Fraser Laboratories, Department of Medicine, McGill University Health Centre and Royal Victoria Hospital, Montreal, Quebec, H3A 1A1, Canada
* Author for correspondence (e-mail: coimbatore.srikant{at}mcgill.ca)
Accepted 30 September 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: DISC, c-FLIP, JNK, Mitochondria, Caspase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fas interacts through the death domain in its intracellular segment with death-effector-domain-containing proteins such as FADD/MORT1 (Boldin et al., 1996; Chinnaiyan et al., 1995
) and procaspase-8 (FLICE, MACH, Mch5) (Kischkel et al., 1995
; Medema et al., 1997
) to form the death-inducing signal complex (DISC) both in the native and activated states (Mundle and Raza, 2002
; Muzio et al., 1996
; Siegel et al., 2000
; Srinivasula et al., 1996
). Autoproteolytic activation of procasapse-8 occurs within DISC in the presence of ligated Fas, a process that is inhibited by the cellular FLICE-inhibitory protein (c-FLIP). Expressed in long (c-FLIPL) and short (c-FLIPS) variants, c-FLIP contains two death effector domains and displays homology to the N-terminal region of procaspase-8 (Irmler et al., 1997
; Shu et al., 1997
). A caspase-like domain that lacks the catalytic activity is also present in c-FLIPL. Recruitment of procasapase-8 to Fas-DISC and its autoproteolysis is inhibited by c-FLIPS. By contrast, c-FLIPL does not completely exclude procaspase-8 from DISC and permits its partial cleavage into a p43 form (Krueger et al., 2001
).
Proteins of the Bcl-2 family act distal to caspase-8 to regulate apoptotic signals at the mitochondria. The anti-apoptotic proteins Bcl-2 and Bcl-XL contain four Bcl-2 homology (BH) domains and a C-terminal membrane-anchoring domain. Homologues of Bcl-2 that possess up to three BH domains but lack the membrane-anchoring domain are proapoptotic. Of these, Bax and tBid (a cleavage product of Bid) migrate to the mitochondria in response to proapoptotic stimuli (Eskes et al., 2000; Wei et al., 2001
; Wolter et al., 1997
). The regulated presence and interactions of Bcl-2 proteins at the mitochondria precedes the disruption of its integrity and the release of cytochrome c (cyt c) into the cytoplasm leading to the activation of caspase-9 (Scaffidi et al., 1998
; Stennicke et al., 1998
). For instance, tBid facilitates proapoptotic activity by inducing Bax-Bak oligomerization and by sequestering Bcl-2 and Bcl-XL (Cheng et al., 2001
). Bad, whose function is governed by serine phosphorylation (residues 112, 136 and 155), acts by inhibiting Bcl-2 and Bcl-XL (Gross et al., 1999
; Virdee et al., 2000
; Zha et al., 1996
).
Stress-activated protein kinases p38 and JNK are induced during apoptosis (Kyriakis and Avruch, 1996) but their roles in regulating cell death have remained controversial. Apoptosis is facilitated by p38 and JNK (Aoshiba et al., 1999
; Bae and Song, 2003
; Vivo et al., 2003
; Yoshino et al., 2001
) in some models but inhibited in others (Assefa et al., 1999
; Nemoto et al., 1998
; Porras et al., 2004
; Zhang et al., 2002
). These kinases have been reported to influence the expression Fas and/or FasL (Hsu et al., 1999
; Ivanov and Ronai, 2000
; Zhang et al., 2000
), interaction of FADD with procaspase-8 or c-FLIP (Park et al., 2001
), mitochondrial proapoptotic activity (Holmstrom and Eriksson, 2000
; Tournier et al., 2000
; Yoshino et al., 2001
) and phosphorylation of Bcl-2 congeners (Deng et al., 2000
; Deng et al., 2001
; Haldar et al., 1994
; Ito et al., 1997
).
In the present study, we observed that the JNK inhibitor SP600125 potentiated Fas-mediated apoptosis to a greater extent than the p38 inhibitor PD169316, distal to caspase-8 in the absence but not in presence of the latter in several tumor cells. Given that PD169316 inhibits p38 isoforms that promote (p38) or suppress (p38ß) apoptosis (Jiang et al., 1996
; Kaiser et al., 2004
; Nemoto et al., 1998
) we analyzed their involvement in regulating Fas-mediated apoptosis in Jurkat T lymphocytes. Here, we demonstrate that p38
, but not p38ß, facilitates Fas-mediated activation of caspase-8 by inhibiting the phosphorylation and presence of c-FLIPS, but not c-FLIPL, in the DISC. Inhibition of Fas-mediated caspase-8 activation in p38
-lacking cells correlated with the expected inhibition of tBid formation, mitochondrial presence of tBid and Bax and dephosphorylation of Bad at Ser112 and Ser155. By contrast, only mitochondrial presence of Bax and Bad dephosphorylation were suppressed in cells lacking p38ß. Inhibition of JNK prevented the phosphorylation of Bad at Ser136 and enhanced type II signaling but only in the presence of p38
-dependent caspase-8 activation and downstream effects of p38
and p38ß.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and transfection
Jurkat cells were grown in RPMI 1640 medium (Invitrogen Canada, Burlington, ON) supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic, 2 mg/ml glucose, 10 mM HEPES, 1mM sodium pyruvate and 2 mM L-Glutamine at 37°C in a humidified atmosphere containing 5% CO2. To achieve targeted ablation of individual kinase isoforms, cells were transfected with annealed ds-siRNA oligonucleotides. Briefly, 3 µl of TransIT reagent was added to 200 µl of serum-free medium maintained at room temperature, mixed with ds-siRNA (60 pmol) and incubated for 30 minutes before adding it to the cells. The efficiency of siRNA-mediated translational silencing of each kinase was monitored by immunoblot analysis as a function of time with maximal suppression being observed at 36 to 48 hours (details not shown).
To generate Jurkat T cells that transiently overexpress p38 and its dominant negative variant p38-
-AGF, we transfected the cells with the plasmids pCMV-Flag-p38-
and pCMV-Flag-p38-
-agf. Experiments were carried out 48 hours after transfection when maximal expression of the transfected proteins was observed.
