Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-01, Japan
IL-1 converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-,
CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by
activation of CPP32-like proteases which is thought to
be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK
and p38, are activated during Fas-induced apoptosis.
Here, we have shown that MKK7, but not SEK1/
MKK4, is activated by Fas as an activator for JNK/
SAPK and that MKK6 is a major activator for p38 in
Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost
all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and
Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the
activation of JNK/SAPK and p38. Interestingly, these
DEVD-type inhibitors did not block the Fas-induced
morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell
death (as assessed by the dye exclusion ability). These
results suggest that the Fas-induced activation of the
JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events
(such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the
cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes,
each of which may be responsible for part of the apoptotic cellular responses.
APOPTOSIS, a mechanism of cell suicide, is an intrinsic
biological event that plays an essential role in various developmental stages and also in immune systems. Apoptosis is characterized by dramatic morphological changes of the cell, including membrane blebbing, cell
shrinkage, chromatin condensation, DNA cleavage, and
fragmentation of the cell into apoptotic bodies. The apoptotic pathway could be divided into four steps, decision to
die, execution of death, engulfment of apoptotic cells by
macrophages, and degradation of apoptotic cells. An insight into the execution machinery of apoptosis has come
from genetical studies in the nematode Caenorhabditis elegans. Three kinds of apoptosis-related genes have been
cloned, ced-3, ced-4, and ced-9. The former two genes are
required for execution of death (Ellis et al., 1991 Fas (Yonehara et al., 1989 Two subfamily members of the MAP kinase superfamily, JNK/SAPK (Derijard et al., 1994 Cell Cultures
Jurkat cells were cultured in RPMI 1640 containing 10% fetal calf serum.
KB cells were cultured in Dulbecco's modified Eagle's medium containing
10% fetal calf serum. These cells were maintained in 5% CO2 at 37°C. KB
cells were transiently transfected using LipofectAMINE (GIBCO BRL,
Gaithersburg, MD) as described previously (Moriguchi et al., 1996a Reagents, Antibodies, and Recombinant Proteins
Ac-YVAD-CHO and Ac-DEVD-CHO were purchased from Peptide Institute, Inc. (Osaka, Japan) and Z-VAD-FK and Z-DEVD-FK from Kamiya Biomedical Co. (Tukwila, WA). Anti-Fas mAb (clone CH-11),
Apo2.7 mAb, anti-actin mAb (A-4700), and anti-JNK1 and anti-p38 polyclonal antibodies were purchased from MBL (Nagoya, Japan), Immunotech (Luminy, France), Sigma Chem. Co. (St. Louis, MO), and Santa Cruz Biotechnology Inc., respectively. His-tagged wild-type SAPK Immunecomplex Kinase Assays
The cell lysate was prepared as described (Moriguchi et al., 1996b Column Chromatography
The cell lysate, obtained from Jurkat cells (200 ml culture) treated with
anti-Fas mAb (CH-11, 150 ng/ml) for 5 h, were loaded onto a HiTrap Q (1 ml; Pharmacia Biotech Inc.) equilibrated with extraction buffer containing
0.01% Brij-35 (buffer A). The column was washed with 3 ml of buffer A
and developed with a 18 ml NaCl gradient (0-0.3 M NaCl). To measure
the activity to activate SAPK Immunodepletion
Jurkat cells were treated with anti-Fas mAb (CH-11, 150 ng/ml) for 5 h
and the cell lysate was prepared as described above. The lysate was incubated for 1 h at 4°C with increasing amounts of anti-MKK6 polyclonal antibody (Moriguchi et al., 1996b Jurkat cells were treated with anti-Fas mAb (CH-11, 200 ng/ml) for 5 h
and the cell lysate was loaded onto a Hitrap Q column as described above.
The flow-through fraction was incubated for 2 h at 4°C with anti-MKK7
polyclonal antibody (Moriguchi et al., manuscript in preparation) and protein A-Sepharose beads, and then centrifuged. The activity to activate
JNK/SAPK, remaining in the supernatant, was measured by using 1 µg
GST-SAPK and 3 µg His-tagged c-Jun.
Cell Staining
Jurkat cells were treated with anti-Fas mAb (CH-11, 150 ng/ml) for 20 h.
