. Cells were grown in a 2-liter fermentor in Superbroth (Digene Diagnostics Inc., Beltsville, MD) supplemented with 100 µg/ml ampicillin and 50 µg/ml kanamycin to an optical density at 600 nm of 9. Expression of the protein was induced by rapidly raising the temperature to 40 °C for 100 min. Full-length caspase-1 was cloned into the E. coli expression vector pMCH-1, maintained in MM294A, and expressed as above.
Purification of the His-tagged caspase-3 was accomplished by resuspending the pelleted cells in 25 ml of buffer containing 50 mM HEPES, 0.2 M NaCl, 10% glycerol, 0.1% CHAPS, 0.2 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, and 10 µM pepstatin at pH 7.5. The cell suspension was stirred at 4 °C until smooth and then lysed in a French press at 18,000 p.s.i. The lysate was cleared by centrifugation at 31,000 × g for 30 min and at 4 °C. The supernatant was stirred with an equal volume of lysis buffer and loaded onto a column packed with 10 ml of Ni-NTA resin (Qiagen, Chatsworth, CA) that had been previously equilibrated in lysis buffer. The column was washed with lysis buffer containing 5 mM Imidazole and then again with lysis buffer containing 25 mM Imidazole. The protein was then eluted with five, 10-ml aliquots of lysis buffer containing 125 mM Imidazole. All fractions were collected and analyzed by SDS-PAGE before pooling peak fractions and quantitating protein concentration and enzyme activity. The enzyme was stored frozen atEnzymatic Activity Assays-- Caspase-3 activity was assayed by monitoring the release of p-nitroaniline from the synthetic substrate Ac-DEVD-pNA at 380 nm. The total reaction volume was 200 µl and contained HGE buffer (100 mM HEPES, pH 7.4, 20% glycerol, 0.1 mM EDTA), 5 mM DTT, 0.05% BSA, 100 µM Ac-DEVD-pNA, and 5 nM caspase-3. Inhibition of caspase-3 activity by PP2A was measured by varying the concentration of PP2A holoenzyme (Upstate Biotechnology Inc., Lake Placid, NY) in each of the wells of a microtiter dish. The reactions were initiated by the addition of the caspase-3. The assays proceeded for 30 min and were linear throughout the entire time course.
Inhibition of Caspase-3-mediated PARP Cleavage by PP2A-- The cDNA for PARP, contained on the T7 driven expression vector pKV, was in vitro transcribed and translated using the TNT T7-coupled reticulocyte lysate system (Promega Corp., Madison, WI) according to the manufacturer's protocol. [35S]Methionine (1000 Ci/mmol, Amersham Pharmacia Biotech) was substituted for cold methionine in the reaction and resulted in the production of approximately 15 µg of 35S-labeled PARP. The labeled PARP was diluted 75-fold with HGE and used without further purification. In a 20-µl reaction, 15 µl of diluted PARP was mixed with 2 µl of caspase-3 (to give a final concentration of 20 nM caspase-3), 0.5 mg/ml BSA in HGE containing various concentrations of PP2A in a total of 3 µl of PP2A buffer (50 mM Tris, 50% glycerol, 0.1 mM EDTA, 0.1% 2-mercaptoethanol, pH 7.5). The reactions were incubated at 30 °C for 30 min and quenched by addition of an equal volume of 2× SDS-PAGE sample buffer (Integrated Separation Systems, Natick, MA) and then heated for 10 min at 95 °C. The quenched reaction was loaded onto a 10-20% denaturing acrylamide gradient gel and electrophoresed in a Tris-Tricine buffer system (Novex, San Diego, CA). The gel was dried and imaged using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the cleavage products were quantitated by densitometry using the PhosphorImager software. Data are expressed as a percent of the amount of the 89- or 25-kDa products found in the control reaction with no PP2A.