Detection of apoptosis
Cells were incubated with 150 ng/ml of Fas-mAb and were pretreated or not with PD169316 and/or SP600125 for 30 minutes. In parallel experiments, we assessed the effect of these inhibitors on apoptosis induced by FasL plus (0.1 µg/ml). Cells were washed and labeled with annexin-V-FITC and propidium iodide (PI) using the annexin-V-FLUOS staining kit according to the manufacturer's instructions. Apoptotic cells were detected by flow cytometry by annexin-V-FITC labeling in the absence of PI uptake. At least 20,000-gated events were counted for each sample and analyzed by WinList software (Verity Software House, ME).
Measurement of mitochondrial membrane potential (m)
The potential sensitive dye DiOC6(3) (10 µM) was added to the cells 30 minutes before the treatment with Fas-mAb was finished. Cells were washed with PBS, excited at 360 nm and the fluorescence emission was captured at 560 nm in a flow cytometer (Liu et al., 2000).
Caspase activity measurements
Activities of caspase-3, caspase-8 and caspase-9 in cell extracts were measured using AMC derivatives of DEVD, IETD and LEHD respectively (Liu et al., 2000). The generation of the fluorogenic product was monitored by exciting the samples at 380 nm and measuring the emission at 460 nm, and quantified against a standard curve generated by using 0-1000 nM AMC. To minimize cross reactivity, inhibitors of caspase-3 (DEVD-CHO) or caspase-8 (IETD-CHO) were included at a concentration of 1.5 µM when measuring the activities of caspase-8 and caspase-3, respectively.
Isolation of mitochondria
Cells were washed with PBS, lysed on ice for 10 minutes with the cytosol extraction buffer (4.3 mM Na2HPO4, 1.4 mM KH2PO4, 250 mM sucrose, 70 mM KCl, 137 mM NaCl, 200 µg/ml digitonin and protease inhibitors) and centrifuged at 3000 g for 5 minutes to obtain the cytosolic fraction. The pellet was washed thrice with cytosol extraction buffer and lysed with 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% v/v Triton X-100, 0.3% NP40, 0.5% sodium deoxycholate and protease inhibitors), incubated on ice for 15 minutes and the mitochondrial fraction was recovered in the supernatant following centrifugation at 10,500 g for 10 minutes at 4°C.
Immunoblot analysis
Cells were lysed in 50 mM Tris-HCl buffer (pH 8.0) containing 1% NP40, 150 mM NaCl, 1 mM EDTA 0.05% SDS and protease inhibitors. Aliquots of lysates containing 40 µg of protein were subjected to immunoblot analysis with antibodies as indicated and the appropriate horseradish peroxidase-conjugated secondary antibodies, and the signals were captured in an Alpha Innotech Imager (San Leandro, CA).
Immunoprecipitation of Fas-associated proteins
Cells were lysed in 10 mM Tris-HCl buffer (pH 7.5) containing 1% v/v Nonidet P-40, 150 mM NaCl and 0.4 mM EDTA and protease inhibitors as described previously (Daigle et al., 2002) with slight modifications. Aliquots of lysates containing 1 mg total protein were pre-cleared using normal mouse IgG and incubated with 2 µg of the non-agonistic anti-Fas antibody in presence of 80 µl of Protein A/G Sepharose beads. To minimize the interference of heavy and light chains of primary antibody during the IP, we employed Seize Classic A&G Immunoprecipitation Kit from Pierce (Rockland, IL). Briefly, cells lysates were incubated with either anti-c-FLIPL or anti-c-FLIPS (Santa Cruz) overnight after which immobilized Protein A suspension, containing a cross linking agent (DSS), was added. Immune complexes were washed, electrophoresed and immunoblotted for the detection of Fas, FADD, c-FLIPL/S and procaspase-8. For detecting c-FLIP phosphorylation, c-FLIP was immunoprecipitated, electrophoresed, probed with ProQ Diamond phosphoprotein gel stain and scanned at 590 nm in a Typhoon 9200 Imager (Amersham Biosciences).
Metabolic labeling and detection of 32P labeled c-FLIPL and c-FLIPS
Endogenous phosphate was first depleted from cells by incubation in phosphate-free minimum essential medium (MEM) for 48 hours. The cells were then exposed to fresh medium supplemented with [32P]sodium o-phosphate (37.5 µCi/well) for 24 hours. The cells were washed and incubated for 4 hours in phosphate-free medium before lysis. Cell lysates containing 1 mg protein aliquots in 0.5 ml lysis buffer was pre-cleared by incubation with 30 ml of Sepharose-CL 4B for 3 hours at 4°C. Labeled c-FLIPL and c-FLIPS were then immunoprecipitated with anti-c-FLIPS/L antibody (10 µg) using the Seize Classic A&G Immunoprecipitation kit. Immune complexes were pelleted by centrifugation for 5 minutes at 14,000 g, washed five times with the lysis buffer and resolved by SDS-PAGE. The gels were dried and the radioactive bands were visualized using the Cyclone Storage Phosphor System (Perkin Elmer, Torrance, CA).
In vitro kinase activity assays
Aliquots of cell extracts containing equal amounts of protein were immunoprecipitated with phospho-p38 or phospho-JNK antibodies. The activities of the immunoprecipitated phosphorylated-kinases were detected by in vitro phosphorylation of ATF-2 (by p38) and JUN (by JNK).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Potentiation of Fas mediated cell death by pharmacological inhibition of p38 and JNK
We first established the specificity of the inhibitors of p38 and JNK by demonstrating that PD169316 inhibited the ability of p38 in Fas-activated cells to phosphorylate ATF-2, whereas SP600125 selectively inhibited JNK-mediated phosphorylation of JUN (Fig. 2, panels A and B, compare lanes 3 and 4 with lane 2). Both kinases were inhibited in cells that had been simultaneously exposed to the two inhibitors (Fig. 2, lane 5). Their activities in native Jurkat cells were below the detection limit under these conditions (Fig. 2, lane 1). The concentration of the inhibitors (15 µM) used in this experiment was determined to be the lowest concentration that effectively inhibited Fas-mediated activation of the respective kinases for up to 24 hours (details not shown).