Cells were fixed and stained with Apo2.7 mAb (Zhang et al., 1996 DNA Fragmentation Assay
Jurkat cells (106 cells) were treated with anti-Fas mAb (CH-11, 150 ng/ml)
for 20 h. Cells were collected, washed once in PBS (20 mM NaH2PO4, 150 mM NaCl, pH 7.4), lysed in 100 µl TE-T buffer (10 mM Tris, 10 mM
EDTA, 0.5% Triton X-100) and then centrifuged at 15,000 rpm for 20 min. The supernatant was subjected to digestion with ribonuclease A (0.4 mg/ml) for 1 h at 37°C followed by incubation with proteinase K (0.4 mg/
ml) for 1 h at 37°C. The sample was then extracted with isopropanol for
overnight at Cell Death Assay
Cell death was measured by two assays, trypan blue staining and MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MTT
assay was done as previously described (Mosmann, 1983 Fas Induces Activation of the MKK7-JNK/SAPK and
MKK6-p38 Protein Kinase Cascades
It was recently reported that two subfamily members of
the MAP kinase superfamily, JNK/SAPK and p38, are activated in Fas-induced apoptosis (Latinis et al., 1996; Wilson et al., 1996
As for the MAP kinase kinase (MAPKK) superfamily,
we could not detect significant activation of SEK1/MKK4,
a well-known activator of JNK/SAPK, during Fas-induced
signaling (Fig. 1 B, left). We have recently identified another activator for JNK/SAPK designated MKK7 (Moriguchi et al., manuscript in preparation). Then, we measured the activity of endogenous MKK7 by the immunecomplex
kinase assay and found that MKK7 was activated by Fas
before the activation of JNK/SAPK (Fig. 1, A and B). To
examine the possibility that MKK7, but not SEK1/MKK4,
is activated by Fas as a major activator for JNK/SAPK, we
subjected the cell lysate obtained from the anti-Fas- treated Jurkat cells to chromatography on Hitrap-Q (Fig.
1 C). The JNK/SAPK activating activity was measured by
increased phosphorylation of c-Jun in the presence of recombinant SAPK. Two peaks of JNK/SAPK activating activity were observed; one large peak in the flow-through (FT) and wash (W) fractions and the other, small peak
eluting at about 0.1 M NaCl (Fig. 1 C). We have previously
shown that MKK7 was eluted in the flow-through and
wash fractions and SEK1/MKK4 in the 0.1 M NaCl fractions on Hitrap-Q (Moriguchi et al., 1995 We found also that MKK6, a specific activator of p38,
was activated markedly by Fas before the activation of p38
(Fig. 1 A and B). In contrast to relatively strong activation
of MKK6, another activator of p38, MKK3, was activated
only weakly (Fig. 1 B, right). To examine whether MKK6
acts as the major activator of p38 in Fas-induced signaling,
we immunodepleted endogenous MKK6 in the cell lysate
obtained from Fas-treated Jurkat cells by using a specific
polyclonal anti-MKK6 antibody (Fig. 1 D). The p38 activating activity remaining in the supernatant was decreased
to less than 15% of the original level by increasing the
amount of anti-MKK6 antibody (Fig. 1 D, upper). Immunoblotting of the supernatants with anti-MKK6 and anti-MKK3 antibodies showed that while the protein level of
MKK6 was decreased in parallel with the decrease in the
p38 activating activity, the protein level of MKK3 in the supernatant was not decreased at all (Fig. 1 D, lower).
Therefore, it may be concluded that the major activator of
p38 in Fas-induced signaling is MKK6, and MKK3 constitutes only part of the p38 activating activity.
Fas-induced Activation of JNK/SAPK and p38 Is
Inhibited by Z-VAD-FK but Not by Ac-DEVD-CHO
or Ac-YVAD-CHO
To analyze the relationship between the ICE family proteases and the JNK/SAPK and p38 kinase cascades, we
used several kinds of peptide inhibitors for ICE family
proteases with different specificities. Z-VAD-FK, an inhibitor for a broad spectrum of ICE family proteases,
blocked the Fas-induced activation of JNK/SAPK and p38
almost completely (Fig. 2 B, left and right). In contrast, Ac-YVAD-CHO, a specific inhibitor for ICE-like proteases, did not block the activation of either kinase (Fig. 2
C). This is not surprising because, as described below,
Ac-YVAD-CHO did not influence any cellular responses
induced by Fas. Importantly, Ac-DEVD-CHO, a specific
inhibitor for CPP32-like proteases, hardly blocked the activation of JNK/SAPK and p38 (Fig. 2 A). This is not due
to insufficiency of the amount of this inhibitor because the
same concentration of the inhibitor blocked the Fas-induced chromatin condensation and DNA fragmentation (see Figs.
3 and 4). In addition, essentially the same results were
obtained by using Z-DEVD-FK, the other specific inhibitor for CPP32-like proteases, that is more permeable than
Ac-DEVD-CHO (data not shown).
Fas-induced Cellular Responses Are Totally Inhibited
by Z-VAD-FK but Only Partly by Ac-DEVD-CHO
To dissect the Fas-induced apoptotic signaling pathways,
we investigated the effects of several kinds of protease inhibitors on the Fas-induced cellular responses. We first examined chromatin condensaton and DNA fragmentation,
typical nuclear phenomena in apoptosis. Jurkat cells were
incubated with several kinds of inhibitors for ICE family
for 1 h followed by treatment with anti-Fas mAb (150 ng/
ml) for 20 h. The cells were fixed and stained with DAPI (Fig. 3, b, e, h, k, and n). In the presence of Z-VAD-FK
the Fas-induced chromatin condensation was completely
inhibited (Fig. 3, k, and Fig. 4 B, VAD) and no change in
cell morphology was induced by Fas (Fig. 3, a, d, and j).