Cleavage of PP2A by Caspase-3--
PP2A holoenzyme (1 µg),
isolated from rabbit skeletal muscle (Upstate Biotechnology Inc, Lake
Placid, New York), or 10 µg of recombinant A subunit (26) was
incubated in 100 µl of modified HGE buffer (containing only 5%
glycerol) and 5 mM DTT and initiated by the addition of 40 nM caspase-3 or caspase-1 (as a negative control) at time 0 or after a 15-min preincubation of the phosphatase. Control reactions
either minus PP2A or minus caspase-3 were also included. The
incubations were carried out at 37 °C for 60 min, and the reaction
was terminated by the addition of 5 volumes of acetone. The samples
were chilled at 20 °C for 2 h and then centrifuged at
14,000 × g for 30 min. The samples were resuspended in
20 µl of 2× SDS-PAGE sample buffer, electrophoresed as above, and
transferred to nitrocellulose or PVDF membranes. For immunostaining,
nitrocellulose membranes were blocked with a mixture of nonfat dried
milk and bovine serum albumin (2% of each) in phosphate-buffered
saline that contained 0.05% Tween 20. The PP2A regulatory A subunit
was visualized by probing with a 10,000-fold dilution of the monoclonal antibody 6G3, followed by enzyme-linked chemiluminescence. Membranes were washed with phosphate-buffered saline that contained 0.05% Tween
20. This antibody was previously demonstrated to recognize the 15th
repeat in the C-terminal portion of the regulatory A subunit (27). For
N-terminal sequencing, the PVDF membranes were stained with Coomassie
Blue, and the 42-kDa band was excised and sequenced by the Parke-Davis
protein sequencing facility.
Phosphatase Assays-- PP2A activity was determined in one of two ways. The effect of caspase-3 on purified PP2A activity from human red blood cells was determined using a Ser/Thr phosphatase assay kit (Upstate Biotechnology Inc, Lake Placid, New York) according to the directions supplied by the manufacturer. In brief, PP2A holoenzyme was incubated in 25 µl of modified HGE buffer (containing only 5% glycerol), 5 mM DTT, and 0.05% BSA and either 40 nM caspase-3 or caspase-1 (as a negative control). In addition, a reaction was done with DEVD-cho-bound caspase-3. Caspase-3 (1 µM) was incubated with 1 nM DEVD-cho for 15 min prior to addition of 1 µl of the enzyme to the 25-µl total reaction volume. The assay was initiated by the addition of phosphorylated hexapeptide substrate and quenched at time 0-20 min by the addition of an acidic malachite green solution. The green color was quantitated spectrophotometrically at 650 nm against a phosphate standard curve.
The second in vitro phosphatase assay used for the detection of phosphatase activity in cell lysates was performed by the method of Cohen et al. (28) and involved the quantitation of trichloroacetic acid-soluble 32Pi released from 32P-labeled phosphorylase a. Treated cells were harvested from a 96-well tissue culture dish by centrifugation and then snap-frozen in liquid nitrogen. The cell pellets were resuspended in 100 µl of assay buffer that contained 50 mM HEPES, pH 7.2, 2 mM EDTA, 2 mg/ml glycogen, 2% 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 µg/ml aprotinin and then lysed by sonication. Duplicate assays were run in the presence and absence of 3 nM okadaic acid. The 10-min reactions were initiated by the addition of [32P]phosphorylase a and quenched by the addition of 90 µl of ice-cold 20% trichloroacetic acid. Samples were chilled for 10 min, and the insoluble protein was cleared by centrifugation at 14,000 × g for 2 min. Half of the soluble fraction was counted in a scintillation counter. All experiments were repeated three times. PP2A activity was determined by subtracting the counts from the okadaic acid-insensitive fraction from the total counts in the soluble fraction. In these experiments, at least 95% of all measured phosphatase activity was due to PP1 and PP2A activity as determined in a preliminary experiment using 500 nM okadaic acid which will totally inhibit both PP1 and PP2A (29).Apoptosis Assays-- Jurkat cells were seeded at a concentration of 1 × 106 cells/ml and grown overnight in RPMI 1640 media supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (10,000 units/ml and 10,000 µg/ml, respectively). Cells were harvested by centrifugation and aliquoted into a 96-well tissue culture dish at a density of 5 × 105 cells per 200 µl. Cells were treated with anti-Fas antibody (63 µg/ml) (Upstate Biotechnology Inc., Lake Placid, NY) with or without DEVD-fmk, FA-FMK (Enzyme Systems Products, Dublin, CA), or DEVD-cho (Bachem Biosciences Inc., King of Prussia, PA) added at time 0, or at various times after anti-Fas addition. Samples were harvested at various times after the addition of anti-Fas antibody. Cells were analyzed for protein phosphatase activity, as described above, and the ability to metabolize the dye AlamarBlue (Alamar Bio-Sciences, Sacramento, CA). After incubation, 10% volume of AlamarBlue dye was added to each well of the 96-well plate. The plates were incubated for 6 h at 37 °C. The reaction product was monitored at an excitation wavelength of 584 nm and an emission wavelength of 612 nm, on a fluorometric plate reader (Molecular Devices, Sunnyvale, CA).