|
The sensitizing effect of these inhibitors on Fas-signaled apoptosis was best observed following a 4-hour treatment with Fas-mAb. A 2.5- and 4-fold increase in apoptosis occurred in the presence of PD169316 and SP600125 (26.9±0.5 and 45.3±0.7%, respectively) compared with 11.4±0.6% observed in their absence (Fig. 3A, left panel, lanes 2-4). When added together, PD169316 reversed the greater potentiating effect SP600125 (lane 5). Following treatment with Fas-mAb, a significant increase in the number of cells with reduced m was observed in the presence of SP600125 (58±4 and 37±2% for presence and absence, respectively) (Fig. 3B). PD169316 inhibited SP600125-induced increase in the number of cells with decreased
m, (28±5%) but not the effect of Fas-mAb itself. On its own, neither inhibitor induced apoptosis or decreased
m (Fig. 3A,B, right panels). Apoptosis and the reduction in
m induced by ligating Fas with its natural ligand FasL were also sensitized to a greater extent by SP600125 in the absence, but not presence, of PD169316 (Fig. 3C,D). The effects of inhibition of p38 on Fas-signaled apoptosis and its sensitization by SP600125 were confirmed with two other pharmacological inhibitors of p38 SB202190 and SB203580 and by the lack of effect of the nonfunctional derivative SB202474 (Fig. 3, panels E and F). The ability of SP600125 and PD169316 to potentiate Fas-mediated apoptosis was not unique to Jurkat cells and was also observed in other tumor cell types such as HeLa, MCF-7 and T47D (Table 1).
|
|
Sensitizing effect of SP600125 is abrogated by PD169316 distal to caspase-8
Fas-mediated activation of caspase-8 was greater in cells treated with SP600125 than with PD169316 (0.51±0.02 and 0.30±0.01 nmol/µg/minute) compared with 0.19±0.006 nmol/µg/minute without either inhibitor (Fig. 4A). PD169316 did not, however, suppress the effect of SP600125 on caspase-8 activation. By contrast, caspase-9 activity was lower in cells incubated with PD169316 than without, but was higher in cells treated with SP600125 (0.30±0.003 vs 0.17±0.04 0.56±0.006 nmol/µg/minute) (Fig. 4B). In the presence of both inhibitors, Fas-induced caspase-9 activation was comparable to that seen with PD169316 alone (0.17±0.04 vs 0.24±0.04 nmol/µg/minute). Likewise, caspase-3 activity was greater in the presence of SP600125 than PD169316 (0.53±0.007 and 0.40±0.02, respectively compared with 0.19±0.005 nmol/µg/min in their absence, Fig. 4C). The observed differences in the activities of these caspases correlated with the increase in the amount of cleaved fragments of procaspase-8 in cells treated with these inhibitors either individually or together (Fig. 4D, blot 1), and of cleaved fragments of caspase-9 and caspase -3 in cells treated with SP600125 in the absence, but not presence of PD169316 (Fig. 4D, blots 3 and 5); they correlated with the generation of tBid, the cleavage product of the caspase-8 substrate Bid, cytosolic accumulation of cyt c and the cleaved fragment of PARP (Fig. 4D, blots 2, 4 and 6, respectively). As shown in these blots, the potentiating effect of SP600125 on the activation of caspase-9 and caspase-3 in Fas-ligated cells was abrogated by PD169316 (Fig. 4D, compare lanes 4 and 5) to the level seen in the presence of PD169316 alone (Fig. 4D, lane 3).
|
Both p38 and p38ß isoforms are activated in Fas-ligated cells
Because PD169316 inhibits both and ß isoforms of p38, we confirmed that both these isoforms are activated in response to Fas-ligation in Jurkat cells. Total p38 was immunoprecipitated with an anti-p38 antibody that recognized both isoforms and immunoblotted with antibodies specific for p38
and p38ß (Fig. 5A, blots 2 and 3). Fas-activation did not alter the levels of either p38
or p38ß. The increase in their phosphorylation in Fas-activated cells was confirmed by immunoprecipitation with specific antibodies against p38
and p38ß and subsequent immunoblot analysis with phospho-p38 antibody (Fig. 5B, blots 1 and 2).
|
Effect of translational silencing of p38 and p38ß on Fas-mediated apoptosis
To elucidate the regulation of apoptotic signaling by these p38 isoforms, we inhibited their expression by using siRNA. Expression of p38, but not that of p38ß, was abrogated in siRNA-p38
(si-p38
) cells (Fig. 5C, compare lane 2 in blots 1 and 2) whereas a significant inhibition of the expression of p38ß, but not p38
, was seen in siRNA-p38ß (si-p38ß) cells (lane 4). As shown in these blots, transfection of a control siRNA (si-C) did not affect the expression of both isoforms of p38 (lane 3). In parallel, we generated cells lacking JNK isoforms 1 and 2 [siRNA-JNK1/2 (si-JNK1/2)] in which the expression of both JNK1 and JNK2 was suppressed (Fig. 5D).
Fas-mediated apoptosis was substantially inhibited in si-p38 cells and enhanced in si-p38ß and si-JNK1/2 cells (5.4±0.4%, 41.3±1% and 46±2.8%, respectively, compared with 12±1.2% in si-C cells, Fig. 6A). The reduction in
m in response to Fas-activation was also much lower in si-p38
cells (11.3±0.6%), but not in si-p38ß cells (36±0.9%), compared with the value of 33±2% in si-C cells (Fig. 6B). Interestingly, the reduction in
m was much greater in si-JNK1/2 cells (74±1.4%). Fas-induced generation of caspases-8, -9 and -3 was diminished in si-p38
cells but was enhanced in si-p38ß cells (Fig. 6C, blots 1, 2 and 3). Abrogation of expression of p38
, but not p38ß, inhibited cyt c release (blot 4). In si-JNK1/2 cells, enhanced presence of cleaved caspases-8, -9 and -3 and cytosolic cyt c was observed (Fig. 6D).