Cellular DNA was extracted and analyzed by electrophoresis to detect DNA fragmentation (Fig. 4 C). The Fas-induced DNA fragmentation was also inhibited by
Z-VAD-FK (Fig. 4 C, VAD). In contrast, in the presence
of Ac-YVAD-CHO, neither chromatin condensation nor
DNA fragmentation was inhibited (Fig. 3, n, Fig. 4 B,
YVAD, and Fig. 4 C, YVAD), and the cells were fragmented into apoptotic bodies by Fas (Fig. 3, m). Although Enari et al. (1995 We next examined Apo2.7 staining. Apo2.7 is a monoclonal antibody obtained by immunizing mice with apoptotic Jurkat cells (Zhang et al., 1996 Fas-induced Cell Death Is Not Inhibited
by Ac-DEVD-CHO
To examine the viability of the anti-Fas-treated cells, we
first used the MTT assay which measures the activity of
the mitochondrial enzyme. As shown in Fig. 5 A, the Fas-induced loss of mitochondrial activity was strongly inhibited by Z-VAD-FK but only weakly inhibited by Ac-DEVD-CHO. We next assayed membrane permeability of
Ac-DEVD-CHO-treated cells by trypan blue dye exclusion. Even in the presence of Ac-DEVD-CHO, which inhibited the Fas-induced chromatin condensation and DNA fragmentation, anti-Fas-treated cells lost thier ability of
dye exclusion (Fig. 5 B), suggesting that the Fas-induced
cell death is not inhibited by Ac-DEVD-CHO.
The Activation of JNK/SAPK and p38, the Changes in
Cell Morphology, and the Cell Death Induced by Fas in
KB Cells Are Not Blocked by Inhibition of
CPP32-like Proteases
To generalize the above observation that a CPP32-like
protease(s) may be specifically involved in the Fas-induced
chromatin condensation and DNA fragmentation, but not
in other apoptotic responses, we then used KB cells. It is
known that the apoptosis of KB cells can be induced by
Fas in the presence of cycloheximide. Cycloheximide treatment alone did not induce any apoptotic responses in KB
cells (data not shown). We then examined the activities of
JNK/SAPK and p38 during Fas stimulation in the presence of cycloheximide (Fig. 6). KB cells were preincubated
with 50 µg/ml cycloheximide for 1 h, and then treated with
anti-Fas mAb (CH-11, 200 ng/ml, at time 0 in Fig. 6 A). Cycloheximide is one of known activators of JNK/SAPK (Kyriakis et al., 1994
We next examined other apoptotic responses in KB
cells. KB cells were preincubated with or without each of
peptide inhibitors, Z-VAD-FK or Z-DEVD-FK, for 1 h in
the presence of 50 µg/ml cycloheximide. The cells were
then treated with anti-Fas mAb (CH-11, 150 ng/ml). In the
absence of peptide inhibitors, dramatic morphological
changes, rounding and shrinkage of the cell and membrane blebbing, were induced 3-4 h after Fas stimulation,
and almost all the cells became detached from the dish and
fragmented into apoptotic bodies within 8 h (Fig. 7 A, anti-Fas). In the presence of Z-VAD-FK, all of the Fas-induced
cellular responses were completely inhibited (Fig. 7, A and
B, Z-VAD-FK + anti-Fas). In the presence of Z-DEVD-FK, cellular shrinkage and membrane blebbing were induced
by Fas stimulation, like in the absence of the inhibitor, but
the cells were not detached from the dish and eventually died
without any fragmentation of the cell (Fig. 7 A, Z-DEVD-FK + anti-Fas). When KB cells were fixed 8 h after Fas
stimulation, and stained with DAPI and phalloidin, the
Fas-induced chromatin condensation and disruption of
actin network were clearly observed (Fig. 7 B, anti-Fas).
Z-VAD-FK completely blocked either response; the cells
exhibited normal nuclear morphology and normal actin
network (Fig. 7 B, Z-VAD-FK + anti-Fas). Importantly, Z-DEVD-FK inhibited strongly the Fas-induced chromatin condensation but did not block the disruption of actin
network; the cells thus showed only partial chromatin condensation and no actin network (Fig. 7 B, Z-DEVD-FK + anti-Fas). Essentially the same results were obtained with
Ac-DEVD-CHO (data not shown).