Cell lysates were also examined for the presence of PP2A and MAP kinase. In brief, cells from two independent wells were pooled and harvested by centrifugation at 1000 × g for 2 min. The cells were resuspended in 2× SDS-PAGE buffer, and proteins from 106 cells were fractionated on denaturing 10-20% Tris-Tricine polyacrylamide gels. Proteins were transferred to nitrocellulose or PVDF (NOVEX, San Diego, CA) membranes and probed as described above for protein phosphatase 2A using an N-terminal recognizing antibody (Upstate Biotechnology, Lake Placid, NY). Total MAP kinase (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and active MAP kinase (Promega Inc., Madison, WI) primary antibodies were used at a dilution of 1:20,000. Visualization of the proteins was accomplished by enzyme-linked chemiluminescence (Amersham Pharmacia Biotech). Quantitation of imaged bands was performed with Bio-Rad Molecular Analyst image analysis software (Bio-Rad). ![]() |
RESULTS |
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Yeast Two-hybrid Screening--
In a study by Estojak et
al. (30), it was demonstrated that protein interactions with
dissociation constants as weak as 1-20 µM could be
detected by yeast two-hybrid methodology. Recently, Margolin et
al. (31) determined that the Km of pre-IL-1 for caspase-1 is 4 µM. Based on these data, an attempt
was made to identify substrates for the members of the caspase family
with the yeast two-hybrid system. Positive interactions in the yeast two-hybrid system activate transcription of the His3 and
LacZ genes. p32 caspase-1 and p29 caspase-3 were expressed
in the pGBT9 vector as the mature Gal4 binding domain fusion protein
and used as the bait in separate screens. Gal4 binding domain fusion
proteins were screened against Gal4 activating domain fusion libraries as well as with themselves cloned into activating domain vectors. Transformation with caspase-1- and caspase-3-containing vectors produced no toxic effects and would routinely transform HF7C with an
efficiency between 104 and 1.5 × 105
colonies/µg DNA. Cotransformation frequencies of caspase and random
libraries varied but were usually between 103 and 2 × 104 colonies/µg DNA.
Confirmation of an in Vitro Interaction between Caspase-3 and PP2A-- The in vitro cleavage of purified PP2A was examined utilizing catalytic quantities of purified, recombinant caspase-3. Since the amount of activated caspase-3 in the cell is probably small, it would not be relevant to use large quantities of enzyme which might artificially cleave any DXXD motif. Therefore, all of the in vitro studies in this report used concentrations of caspase-3 in the range of 4-40 nM.
Caspase-3 Cleaves PP2A--
To examine the ability of caspase-3 to
cleave PP2A, 40 nM caspase-3 was incubated with PP2A for
1 h. Caspase-1 was used as a negative control, since this caspase
family homolog prefers a hydrophobic amino acid in the p4 pocket and
thus should not cleave the regulatory subunit A at this site. In
preliminary experiments, it was determined that maximal cleavage of
PP2A by caspase-3 was observed within 2 h. In addition, it was
observed that the order of addition of the phosphatase and the protease was important. Thus the phosphatase was preincubated for 15 min in the
absence of caspase. When visualized with antibodies directed against
the 65-kDa regulatory subunit, a 42-kDa C-terminal piece of the
regulatory subunit could be observed (Fig.
1). The 42-kDa piece is the correct
calculated size for the C-terminal fragment of the regulatory subunit.
This cleavage is consistent with cleavage occurring at the putative
cleavage site, DEQD
S. The regulatory subunit of PP2A purified from
rabbit skeletal muscle also cleaves at this same point (data not
shown), thus the cleavage site is present in at least one other
species. N-terminal sequencing of the 42-kDa cleavage piece yielded the
sequence, 219SVRLLAVEACVNIAQ233, which confirms
the site of digestion of the A subunit by caspase-3. In addition
sequence analysis demonstrated that cleavage also occurred at the N
terminus of the protein after Asp8. Whereas preincubation
of the PP2A holoenzyme was necessary in order for appreciable cleavage
to occur, this was not true for the recombinant A subunit. The likely
reason is that DEQDS is covered by the regulatory B subunit in the
holoenzyme that binds to repeats 1-10. Since caspase-3 can only cleave
the site when it is uncovered, the preincubation gives the B subunit
time to dissociate. Caspase-1 did not cleave the regulatory subunit
with or without the preincubation of PP2A.