|
Ectopically introduced p38 and its inactive mutant p38
-AGF exert opposing effects on Fas-signaling
The selective requirement of p38 for Fas-mediated apoptosis was confirmed by comparing the effects of ectopically introduced HA-tagged wild-type p38
and its dominant negative mutant p38
-AGF. The expression of the Flag-tagged proteins was verified by immunoblot analysis using anti-p38
antibody (Fig. 7, lanes 1-3) and anti-Flag antibody (lanes 4-6). Four-hour treatment with agonistic anti-Fas antibody induced greater apoptosis in cells expressing Flag-p38
compared with native Jurkat cells (31±1.3 vs 11.4±0.6%) and reduction in
m (64±2.1 vs 37±2%, Fig. 7C). By contrast, Fas-ligation failed to induce apoptosis and reduction in
m in cells expressing FLAG-p38
-AGF. The basal level of apoptosis in Jurkat cells was unaffected by the ectopically expressed wild type p38
or its mutant p38
-AGF.
|
Effect of p38 and p38ß on c-FLIPS phosphorylation and DISC assembly
Depletion of p38, but not p38ß or JNK1/2, diminished Fas-mediated caspase-8 activation. Hence, we compared the effects of chemical inhibitors and siRNA-mediated knock down of these kinases on DISC assembly in the native and Fas-activated Jurkat cells. An increase in Fas-associated FADD and procaspase-8 and a concomitant decrease in the presence of c-FLIPS was evident in the native cells treated with PD169316 and/or SP600125 (Fig. 8A, top left panel). These changes became more pronounced in cells incubated with Fas-mAb for 1 hour (when there was no detectable formation of active caspase-8 fragments) in the presence of PD169316 and SP600125 (top right panel). Strikingly, no change in the level of DISC-associated c-FLIPL was seen in presence of either inhibitor alone or both inhibitors together. In parallel experiments, we observed that a marked reduction in the presence of c-FLIPS and a concomitant increase in FADD and procaspase-8 was seen in si-p38ß cells (Fig. 8B, lane 3, top panel) compared with native Jurkat cells (lane 1) and si-p38
cells (lane 2). Increased presence of FADD and procaspase-8 was also observed in si-JNK1/2 cells (lane 4). Treatment of the cells with PD169316 and SP600125 or silencing of these kinases did not alter the levels of Fas (Fig. 8A and B, top panels), or of total FADD, procaspase-8, c-FLIPL and c-FLIPS (bottom panels).
|
Phosphorylation of c-FLIPS, but not c-FLIPL, is increased in the cells lacking p38
To determine whether the striking difference in the presence of c-FLIPS in the DISC is because of an altered phosphorylation status in Jurkat cells lacking p38, we immunoprecipitated c-FLIPS, electrophoresed and probed the gel with Pro-Q Diamond stain for the detection of phosphorylated proteins. Phosphorylation of c-FLIPS was found to be greater in si-p38
than in si-C cells (Fig. 9A, bottom panel, compare lanes 1 and 2). By contrast, phosphorylation of c-FLIPS was undetectable in si-p38ß or si-JNK1/2 cells (lanes 3 and 4). The levels of immunoprecipitated c-FLIPS in these cells were comparable as demonstrated by Sypro Ruby protein staining (Fig. 9A, bottom panel). These findings were further confirmed by monitoring the phosphorylation of c-FLIP in Jurkat cells metabolically labeled with radioactive 32P. The increase in phosphorylation of c-FLIPS seen in cells transfected with si-p38
was higher than that in cells transiently expressing Flag-p38
-AGF (Fig. 9B, compare lanes 3 and 4). Enforced expression of p38
suppressed c-FLIPS phosphorylation (lane 5) to the level seen in the native and in control siRNA transfected Jurkat cells (lanes 1 and 2). By contrast, such manipulation of p38
activity did not affect the phosphorylation status of c-FLIPL.
|
Potentiating effect of SP600125 on Fas-mediated apoptosis in siRNA transfected cells
In si-p38 cells, treatment with PD169316 enhanced the apoptotic responsiveness to Fas-ligation fivefold (30.3±2.5 vs 5.4±0.9%) and 2.5-fold in si-C cells (30.5±1.1 vs 12±1.2%). By contrast, it decreased the extent of apoptosis by 50% in si-p38ß cells (22.9±1.5 vs 41.3±1.1%). Likewise, the proportion of cells with reduced
m was lower in si-p38
cells (11.3±0.6) but was higher in si-p38ß cells (36±1%) compared with that in si-C cells (32.9±2%, Fig. 10B). Fas-mediated apoptosis was higher in presence of SP600125 than in its absence in si-C cells (48.7±0.8% vs 12±1.2%) and si-p38
cells (18.5±0.8% vs 5.4±0.4%), but not si-p38ß cells (45.3±1.1% vs 41.3 ±1%, Fig. 10A). Similarly, the reduction in
m was higher in the presence of SP600125 than in its absence in si-C (68±1.9% vs 33±2.1%) and si-p38a (27.9±1.8% vs 11.3±0.6%), but not in si-p38ß cells (31.2±2.8% vs 36±0.9%).
|
A significantly higher activation of caspase-8 (0.53±0.02 vs 0.2±0.02 nmol/µg/min) and caspase-3 (0.55±0.03 vs 0.21±0.02 nmol/µg/min) but not caspase-9 (0.25±0.03 vs 0.34±0.03 nmol/µg/min) was seen in si-p38ß cells compared with si-C cells, whereas these caspases were only minimally induced in si-p38 cells. Fas-mediated activation of caspase-8 was potentiated by PD169316 in si-C and si-p38
, but not p38ß, cells. The effect of PD169316 on Fas-mediated activation of caspase-3 revealed a similar pattern, being higher in si-C and p38
cells and lower in si-p38ß cells compared with the values observed following Fas-activation in the absence of this inhibitor. By contrast, caspase-9 was activated in si-C and si-p38ß, but not in si-p38
cells. Of particular interest was the finding that PD169316 completely abrogated Fas-mediated activation of caspase-9 in si-p38ß cells (from 0.25±0.04 to 0.06±0.01 nmol/µg/minute) but only partially in si-C cells (0.21±0.01 from 0.34±0.04 nmol/µg/minute). As expected, SP600125 did not exert any effect in si-JNK cells. Moreover, PD169316 inhibited the sensitizing effect of SP600125 on Fas-mediated activation of all three caspases in si-p38
cells and si-JNK1/2 cells but not in si-p38ß cells.