It was previously reported that actin (45 kD) was proteolytically cleaved by ICE family proteases into 15- and
30-kD fragments in several types of apoptosis (Mashima et
al., 1995 Apoptosis or a programmed cell death is a mechanism of
cell suicide that is conserved in many kinds of species. It
has been shown that ICE and/or ICE-related proteases
play a central role in execution of cell death in many types
of apoptosis. The ICE-related proteases, now designated
caspase-1 to -10, are classified into three groups; ICE-,
CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, it has been suggested that the activation of ICE-like proteases is followed by the activation of CPP32-like proteases and that CPP32-like proteases are essential for Fas-induced apoptotic responses. In addition, a number of
molecules other than proteases have been suggested to be
involved in Fas-induced apoptotic signaling, including ceramide, reactive oxygen species, protein kinases, protein
phosphatases, and cytochrome c. But, the relationship between the ICE-related proteases and the other molecules
has not been fully understood. Further, how these molecules are related to apoptotic cellular responses has not
been analyzed in detail. To address these questions, we
first focused on the MAP kinase superfamily molecules,
and found that slow and sustained activation of both the
MKK7-JNK/SAPK cascade and the MKK6-p38 cascade
occurs during Fas-induced apoptosis in an ICE family protease(s)-dependent manner. This extends previous observations showing activation of JNK/SAPK (Kevin et al.,
1996; Wilson et al., 1996 The close examination of the anti-Fas-treated Jurkat cells
in the presence of the inhibitor for CPP32-like proteases
revealed that a number of apoptotic responses in cells occur
independent of CPP32-like proteases, including cell shrinkage and induction of Apo2.7 staining in mitochondria.
More importantly, even the cell death itself, as assessed by
the dye exclusion ability, can be induced by Fas stimulation in the presence of the inhibitor for CPP32-like proteases.
In this case, the cells die without chromatin condensation
and DNA fragmentation.
In KB cells that are cultured as mono-layer on a dish,
Fas stimulation induces apoptosis in the presence of cycloheximide. The Fas-induced morphological changes are
quite conspicuous in KB cells and thus are easily detected.
Inhibition of CPP32-like proteases did not block these dramatic morphological changes of KB cells such as rounding
and shrinkage and membrane blebbing, but did inhibit
chromatin condensation. These anti-Fas-treated KB cells
with the inhibitor for CPP32-like proteases eventually died
and became detached from the dish, although the detachment was markedly delayed as compared with the cells
that were treated with anti-Fas in the absence of the inhibitor. Therefore, Fas stimulation is able to induce the death
of KB cells without chromatin condensation when CPP32-like proteases are inhibited. Thus, in both Jurkat cells and
KB cells there are CPP32-like proteases-dependent and
-independent pathways; the former leads to chromatin
condensation and DNA fragmentation and the latter may
lead to other apoptotic responses such as morphological
changes. A hypothetical model of our proposed signaling
pathways in Fas-induced apoptosis is shown in Fig. 8. Differential involvement of different proteases in different
apoptotic responses has also been suggested recently (Cryns et al., 1996
The present finding that both the Fas-induced morphological changes (cell shrinkage and membrane blebbing)
and the Fas-induced activation of the MAPK superfamily
molecules (JNK/SAPK and p38) are independent of CPP32-like proteases might suggest the possibility of involvement
of the MAPK superfamily in the morphological changes. In our preliminary experiments, however, expression of
a dominant negative form of MKK7 or a MAPK phosphatase-1 (MKP-1 or CL100) did not affect the Fas-induced
cell shrinkage and membrane blebbing in the presence of
Z-DEVD-FK, the inhibitor for CPP32-like proteases. Thus,
the MAPK superfamily molecules may not be directly responsible for the Fas-induced morphological responses.
We may hypothesize that the Fas signaling pathway diverges into multiple, separate biochemical and cellular
processes, each of which may be responsible for each part
of the apoptotic cellular responses. The molecular mechanisms for the Fas-induced morphological changes as well
as the cellular events elicited by the Fas-induced activation
of the MAPK superfamily molecules are to be elucidated
in future studies.
; Yuan
and Horvitz, 1992
), whereas ced-9 prevents cell death
(Hengartner et al., 1992
). Mammalian homologues of ced-3
and ced-9 have been identified as IL-1
converting enzyme (ICE)1 cysteine protease (Yuan et al., 1993
) and oncogene bcl-2 (Hengartner and Horvitz, 1994
), respectively.
A number of ICE-related proteases have been cloned including ICE (caspase-1), ICH-1/Nedd-2 (caspase-2) (Kumar et al., 1994
; Wang et al., 1995
), CPP32/Yama/apopain
(caspase-3) (Fernandes-Alnemri et al., 1994
; Nicholson et
al., 1995
; Tewari, 1995), TX/ICErel-II/ICH-2 (caspase-4)
(Faucheu et al., 1995
; Kamens et al., 1995
; Munday et al.,
1995
), ICErel-III/TY (caspase-5) (Faucheu et al., 1995
;
Munday et al., 1995
), Mch2 (caspase-6) (Fernandes-
Alnemri et al. 1995a), Mch3/ICE-LAP3/CMH-1 (caspase-7) (Fernandes-Alnemri et al., 1995b
; Duan et al., 1996a
; Lippke
et al., 1996
), FLICE/MACH/Mch5 (caspase-8) (Boldin et
al., 1996
; Fernandes-Alnemri et al., 1996
; Muzio et al.,
1996
), ICE-LAP6/Mch6 (caspase-9) (Duan et al., 1996b
;
Srinivasula et al., 1996
), and Mch4 (caspase-10) (Fernandes-
Alnemri et al., 1996). These ICE-related proteases are
classified into three groups; ICE-, CPP32-, and ICH-1-like
proteases.