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PP2A Competes as a Substrate in Two Caspase-3 Assay Systems-- In a caspase-3 assay which utilizes the synthetic substrate Ac-DEVD-pNA at a concentration of 100 µM (Km = 11 µM), 20 nM PP2A holoenzyme inhibited the hydrolysis of the peptide by 40%. This suggests that PP2A competes reasonably well as a substrate for caspase-3. In order to assess whether PP2A can compete against a protein substrate for caspase-3, the effect of PP2A on caspase-3 cleavage of the well characterized caspase-3 substrate, PARP, was examined. The cleavage of [35S]-PARP by caspase-3 was determined by densitometry following SDS-PAGE. The appearance of the 31-kDa PARP cleavage product was inhibited by 50% at 20 nM PP2A. This result is consistent with the results obtained from studies using Ac-DEVD-pNA as the substrate. These data indicate that PP2A competes reasonably well as a substrate for caspase-3 against either small synthetic peptide substrates or full-length protein substrates.
Caspase-3 Stimulates PP2A Activity--
If caspase-3 is indeed
cleaving the regulatory subunit A of PP2A, then an increase in PP2A
catalytic activity should be observed after incubation with caspase-3,
provided a suitable substrate is being used. In this assay, PP2A
activity was measured by the release of Pi from the
synthetic PP2A substrate KIpTIRR. The phosphatase activity assay was
linear over 30 min with this substrate (data not shown). Upon
incubation of 40 nM caspase-3 with PP2A for 20 min, a 40%
stimulation of PP2A activity over the basal level was observed (Fig.
2). This stimulation was not observed by
incubation with 40 nM caspase-1. Prebinding caspase-3 with
the inhibitor Ac-DEVD-cho inhibited the stimulation observed by
addition of caspase-3. The inhibitor did not influence phosphatase
activity by itself. It should be noted that even in the presence of the regulatory subunit, there is a significant basal level of phosphatase activity. These data are consistent with the cleavage and inactivation of a regulatory subunit A
by caspase-3.
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The Interaction between Caspase-3 and PP2A during Apoptosis-- In order to determine if the interaction between PP2A and caspase-3 is biologically relevant, a series of experiments in Jurkat T cells, induced to undergo apoptosis by ligation of the Fas receptor by anti-Fas antibody, were performed. Jurkat cells were used because caspase-3 is highly expressed and has been shown to be activated in cells of lymphocytic origin (37, 38). This suggests the potential for caspase-3 playing a pivotal role in cells induced to undergo apoptosis. The caspase-3 inhibitors DEVD-fmk and DEVD-cho were used to link the interaction of caspase-3 with PP2A in this cell-based system. YVAD-cho and FA-FMK were used as negative control peptides. The first set of peptides have been demonstrated previously to inhibit anti-Fas-induced apoptosis in Jurkat cells (39, 40). In these studies, apoptosis was monitored by metabolism of the dye AlamarBlue (41). Recently, Vasilakos et al. (42), demonstrated that cell death data obtained with AlamarBlue correlated well with data obtained in the same experiment by monitoring trypan blue exclusion as an index of cell viability. In addition, positive correlation between AlamarBlue and several other parameters of apoptosis, such as caspase-3 activation, DNA laddering, and nuclear condensation, were demonstrated.
The data presented in Fig. 3 are representative of three separate experiments. After 6, 12, and 24 h, cells treated with anti-Fas antibody (63 ng/ml) demonstrated a significant decrease in the ability to metabolize AlamarBlue dye (30, 50, and 70%, respectively) (Fig. 3A). This apoptosis was totally inhibited by 20 µM DEVD-fmk and 100 µM DEVD-cho at 6 and 12 h. Little protective effect was seen in cells incubated with the control peptide FA-FMK; thus, this is not a nonspecific effect of an irreversible modifying agent. Little decrease in the ability to metabolize the dye could be seen at the 3-h time point (data not shown). This suggests that in this model system, cell death begins between 3 and 6 h after treatment with anti-Fas antibody. By 24 h, DEVD-cho showed a decrease in its effectiveness at blocking apoptosis. Since DEVD-fmk is an irreversible inhibitor, it is possible that it is modifying other caspase family members inside the cell, which could account for the differential effectiveness between DEVD-cho and DEVD-fmk. In the absence of anti-Fas antibody, the inhibitors by themselves did not appear to have any negative effect on the ability of the cells to metabolize the dye. In fact at 6 h post anti-Fas treatment, the inhibitors actually appeared to significantly stimulate the metabolism of the AlamarBlue dye.