Influence of the inhibition of p38, p38ß and JNK on Fas-induced alterations in the biochemical and cellular localization properties of Bcl-2 family proteins
The differential regulatory effects of p38, p38ß and JNK1/2 on mitochondrial function in apoptosis prompted us to investigate their effects on the biochemical and subcellular localization properties of Bcl-2 family of proteins. Immunoblot analysis of the mitochondrial and cytosolic fractions revealed that PD169316, but not SP600125, prevented the mitochondrial localization of Bax. The ability of PD169316 to inhibit the mitochondrial presence of Bax was not affected by SP600125 (Fig. 11A, blots 1 and 2, compare lanes 2-5). The presence of tBid in the mitochondria was seen in Fas-ligated cells in the absence and presence of SP600125, but not PD169316 (Fig. 11A, blot 3). However, PD169316 did not inhibit the mitochondrial presence of tBid in presence of SP600125. The presence of Bak in mitochondrial fraction was unaffected under these experimental conditions (Fig. 11A, blot 4). In Fas-activated cells, there was a loss of Bad phosphorylation at Ser112 and Ser155, but not at Ser136. Whereas PD169316 reversed the loss of Bad phosphorylation at Ser112 and Ser155, SP600125 promoted the loss of Ser136 phosphorylation (Fig. 11A, blots 5, 6 and 7, compare lanes 2,3 and 4). These changes in Bad phosphorylation occurred in the absence of any change in the total levels of Bad (Fig. 11A, blot 8). Ser70 phosphorylation of mitochondrial Bcl-2 was inhibited in the presence of PD169316, but not SP600125 (Fig. 11A, blot 9, compare lanes 2,3 and 4). These inhibitors did not affect the mitochondrial presence of Bcl-2 (blot 10). The efficacy of subcellular fractionation was confirmed by the detection of the mitochondrial marker TOM-20 exclusively in mitochondrial fractions (Fig. 11A, blot 11).
|
Mitochondrial translocation of Bax failed to occur in Fas-activated si-p38 and si-p38ß cells (Fig. 11B, blots 1 and 2, compare lanes 3, 4 with lane 2). However, the mitochondrial localization of Bax in si-JNK1/2 cells was higher than in si-C cells (Fig. 11B, blots 1 and 2, compare lanes 2 and 5). Cleavage of Bid into tBid (Fig. 11B, blot 3) and translocation of tBid to the mitochondria occurred in si-C, si-p38ß and si-JNK1/2 cells, but not in si-p38
cells (Fig. 11B, blot 4). Fas-induced loss of phosphorylation of Bad at Ser112 and Ser155 was prevented in both si-p38
and si-p38ß cells (Fig. 11B, blots 5 and 7, compare lanes 2, 3 and 4). Bad phosphorylation at Ser136 was absent uniquely in si-JNK1/2 cells (Fig. 11B, blot 6, lane 5). Total Bad levels were comparable in these cells (Fig. 11B, blot 8). In the case of Bcl-2, Ser70 phosphorylation was abrogated in si-p38
, but not si-p38ß or si-JNK1/2 cells (Fig. 11B, blot 9) without affecting its presence at the mitochondria (Fig. 11B, blot 10).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Caspase-8 is generated by the autoproteolytic cleavage of procaspase-8 within the DISC in the presence of ligand-activated Fas and the adapter protein FADD (Kischkel et al., 1995; Medema et al., 1997
). c-FLIPS and c-FLIPL bind to FADD through its DED domains and thus block the recruitment and/or the autoproteolysis of procaspase-8 (Krueger et al., 2001
). Here, we demonstrated that the phosphorylation and DISC localization of c-FLIPS but not c-FLIPL is attenuated by p38
, an effect that is enhanced by ectopically introduced Flag-tagged wild-type p38
and inhibited by the depletion of p38
by siRNA-mediated translational silencing and by the dominant negative effect of Flag-tagged inactive mutant p38
-AGF. This, to our knowledge, constitutes the first evidence for selective post-translational modification of c-FLIPS by p38
-dependent phosphorylation. The identity of putative, p38
-regulated kinase(s) that act solely on c-FLIPS remains to be discovered. In previous reports, calcium-calmodulin-dependent kinase and PKC were identified as capable of phosphorylating c-FLIP. Calcium/calmodulin-dependent protein kinase II was shown to phosphorylate c-FLIPL but not c-FLIPS, increase its presence in the DISC and inhibit Fas-induced apoptosis in glioma cells (Yang et al., 2003
). However, PKC-dependent increase in the phosphorylation and presence of both c-FLIPS and c-FLIPL in TRAIL-DISC was shown to promote TRAIL-mediated apoptosis in bile-acid-treated hepatocytes (Higuchi et al., 2003
). The presence of multiple phosphorylation sites in the DED domains necessitates further studies aimed at the identification of the sites of phosphorylation that are sensitive to different kinases, and in particular the sites targeted in a p38
-dependent manner. The site-specific phosphorylation of the DED domain of c-FLIP variants may account for the differential regulation of DISC activation by different death-receptor ligands. To date, regulation of c-FLIPS/L phosphorylation by phosphatase(s) has not been shown. The possibility that p38
protects c-FLIPS phosphorylation by inhibiting phosphatase(s) cannot be excluded and remains to be examined.