) is a type-I membrane protein and belongs to the TNF/NGF receptor superfamily
(Itoh et al., 1991
). The death domain localized in the cytoplasmic region of Fas is essential for transducing apoptotic signals to cytoplasm (Itoh and Nagata, 1993
). Upon
crosslinking with its ligand or anti-Fas antibody, Fas trimerizes and binds to FADD/MORT1 (Boldin et al., 1995
;
Chinnaiyan et al., 1995
, 1996
) through its death domain. FADD/MORT1 binds to the NH2-terminal region of
FLICE/MACH/Mch5 (Boldin et al., 1996
; Muzio et al.,
1996
). Then FLICE/MACH/Mch5 may be activated, and
other members of ICE-related proteases might be sequentially activated. It has been shown that activation of CPP32-like proteases follows the activation of ICE-like
proteases (Enari et al., 1995
, 1996
; Los et al., 1995
) and inhibition of CPP32-like proteases prevents Fas-induced
apoptosis (Schlegel et al., 1996
). Therefore, CPP32-like
proteases are thought to be essential for Fas-induced apoptosis. It remains, however, unclear how these ICE-related
proteases are involved in each of the apoptotic cellular responses.
; Kyriakis et al., 1994
)
and p38 (Han et al., 1994
; Lee et al., 1994
; Rouse et al.,
1994
), have recently been implicated in several types of
apoptosis, such as apoptosis of differentiated PC12 cells
induced by NGF-deprivation (Xia et al., 1995
) and apoptosis of different kinds of cells induced by ceramide (Verheij et al., 1996
), UV and
irradiation (Chen et al., 1996
).
In the Fas-induced signaling pathway also, activation of
JNK/SAPK and p38 has been observed (Latinis et al.,
1996; Wilson et al., 1996
; Juo et al., 1997
; Lenczowski et al.,
1997
). However, little is known about upstream activators
for these kinases in this pathway, and the relationship between these kinases and the ICE-related proteases remains unclear. Here we have shown first that MKK7 (= a
newly identified JNK/SAPK activator; Moriguchi et al.,
1997
), but not SEK1/MKK4 (Sanchez et al., 1994
; Derijard et al., 1995
), is activated by Fas as an activator for JNK/
SAPK and that MKK6 (Cuenda et al., 1996
; Han et al.,
1996
; Moriguchi et al., 1996a
; Raingeaud et al., 1996
; Stein
et al., 1996
) acts as a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by
Fas, we used several kinds of inhibitors for ICE-related
proteases with different specificities; Z-VAD-FK as an inhibitor for a broad spectrum of ICE-related proteases, Ac-DEVD-CHO and Z-DEVD-FK as specific inhibitors for
CPP32-like proteases, and Ac-YVAD-CHO as a specific inhibitor for ICE-like proteases. Z-VAD-FK blocked both
the cell death and the activation of JNK/SAPK and p38,
but Ac-YVAD-CHO did not block either response. Importantly, Ac-DEVD-CHO and Z-DEVD-FK did block
the chromatin condensation and DNA fragmentation almost completely, but did not block the activation of JNK/
SAPK and p38. Moreover, these inhibitors did not block
the Fas-induced membrane blebbing, cell shrinkage or other
apoptotic events of the cytoplasm. Thus, in the presence of
the inhibitors for CPP32-like proteases, Fas causes the cell
to die without any chromatin condensation. These results suggest that Fas can induce at least two separate signaling
pathways; one is CPP32-like proteases-dependent and the
other is independent and that the former pathway is essential for some of the apoptotic nuclear events such as the
drastic changes in chromatin and DNA, but is dispensable
for the apoptotic cytoplasmic events, the activation of
JNK/SAPK and p38, or the cell death.
Materials and Methods
).
, kinase
negative MPK2, c-Jun, and ATF-2 were expressed in Escherichia coli and
purified using a Ni+ affinity column (Moriguchi et al., 1996b
). GST-kinase
negative SAPK
, GST wild-type SAPK
, and GST-p38 were purified by
affinity chromatography on glutathione-Sepharose 4B (Pharmacia LKB
Biotech Inc., Piscataway, NJ) (Moriguchi et al., 1996b
).