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DISCUSSION |
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These studies were stimulated by the desire to know and understand more about the biology and the natural substrates for several of the members of the caspase family. There have been many reports of substrates for caspase-3, with the most common one being PARP. The evidence supporting this, however, is indirect. During programmed cell death, the cell undergoes changes in morphology as well as metabolism. What has not been elucidated to date is the link between the caspase family and the signal transduction process that could lead to the changes observed in the cell during apoptosis. Since there is a delicate balance in the cell between life and death, it seems possible that any protein with a role in cell proliferation might also, if stimulated or destroyed, play a role in apoptosis.
A recent report by Martins et al. (47) indicates that
caspase-3 may be found in the nucleus of the cell during apoptosis. This translocation to the nucleus may be the method by which PARP is
cleaved. However, the cleavage of this DNA repair enzyme may not be
sufficient to cause the morphological and metabolic changes that the
cell undergoes during apoptosis. Indeed, mice that have had the PARP
gene deleted appear to develop normally, unlike the mice where
caspase-3 has been deleted (9). Ghayur et al. (48) reported
that caspase-3 cleaved protein kinase C-, which resulted in
up-regulation of its activity. Overexpression of the active catalytic
kinase fragment in cells is associated with chromatin condensation,
nuclear fragmentation, induction of sub-G1 phase DNA, and
lethality. These pieces of information would indicate that caspase-3
may interact in a pathway that is more central to cellular metabolism.
In order to answer the question of what additional cellular substrates
caspase-3 might interact with directly, a random yeast two-hybrid
screen was performed with caspase-3 acting as the bait.
Until now, substrates of caspase-3 have been identified mostly by indirect evidence indicating that they are cleaved during apoptosis (49). The best characterized interaction is that between PARP and caspase-3, detailed in recent publications by Margolin et al. (31) and Nicholson et al. (6). DNA-dependent protein kinase catalytic subunit has also been demonstrated to be cleaved by caspase-3 in the cell (8, 50). Additional putative substrates are comprehensively reviewed in a recent article by Schwartz and Milligan (51). The yeast two-hybrid system, however, is a random selection method that should be able to directly find proteins that interact with caspases.
When caspase-3 was used as the bait in several random screens, an
interaction between this protease and the regulatory subunit A of
protein phosphatase 2A was found. Upon examination of the amino acid
sequence for this protein, the recognition sequence for a caspase-3
cleavage site (DXXD) was found (DEQD specifically). This
sequence is similar to the cleavage sequence found in PARP (DEVD) and
many of the other putative caspase-3 substrates. This sequence is also
comparable to the optimal cleavage sequence suggested by the work of
Talanian et al. (36). Upon analysis of all entries in the
Entrez protein data base for the PP2A 65-kDa regulatory subunit A
,
the DXXD cleavage site is conserved in frog and pig as well
as in rabbit and human.
The data presented in this report demonstrate that PP2A is a substrate
for caspase-3. PP2A will compete with either small synthetic substrates
(like Ac-DEVD-pNA) or protein substrates (like PARP) for caspase-3.
Additionally, caspase-3 was shown to cleave the PP2A regulatory A
subunit (Fig. 1). This cleavage resulted in approximately a 40%
increase in phosphatase activity in vitro after 20 min. The
PP2A core and holoenzymes are similarly abundant in cells but differ in
substrate specificity (27). The holoenzyme is more active than the core
enzyme toward substrates phosphorylated by cyclin-dependent
kinases, and the core enzyme is more active against these substrates
than the free C subunit. Thus, with the cyclin-dependent
kinase substrates, the A and B subunits have a stimulatory effect on
the C subunit. On the other hand, the core enzyme is equally or more
active than the holoenzyme toward most other substrates, indicating
that the B subunit (B being the most frequently studied) is
inhibitory (52-56). With respect to core enzyme and free catalytic
subunit, the situation is also complex. Depending on the substrate
size, type of substrate, and reaction conditions, the A subunit either
inhibits or stimulates the catalytic subunit (57).