Fas-mediated activation of caspase-8 was potentiated by SP600125 in si-C cells and si-p38ß cells but not si-p38 cells. However, enhanced activation of caspase-9 in the presence of SP600125 was seen in Fas-activated si-C cells but not si-p38
cells or si-p38ß cells. Moreover, PD169316 diminished Fas-mediated caspase-9 activation in si-JNK1/2 cells. Taken together, these findings suggest that p38
promotes caspase-3 activation via type I signaling and that p38
and p38ß both promote type II signaling, whereas JNK1/2 selectively inhibits type II signaling. The greater reduction in
m, cyt c release and caspase-9 activation induced by Fas-activation in JNK-inhibited cells correlated with the loss of Ser136-phosphorylation in Bad and depended on p38
-p38ß-regulated loss of phosphorylation at Ser112 and Ser155 and also mitochondrial localization of Bax and tBid. Fas-induced targeting of Bax and tBid and the loss of phosphorylation at Ser112 and Ser155 in Bad were prevented by inhibition of p38, irrespective of the presence or absence of JNK activity. The ability of SP600125 to sensitize mitochondrial proapoptotic activity was seen in si-p38ß cells, but not si-p38
cells. Moreover, the degree of sensitizing effect of JNK inhibition was significantly lower in si-p38ß cells compared with that seen in si-JNK1/2 or si-C cells (Fig. 9). The stimulatory effect of p38
and the inhibitory effect of p38ß on Fas-mediated caspase-8 activation largely account for the observed differences in the proapoptotic events distal to caspase-8. As expected, Fas-induced mitochondrial presence of tBid and Bax, and loss of phosphorylation of Ser70 in Bcl-2 and, the phosphorylation of Ser112 and Ser155 in Bad were not seen in si-p38
cells. Likewise, in si-p38ß cells, which were more sensitive to Fas-signaling because of the presence and activation of p38
, we observed the expected presence of tBid at the mitochondria, reduction in
m, cyt c release and, activation of caspase-9 and caspase-3. However, Bax was excluded from the mitochondria despite the presence of tBid at this organelle in si-p38ß cells. The failure of Bax to localize to the mitochondria despite the presence of tBid in cells lacking p38ß may, therefore, be owing to the lack of Bad dephosphorylation at Ser112 and Ser155 (Yusta et al., 2000
). In its unphosphorylated form Bad can interact with Bcl-2 or Bcl-XL (Bae et al., 2001
; Harada et al., 1999
) and dephosphorylation of all three Ser moieties is essential for the ability of Bad to sequester Bcl-2 and Bcl-XL (Bae and Song, 2003
; Datta et al., 2002
) and to facilitate tBid-induced molecular interaction between the proapoptotic partners Bax and Bak (Wei et al., 2001
). Ser155 phosphorylation is known to promote the dissociation of Bad from Bcl-XL (Klumpp and Krieglstein, 2002
), suggesting that p38
and/or p38ß may influence mitochondrial dysfunction by regulating the molecular interaction between pro- and anti-apoptotic Bcl-2 proteins. Direct regulation of Bad phosphorylation by p38
, p38ß and JNK has not been shown, raising the possibility that it may be mediated via other kinases including p21-activated kinase, PKA, PKC, Raf-1, Rsk and Akt/PKB (Gnesutta et al., 2001
; Jones et al., 2002
; Schurmann et al., 2000
; Wolf et al., 2001
), and phosphatases such as PP1, PP2A and PP2B (Ayllon et al., 2000
; Chiang et al., 2001
). An additional mechanism may involve Bcl-2 dependence of Bax recruitment. Such a regulation has been reported to result from altered Bcl-2 phosphorylation (Ishikawa et al., 2003
). Absence of Bax at the mitochondria correlated with reduced Bcl-2 (Ser70) phosphorylation in cells treated with PD169316 and in si-p38
cells, but not si-p38ß cells. Hence, p38
-dependent phosphorylation of Bcl-2 may be necessary to facilitate mitochondrial localization of Bax in cells undergoing apoptosis. Recently, p38 was shown to sequester in the mitochondria in apoptotic cells, raising the possibility that it directly regulates the disruption of this organelle (Tikhomirov and Carpenter, 2004
). It remains to be determined, whether p38
, p38ß or both localize to this organelle and affect its integrity through event(s) that are independent of their effects on events upstream of mitochondria.
PD169316 treatment enhanced Fas-mediated apoptosis more than fivefold from 5.4% to 30% in si-p38 cells and by 2.5-fold from 12% to 30.5% in si-C cells. By contrast, its effect was inhibitory in si-p38ß and si-JNK1/2 cells. Caspase-8 activation was unaffected by PD169316 in si-JNK1/2 cells, enhanced in si-C cells and si-p38
cells, and inhibited in si-p38ß cells. SP600125 was able to potentiate caspase-8 activation and apoptotic responsiveness in si-p38ß cells but not in si-p38
cells. These data indicate that in Jurkat cells both p38ß and JNK inhibit caspase-8 activation at the level of DISC in a non-additive manner and their effects are offset by p38
-mediated DISC activation. Additionally, mitochondrial presence of tBid was inhibited by PD169316 despite the potentiation of caspase-8 and the cleavage of Bid into tBid in si-C and Jurkat cells, and its mitochondrial presence was inhibited uniquely in Fas-activated p38
cells. Thus, its localization to the mitochondria depends on the presence and activation of p38
. The mechanism involved in such a regulation is not known but may be modulated by the effect of p38
on other BH-domain-bearing proteins such as Bfl1/A1, which was recently implicated in the mitochondrial targeting of tBid (Werner et al., 2002
).
The present findings have thus demonstrated the opposing effects of p38 and p38ß on caspase-8 activation, similar effects on Bad phosphorylation and mitochondrial targeting of Bax and p38
-dependent mitochondrial localization of tBid. Finally, our findings also reveal that p38
, but not p38ß can promote the direct caspase activation cascade as evidenced by the increased activation of caspase-8 and caspase-3, but not caspase-9, in si-p38ß cells. Such isoform-specific effects of p38
and p38ß could not be discerned in studies employing the pyridylimidazole inhibitors and underscore the need to interpret again the previously reported conclusions derived from the pharmacological inhibition of p38.