). In
brief, Jurkat cells were lysed in lysis buffer (20 mM Hepes, pH 7.35, 12.5 mM 2-glycerophosphate, 150 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 2 mM DTT, 1 mM sodium vanadate, 1 mM
PMSF, and 20 µg/ml aprotinin) and then centrifuged at 15,000 rpm for 15 min. JNK/SAPK, p38, SEK1/MKK4/XMEK2, MKK3, MKK6, and MKK7
were precipitated from the cell lysate by incubation with specific antibodies and protein A-Sepharose beads (Pharmacia Biotech Inc.) as described
previously (Moriguchi et al., 1996a
,b; Moriguchi et al., 1997
). Each precipitate was washed three times with TBS-T (20 mM Tris, pH 7.5, 0.15 M
NaCl, 0.2% Tween-20), and once with extraction buffer (20 mM Tris, pH
7.2, 25 mM 2-glycerophosphate, 2 mM EGTA, 2 mM DTT, 1 mM sodiume
vanadate) and then mixed with 3 µg of each substrate (His-tagged c-Jun
for JNK/SAPK, GST-kinase negative SAPK for SEK1/MKK4/XMEK2
and MKK7, His-tagged ATF2 for p38, and His-tagged kinase negative
MPK2 for MKK3 and MKK6), 50 µM ATP, 15 mM MgCl2, and 2 µCi of
[
-32P]ATP and incubated for 20 min at 30°C in a final volume of 15 µl.
The reaction was stopped by addition of Laemmli's sample buffer and
boiling. The reaction mixture was resolved by SDS-PAGE analysis.
, each fraction (7 µl) was first incubated for
30 min at 30°C with or without 1 µg His-tagged SAPK
, 100 µM ATP, and
20 mM MgCl2 and subsequently incubated for 20 min at 25°C with 3 µg of
His-tagged c-Jun and 1 µCi [
-32P]ATP (final volume, 15 µl). The reaction
was stopped by addition of Laemmli's sample buffer and boiling.
) and protein A-Sepharose beads, and then
centrifuged. To measure the activity of the supernatant to activate p38,
the samples were first incubated for 30 min at 30°C with 1 µg GST-p38, 50 µM ATP, and 15 mM MgCl2 followed by incubation for 1 h at 4°C with glutathione-Sepharose 4B. The precipitated GST-p38 was washed three
times with TBS-T and once with extraction buffer, and then incubated for
20 min at 20°C with 3 µg His-tagged ATF2, 50 µM ATP, and 15 mM
MgCl2, and 2.5 µCi [
-32P]ATP. The reaction was stopped by addition of
Laemmli's sample buffer and boiling. Each of the supernatants was analyzed by immunoblotting with anti-MKK6 mAb (Moriguchi et al., 1996b
)
and anti-MKK3 polyclonal antibody (C-19; Santa Cruz Biotechnology Inc.).
) by the
slightly modified method. In brief, cells were fixed in 1% paraformaldehyde solution in PBS for 2 min on ice and washed twice with PBS followed
by incubation for 15 h on ice with Phycoerythrin-labeled Apo2.7 mAb
(Immunotech), 40 µg/ml digitonin, and 0.1 mg/ml DAPI in blocking
buffer (1 mg/ml goat IgG, 10 mg/ml BSA in PBS). KB cells were treated
with anti-Fas mAb (CH-11, 150 ng/ml) for 8 h and fixed in 3.7% formalin
solution in PBS for 10 min at 37°C and permeabilized by incubation in
0.5% Triton X-100 in PBS for 10 min. Cells were stained with 0.1 mg/ml DAPI and FITC-labeled phalloidin. Cells were observed under fluorescent microscope (Axiophot).
20°C. DNA was precipitated and resuspended in 20 µl of
TE buffer and analyzed by electrophoresis on a 0.7% agarose gel in the
presence of 0.5 µg/ml ethidium bromide.
). In brief, Jurkat
cells (5 x 103 cells) were incubated overnight in 96-well plate and treated with anti-Fas mAb (CH-11, 150 ng/ml) for 20 h in the presence or absence
of various inhibitors. 10 µl of a 5 mg/ml MTT (Sigma) stock solution was
added per well and incubated for 4 h at 37°C. Cells were lysed and the difference between the absorbance at 562 and 650 nm of each sample was
measured. The value of the sample without anti-Fas mAb was regarded as
100% survival. The percentage of cell death was expressed as: 100
(%
survival).
Results
; Juo et al., 1997
; Lenczowski et al., 1997
).
We confirmed the activation of both JNK/SAPK and p38
in Fas-induced signaling by measuring the activities of endogenous JNK/SAPK and endogenous p38 in Jurkat T
cells at various time points after treatment with anti-Fas
mAb (CH-11). The activation was relatively slow but sustained; the increase in the kinase activities was detected 2 h
after Fas stimulation and the maximal activation was attained at about 6 h (Fig. 1 A). In this series of experiments
the apoptotic bodies began to appear under microscope 4 h
after Fas stimulation.
Fig. 1.