Since the various forms of PP2A differ so markedly in substrate specificity, it is important to discuss how accessible the core and holoenzymes are to cleavage by caspase-3. According to the previous model of PP2A (58, 59), the B subunit binding region on the A subunit includes the caspase-3 recognition sequence DEQD in repeat 6 of the A subunit. This was suggested by the finding that mutation of this sequence destroyed binding of the B subunit to the A subunit, whereas binding of the C subunit was intact (58). Therefore, we would predict that in holoenzyme, the A subunit is protected by the B subunit from cleavage by caspase-3, whereas in core enzyme it can be cleaved. This explains the observation that cleavage of the holoenzyme, but not of free A subunit, by caspase-3 required preincubation, resulting in dissociation of the B subunit from the holoenzyme and generation of core enzyme. The finding that the A subunit completely disappeared from cells after induction of apoptosis, whereas C subunit was stable, suggests that holoenzyme was slowly converted to core enzyme and that all core enzyme was cleaved by caspase-3. It may not be surprising that the generation of a high amount of free C subunit, which normally does not exist in cells, has dramatic effects on cell growth, signal transduction, and apoptosis.
In order to answer the question of relevancy in the cell, we examined the activity of phosphatases during anti-Fas antibody-induced apoptosis in Jurkat cells. The ability of several caspase family inhibitors to block apoptosis was correlated with their ability to block the increase in PP2A activity. These data show that by 6 h of anti-Fas treatment, cells had reduced ability to metabolize AlamarBlue and PP2A activity had increased 4.5-fold when compared with untreated control cells. In fact, after 6 h, cells treated with only inhibitor demonstrated a significant increase in their ability to metabolize the dye. This suggests that these cells may have a basal amount of apoptosis which is inhibited by the caspase-3 inhibitors. The fact that this increase in phosphatase activity is completely inhibited by both DEVD-fmk and DEVD-cho suggests the involvement of caspase family members in this up-regulation of PP2A activity. In this cell model, PP1 activity was not up-regulated by either anti-Fas or inhibitor.
These data partially agree with the data of Morana et al. (20), demonstrating that several inhibitors of serine/threonine phosphatases, including okadaic acid, will also completely inhibit etoposide-induced apoptosis. However, they concluded that a decrease in phosphorylation of a PP1 substrate, Rb protein, during apoptosis was not due to up-regulation of PP1 activity. They did not observe an increase in PP2A activity either. In addition they did observe a decrease in the level of PP1 protein during apoptosis without subsequent loss of PP1 activity. It is possible that the one time point at which they examined phosphatase activity was insufficient, since this is likely a temporal response and relies on the model system being tested. In fact, in the studies reported here, by 24 h after anti-Fas administration, the PP2A activity was not significantly elevated compared with control. Song and Lavin (19) have also demonstrated that inhibitors of PP1 and PP2A (calyculin A and okadaic acid) will prevent apoptosis in a Burkitt's lymphoma cell line. The authors concluded that there was a role for up-regulation of phosphatase activity in apoptosis. Wolf et al. (60) reported a correlation between Rb dephosphorylation as an index of phosphatase activity and apoptosis. In that report, PARP cleavage, intracellular acidification, and DNA digestion, all indicators of apoptosis, were inhibited by the caspase family inhibitor, benzyloxycarbonyl-VAD-fmk. However, Rb dephosphorylation was not prevented by this same inhibitor. This observation suggested that the phosphatases play a role upstream of the caspases. However, since VAD-fmk is not a specific caspase-3 inhibitor, this does not rule out the ability of caspase-3 to affect directly or indirectly Rb protein and thus lie upstream of the phosphatases. Although many reports have suggested the connection between phosphatases and caspase family members, no direct link has been identified. Therefore, the interaction between caspase-3 and PP2A is significant.