In summary, we have shown that p38 facilitates Fas-induced DISC activation by modulating c-FLIPS, caspase-8-mediated direct caspase-cascade (type I signaling) and also the mitochondrial arm of the (type II) apoptotic signaling, by multiple effects that promote mitochondrial localization of tBid and Bax, and inhibition of Bad (Ser112 and Ser155) phosphorylation but not that of Bcl-2 (Ser70). However, p38ß controls the sensitivity of type II signaling solely by regulating Ser112 and Ser155 phosphorylation in Bad. JNK1/2 attenuate the antiapoptotic activity of Bad by inhibiting dephosphorylation of Ser136. These findings highlight the need to assess again the conclusions drawn from studies using p38 inhibitors that target multiple isoforms and point to the need for identifying inhibitors of individual p38 isoforms with exclusive selectivity. The development of such compounds should permit selective inhibition of p38ß and JNK1/2 to optimally exploit the proapoptotic effect of p38
in sensitizing tumor cells to Fas-mediated apoptosis.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aoshiba, K., Yasui, S., Hayashi, M., Tamaoki, J. and Nagai, A. (1999). Role of p38-mitogen-activated protein kinase in spontaneous apoptosis of human neutrophils. J. Immunol. 162, 1692-1700.
Assefa, Z., Vantieghem, A., Declercq, W., Vandenabeele, P., Vandenheede, J. R., Merlevede, W., de Witte, P. and Agostinis, P. (1999). The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with Hypericin. J. Biol. Chem. 274, 8788-8796.
Ayllon, V., Martinez, A. C., Garcia, A., Cayla, X. and Rebollo, A. (2000). Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. EMBO J. 19, 2237-2246.
Bae, J., Hsu, S. Y., Leo, C. P., Zell, K. and Hsueh, A. J. (2001). Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis. Apoptosis 6, 319-330.[CrossRef][Medline]
Bae, M.-A. and Song, B. J. (2003). Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells. Mol. Pharmacol. 63, 401-408.
Boldin, M. P., Goncharov, T. M., Goltsev, Y. V. and Wallach, D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803-815.[Medline]
Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T. and Korsmeyer, S. J. (2001). BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell 8, 705-711.[CrossRef][Medline]
Chiang, C. W., Harris, G., Ellig, C., Masters, S. C., Subramanian, R., Shenolikar, S., Wadzinski, B. E. and Yang, E. (2001). Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin-3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation. Blood 97, 1289-1297.
Chinnaiyan, A. M., O'Rourke, K., Tewari, M. and Dixit, V. M. (1995). FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81, 505-512.[Medline]
Daigle, I., Yousefi, S., Colonna, M., Green, D. R. and Simon, H. U. (2002). Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils. Nat. Med. 8, 61-67.[CrossRef][Medline]
Datta, S. R., Ranger, A. M., Lin, M. Z., Sturgill, J. F., Ma, Y. C., Cowan, C. W., Dikkes, P., Korsmeyer, S. J. and Greenberg, M. E. (2002). Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis. Dev. Cell 3, 631-643.[Medline]
Deng, X., Kornblau, S. M., Ruvolo, P. P. and May, W. S., Jr (2000). Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance. J. Natl. Cancer Inst. Monogr. 28, 30-37.
Deng, X., Xiao, L., Lang, W., Gao, F., Ruvolo, P. and May, W. S., Jr (2001). Novel role for JNK as a stress-activated Bcl2 kinase. J. Biol. Chem. 276, 23681-23688.
Eskes, R., Desagher, S., Antonsson, B. and Martinou, J. C. (2000). Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol. 20, 929-935.
Fulda, S., Meyer, E., Friesen, C., Susin, S. A., Kroemer, G. and Debatin, K. M. (2001). Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis. Oncogene 20, 1063-1075.[CrossRef][Medline]
Gnesutta, N., Qu, J. and Minden, A. (2001). The serine/threonine kinase PAK4 prevents caspase activation and protects cells from apoptosis. J. Biol. Chem. 276, 14414-14419.
Gross, A., McDonnell, J. M. and Korsmeyer, S. J. (1999). BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899-1911.
Haldar, S., Jena, N. and Croce, C. M. (1994). Antiapoptosis potential of bcl-2 oncogene by dephosphorylation. Biochem. Cell Biol. 72, 455-462.[Medline]
Harada, H., Becknell, B., Wilm, M., Mann, M., Huang, L. J., Taylor, S. S., Scott, J. D. and Korsmeyer, S. J. (1999). Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A. Mol. Cell 3, 413-422.[Medline]
Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770-776.[CrossRef][Medline]
Higuchi, H., Yoon, J.-H., Grambihler, A., Werneburg, N., Bronk, S. F. and Gores, G. J. (2003). Bile acids stimulate cFLIP phosphorylation enhancing TRAIL-mediated apoptosis. J. Biol. Chem. 278, 454-461.
Holmstrom, T. H. and Eriksson, J. E. (2000). Phosphorylation-based signaling in Fas receptor-mediated apoptosis. Crit. Rev. Immunol. 20, 121-152.[Medline]
Hsu, S. C., Gavrilin, M. A., Tsai, M. H., Han, J. and Lai, M. Z. (1999). p38 mitogen-activated protein kinase is involved in Fas ligand expression. J. Biol. Chem. 274, 25769-25776.
Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C. et al. (1997). Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195.[CrossRef][Medline]
Ishikawa, Y., Kusaka, E., Enokido, Y., Ikeuchi, T. and Hatanaka, H. (2003). Regulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis. Mol. Cell. Neurosci. 24, 451-459.[CrossRef][Medline]
Ito, T., Deng, X., Carr, B. and May, W. S. (1997). Bcl-2 phosphorylation required for anti-apoptosis function. J. Biol. Chem. 272, 11671-11673.
Ivanov, V. N. and Ronai, Z. (2000). p38 protects human melanoma cells from UV-induced apoptosis through down-regulation of NF-kappaB activity and Fas expression. Oncogene 19, 3003-3012.[CrossRef][Medline]
Jiang, Y., Chen, C., Li, Z., Guo, W., Gegner, J. A., Lin, S. and Han, J. (1996). Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta). J. Biol. Chem. 271, 17920-17926.
Jones, R. G., Elford, A. R., Parsons, M. J., Wu, L., Krawczyk, C. M., Yeh, W.-C., Hakem, R., Rottapel, R., Woodgett, J. R. and Ohashi, P. S. (2002). CD28-dependent activation of protein kinase B/Akt blocks Fas-mediated apoptosis by preventing death-inducing signaling complex assembly. J. Exp. Med. 196, 335-348.