(A) Activation of JNK/SAPK and MKK7 (top) and p38 and MKK6 (bottom) during Fas-induced apoptosis. Jurkat cells were
incubated with anti-Fas mAb (CH-11, 250 ng/ml) for various periods, and were collected and lysed. The activities of JNK/SAPK (top),
MKK7 (top), p38 (bottom), and MKK6 (bottom) in the cell lysate were determined by immunecomplex kinase assays. Each kinase activity was expressed as percent activation, relative to the maximal activation. The autoradiographed images are presented beneath the
graph. (B) The activities of SEK1/MKK4 and MKK3 during Fas-induced apoptosis. Jurkat cells were left untreated (untreated), or were
incubated with anti-Fas (CH-11, 250 ng/ml) for 5 h (anti-Fas), or exposed to 0.7 M NaCl for 30 min (NaCl), and were collected and lysed. The activities of MKK7 (left), SEK1/MKK4(left), MKK6 (right), and MKK3 (right) in the cell lysate were determined by immunecomplex kinase assays. (C) Fractionation of JNK/SAPK activating activity. Jurkat cells (200 ml culture) were incubated with anti-Fas mAb (CH-11, 150 ng/ml) for 5 h, and collected and lysed. The cell lysate was subjected to Hitrap-Q chromatography, and each fraction was assayed for JNK/SAPK activating activity by measuring c-Jun phosphorylating activity in the presence (SAPK+) or absence (SAPK) of
His-tagged SAPK. (Inset) A portion (300 µl) of the unadsorbed fraction (FT) was subjected to immunoprecipitation without (
) or with
(+) anti-MKK7 antibody (10 µl). JNK/SAPK activating activity remaining in the supernatant was measured by c-Jun phosphorylating activity in the presence of GST-SAPK. (D) Immunodepletion of MKK6. Jurkat cells were incubated with anti-Fas mAb (CH-11, 250 ng/ ml) for 5 h, and collected and lysed. A portion of the cell lysate was subjected to immunoprecipitation with increasing amounts of anti-MKK6 pAb. p38 activating activity remaining in the supernatant was measured by ATF-2 phosphorylating activity in the presence of
GST-p38 (top, closed circles). Preimmune serum was used for a control experiment (top, open circles). Each of the supernatants was analyzed by immunoblotting with anti-MKK6 and anti-MKK3 antibodies (bottom).
[View Larger Versions of these Images (23 + 18 + 18 + 17K GIF file)]
). We fractionated this large peak of the unadsorbed fractions of Hitrap-Q
by heparin-Sepharose and found that elution of the JNK/ SAPK activating activity coincided with the elution of the
MKK7 polypeptide detected by immunoblotting (data not
shown). Furthermore, >50% of the JNK/SAPK activating
activity in the unadsorbed fraction of the Hitrap-Q chromatography could be immunoprecipitated with anti-MKK7
antibody (Fig. 1 C, inset). Although we could not rule out
the possibility that there is another activator for JNK/
SAPK in the unadsorbed fraction since some activity remained in the antibody-treated supernatant, this might result from the low titer of the antibody. Taken together,
these results suggested that MKK7, but not SEK1/MKK4,
acts as a major activator for JNK/SAPK in Fas-induced
signaling.
Fig. 2.
Effect of ICE-family protease inhibitors on Fas-induced
activation of JNK/SAPK (left) and p38 (right). Jurkat cells were
preincubated with ICE-family protease inhibitors (A, Ac-DEVD-CHO, 120 µM; B, Z-VAD-FK, 40 µM; C, Ac-YVAD-CHO, 120 µM) (closed circles) or with the solvent DMSO (open circles) as
control for 1 h and then treated with anti-Fas mAb (CH-11, 150 ng/ml) for various periods. Cell lysates were prepared, and the
activities of endogenous JNK/SAPK (left panels) and endogenous p38 (right panels) were determined by immunecomplex kinase assays. The kinase activity was expressed as percent activation, relative to the maximal activation in control cells at 6 h
incubation as 100%.
[View Larger Version of this Image (22K GIF file)]
Fig. 3.
Effect of ICE-family protease inhibitors on Fas-induced
cellular responses. Jurkat cells were treated with anti-Fas mAb
(CH-11, 150 ng/ml) for 20 h after a 1-h preincubation without
(d-f) or with ICE-family protease inhibitors; Ac-DEVD-CHO,
120 µM (g-i); Z-VAD-FK, 40 µM (j-l); Ac-YVAD-CHO, 120 µM
(m-o). Cells were fixed and double stained with DAPI (middle
panels) and Apo2.7 (right panels). Left panels show phase contrast
images of cells before fixation.
[View Larger Version of this Image (71K GIF file)]
Fig. 4.
Inhibition of CPP32-like proteases did block Fas-induced chromatin condensation and DNA fragmentation but did not block
induction of Apo2.7 staining. Jurkat cells were treated as in Fig. 3, and the cells that were stained with Apo2.7 (A) and the cells with apoptotic condensed nuclei (B) were counted. (C) Cellular DNA was extracted and analyzed by electrophoresis on a 1.7% agarose gel to detect DNA fragmentation.