Analysis of cell lysates by antibodies directed to the PP2A regulatory A subunit demonstrated the loss of this subunit after 12 h. In inhibitor-treated cells, there was no loss of this protein subunit. At the same time, there was no decrease in the amount of the catalytic subunit. These data strongly indicate that the up-regulation of PP2A activity is due to removal of the regulatory A subunit. Cleavage and subsequent proteolytic degradation is one explanation for these data. However, due to the lack of visualization of a cleavage fragment, other mechanisms cannot be ruled out. It also suggests that the catalytic subunit is stable in cells in the absence of the regulatory A subunit.
If the effect on PP2A activity observed has biological relevance, a corresponding change should be seen in the phosphorylation state of a PP2A substrate. The caspase-3 inhibitable decrease in the phosphorylation state of only the activated MAP kinase satisfies this requirement. In a recent report by Cardone et al. (61), it was demonstrated that caspases were required to activate apoptosis via the JNK pathway, by cleavage and activation of MEKK-1. These data, in combination with the data reported here, provide convincing support for the involvement of caspases in the regulation of cell signaling events. Caspases appear to work in concert to turn on the stress-activated and turn off the growth factor-activated cell signaling pathways.
Involvement of PP2A in apoptosis would directly link two lines of
evidence suggesting that both regulation of the cell cycle and the
caspase family play a role in apoptosis. This would also suggest an
upstream role in the induction of cell death. Recently, Dou et
al. (62) have reported that induction of a protein
serine/threonine phosphatase is responsible for the anti-cancer
drug-induced Rb hypophosphorylation and consequent G1
arrest and apoptosis in two p53-null human leukemic cell lines, HL60
and U937. Cells unable to hypophosphorylate Rb protein were resistant
to drug-induced cell death. In a subsequent paper, An and Dou (63)
reported that hypophosphorylated Rb protein is cleaved during DNA
damage-induced apoptosis by a caspase-3-like protease. These data
strongly suggest a relationship between the caspase-3-like subfamily
members and protein phosphatases at both the level of dephosphorylation
and cleavage of dephosphorylated substrates. It is intriguing to note that many of the caspase-3 substrates have cleavage sites containing a
serine either in or near the P1' position of the cleavage sequence. These substrates include the regulatory subunit of PP2A, (DEQDS), Rb
protein (DSIDS and DEADGS), protein kinase C (DMQDNS), and GDP
dissociation inhibitor protein (DELDS).
The interaction between PP2A and caspase-3 may provide a partial explanation for the phenotype of the caspase-3 knock-out mouse, since the proportion of PP2A in brain tissue is much higher in the developing brain than in the adult brain (64). Thus the interaction between caspase-3 and PP2A may be more important in developing brains than in other tissues.
In summary, this is one of the first reports of direct evidence for a caspase-3 substrate. These data support a model where caspase-3 may also act as an upstream initiator of apoptosis. Activation of caspase-3 then causes up-regulation of PP2A activity. This increase in phosphatase activity is carried out by cleavage and inactivation of the regulatory A subunit of PP2A. Our data suggest this causes the hypophosphorylation of the PP2A substrate, MAP kinase. However, this is just one PP2A substrate. As the role of PP2A in the cell is further elucidated, the significance of the interaction with caspase-3 should become apparent. Further work is needed to identify additional critical substrates for both proteolysis and dephosphorylation and to determine what role phosphatase activation plays in the commitment of the cells to apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Michael Skidmore for providing preliminary data. We also thank Frank Bourbonais for providing PP2A substrate and Drs. Jack Dixon, Hamish Allen, and Daniel Tracey for critically reviewing this manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§§ To whom correspondence should be addressed: Biochemistry Dept., Parke-Davis Research, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-5844; Fax: 734-622-5875; E-mail: Giegeld{at}aa.wl.com.
1 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; PP1, protein phosphatase 1; CHAPS, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; BSA, bovine serum albumin; Ac-DEVD-pNA, Ac-Asp-Glu-Val-Asp-p-nitroanilide; DEVD-fmk, Asp-Glu-Val-Asp-fluoromethyl ketone; FA-FMK, Phe-Ala-fluoromethyl ketone; DEVD-cho, Ac-Asp-Glu-Val-Asp aldehyde; YVAD-cho, Ac-Tyr-Val-Ala-Asp aldehyde; VAD,Val-Ala-Asp; Rb, retinoblastoma associated protein; PP2A, protein phosphatase 2A; MAP kinase, mitogen activated protein; PVDF, polyvinylidene difluoride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PCR, polymerase chain reaction; IL, interleukin.
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REFERENCES |
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