Kaiser, R. A., Bueno, O. F., Lips, D. J., Doevendans, P. A., Jones, F., Kimball, T. F. and Molkentin, J. D. (2004). Targeted inhibition of p38 mitogen-activated protein kinase antagonizes cardiac injury and cell death following ischemia-reperfusion in vivo. J. Biol. Chem. 279, 15524-15530.
Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P. H. and Peter, M. E. (1995). Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579-5588.[Abstract]
Klumpp, S. and Krieglstein, J. (2002). Serine/threonine protein phosphatases in apoptosis. Curr. Opin. Pharmacol. 2, 458-462.[CrossRef][Medline]
Krueger, A., Schmitz, I., Baumann, S., Krammer, P. H. and Kirchhoff, S. (2001). Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. J. Biol. Chem. 276, 20633-20640.
Kyriakis, J. M. and Avruch, J. (1996). Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18, 567-577.[Medline]
Liu, D., Martino, G., Thangaraju, M., Sharma, M., Halwani, F., Shen, S. H., Patel, Y. C. and Srikant, C. B. (2000). Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis. J. Biol. Chem. 275, 9244-9250.
Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Mann, M., Krammer, P. H. and Peter, M. E. (1997). FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794-2804.
Mundle, S. D. and Raza, A. (2002). Defining the dynamics of self-assembled Fas-receptor activation. Trends Immunol. 23, 187-194.[CrossRef][Medline]
Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R. et al. (1996). FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817-827.[Medline]
Nemoto, S., Xiang, J., Huang, S. and Lin, A. (1998). Induction of apoptosis by SB202190 through inhibition of p38beta mitogen-activated protein kinase. J. Biol. Chem. 273, 16415-16420.
Park, S. J., Kim, Y. Y., Ju, J. W., Han, B. G., Park, S. I. and Park, B. J. (2001). Alternative splicing variants of c-FLIP transduce the differential signal through the Raf or TRAF2 in TNF-induced cell proliferation. Biochem. Biophys. Res. Commun. 289, 1205-1210.[CrossRef][Medline]
Porras, A., Zuluaga, S., Black, E., Valladares, A., Alvarez, A. M., Ambrosino, C., Benito, M. and Nebreda, A. R. (2004). p38{alpha} mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol. Biol. Cell 15, 922-933.
Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H. and Peter, M. E. (1998). Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17, 1675-1687.
Schurmann, A., Mooney, A. F., Sanders, L. C., Sells, M. A., Wang, H. G., Reed, J. C. and Bokoch, G. M. (2000). p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis. Mol. Cell. Biol. 20, 453-461.
Shu, H. B., Halpin, D. R. and Goeddel, D. V. (1997). Casper is a FADD- and caspase-related inducer of apoptosis. Immunity 6, 751-763.[Medline]
Siegel, R. M., Frederiksen, J. K., Zacharias, D. A., Chan, F. K., Johnson, M., Lynch, D., Tsien, R. Y. and Lenardo, M. J. (2000). Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 288, 2354-2357.
Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., Litwack, G. and Alnemri, E. S. (1996). Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93, 14486-14491.
Stennicke, H. R., Jurgensmeier, J. M., Shin, H., Deveraux, Q., Wolf, B. B., Yang, X., Zhou, Q., Ellerby, H. M., Ellerby, L. M., Bredesen, D. et al. (1998). Pro-caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem. 273, 27084-27090.
Tikhomirov, O. and Carpenter, G. (2004). Ligand-induced, p38-dependent apoptosis in cells expressing high levels of EGF receptor and ErbB-2. J. Biol. Chem. 279, 12988-12996.
Tournier, C., Hess, P., Yang, D. D., Xu, J., Turner, T. K., Nimnual, A., Bar-Sagi, D., Jones, S. N., Flavell, R. A. and Davis, R. J. (2000). Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 288, 870-874.
Virdee, K., Parone, P. A. and Tolkovsky, A. M. (2000). Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr. Biol. 10, 1151-1154.[CrossRef][Medline]
Vivo, C., Liu, W. and Broaddus, V. C. (2003). c-Jun N-terminal kinase contributes to apoptotic synergy induced by tumor necrosis factor-related apoptosis-inducing ligand plus DNA damage in chemoresistant, p53 inactive mesothelioma cells. J. Biol. Chem. 278, 25461-25467.
Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B. and Korsmeyer, S. J. (2001). Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727-730.
Werner, A. B., de Vries, E., Tait, S. W., Bontjer, I. and Borst, J. (2002). Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax. J. Biol. Chem. 277, 22781-22788.
Wolf, D., Witte, V., Laffert, B., Blume, K., Stromer, E., Trapp, S., d'Aloja, P., Schurmann, A. and Baur, A. S. (2001). HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals. Nat. Med. 7, 1217-1224.[CrossRef][Medline]
Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G. and Youle, R. J. (1997). Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281-1292.
Yang, B. F., Xiao, C., Roa, W. H., Krammer, P. H. and Hao, C. (2003). Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of fas-mediated signaling in malignant glioma cells. J. Biol. Chem. 278, 7043-7050.
Yoshino, T., Kishi, H., Nagata, T., Tsukada, K., Saito, S. and Muraguchi, A. (2001). Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes. Eur. J. Immunol. 31, 2702-2708.[CrossRef][Medline]
Yusta, B., Boushey, R. P. and Drucker, D. J. (2000). The glucagon-like peptide-2 receptor mediates direct inhibition of cellular apoptosis via a cAMP-dependent protein kinase-independent pathway. J. Biol. Chem. 275, 35345-35352.
Zha, J., Harada, H., Yang, E., Jockel, J. and Korsmeyer, S. J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 87, 619-628.[Medline]
Zhang, H., Shi, X., Zhang, Q. J., Hampong, M., Paddon, H., Wahyuningsih, D. and Pelech, S. (2002). Nocodazole-induced p53-dependent c-Jun N-terminal kinase activation reduces apoptosis in human colon carcinoma HCT116 cells. J. Biol. Chem. 277, 43648-43658.
Zhang, J., Gao, J. X., Salojin, K., Shao, Q., Grattan, M., Meagher, C., Laird, D. W. and Delovitch, T. L. (2000). Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase. J. Exp. Med. 191, 1017-1030.