[View Larger Versions of these Images (46 + 40 + 92K GIF file)]
, 1996
) and Los et al. (1995)
showed previously that Fas-induced apoptosis in W4 cells or SKW 6.4 cells is blocked by YVAD-type inhibitors, we could not
see any inhibitory effect of Ac-YVAD-CHO on the Fas-induced apoptosis in Jurkat cells even at the concentration
of 200 µM. This discrepancy may be due to the difference
of cell types used as several groups also demonstrated the
lack of inhibitory effect of YVAD-type inhibitors on the
Fas-induced apoptosis in Jurkat cells (Martin et al., 1996
;
Schlegel et al., 1996
). The effect of Ac-DEVD-CHO was
interesting. In the presence of Ac-DEVD-CHO, the Fas-induced chromatin condensation and DNA fragmentation
were almost completely inhibited (Fig. 3, h, Fig. 4 B, DEVD,
and Fig. 4 C, DEVD), but a marked change in the cell morphology, cell shrinkage, was induced by Fas (Fig. 3, g).
). It has been shown
that Apo2.7 recognizes a 38-kD protein which is expressed
on mitochondrial membrane in apoptotic Jurkat cells and
that Apo2.7 reacts only with apoptotic Jurkat cells but not
with nonapoptotic cells (Zhang et al., 1996
). In fact,
Apo2.7 staining was detected in anti-Fas-treated Jurkat cells but not in untreated cells (Fig. 3, f and c, and Fig. 4 A). Z-VAD-FK inhibited the appearance of this Apo2.7
staining (Fig. 3, l and Fig. 4 A, VAD). In contrast, Ac-YVAD-CHO did not inhibit the induction of the Apo2.7
staining (Fig. 3, o and Fig. 4 A, YVAD) nor apoptotic morphological changes (Fig. 3, m and n), confirming that Ac-YVAD-CHO cannot inhibit Fas-induced apoptosis. Interestingly, Ac-DEVD-CHO-treated cells which, after Fas
stimulation, exhibited normal nuclear morphology were
strongly positive with Apo2.7 (Fig. 3, i and Fig. 4 A,
DEVD). The same result was obtained by using Z-DEVD-FK, another inhibitor for CPP32-like proteases (data not
shown). These results were rather surprising because a
CPP32-like protease(s) was thought to be essential for
Fas-induced apoptotic signaling. So we next examined
whether these cells were dead or not.
Fig. 5.
Inhibition of CPP32-like proteases did not block Fas-induced cell death. (A) Jurkat cells were treated as in Fig. 3, and
the percentage of cell death was determined by MTT assay. (B)
Jurkat cells were preincubated with or without Ac-DEVD-CHO
(120 µM) and then treated with anti-Fas mAb (CH-11, 150 ng/
ml) for 20 h. Cell death was measured by using trypan blue dye
exclusion, and is expressed as a percentage of cell death.
[View Larger Versions of these Images (42 + 44K GIF file)]
) and p38 (Moriguchi et al., 1996b
), and
here we detected slight activation of JNK/SAPK and p38,
peaking at ~1 or 2 h after the addition of cycloheximide in
the absence of anti-Fas (Fig. 6 A, open circles). Then,
strong activation of JNK/SAPK and p38 occurred 3 to 5 h
after the treatment with anti-Fas (Fig. 6 A, closed circles),
when the cycloheximide-induced activation of JNK/SAPK
and p38 was decreased to the basal level (Fig. 6 A, open
circles). Preincubation with Z-DEVD-FK, an inhibitor of
CPP32-like proteases, did not block significantly the Fas-induced activation of JNK/SAPK and p38 in the cycloheximide-treated KB cells (Fig. 6 B). We then examined the
effect of expression of dominant negative MKK7 (KL-MKK7) on the Fas-induced activation of JNK/SAPK (Fig.
6 C). KB cells were transiently cotransfected with HA-tagged
SAPK/JNK and KL-MKK7 (or a vacant plasmid) and then
treated with anti-Fas mAb (CH-11, 250 ng/ml). After 2 h, HA-tagged SAPK/JNK was precipitated with anti-HA antibody and assayed for the kinase activity. The Fas-induced
activation of SAPK/JNK was almost completely inhibited
by KL-MKK7 (Fig. 6 C). The result is consistent with our
conclusion that the major activator of JNK/SAPK during
Fas signaling is MKK7.
Fig. 6.
(A) Fas induced the activation of JNK/SAPK and p38
in KB cells. KB cells were preincubated with 50 µg/ml cycloheximide for 1 h (1 to 0 h) and then treated with (closed circles) or
without (open circles) anti-Fas mAb (CH-11, 200 ng/ml) for various periods. Cell lysates were prepared, and the activities of endogenous JNK/SAPK (left panel) and endogenous p38 (right
panel) were determined by immunecomplex kinase assays. The
activity was expressed as fold-increase relative to that in time
1 h.
(B) Fas-induced activation of JNK/SAPK and p38 is not dependent
on CPP32-like proteases in KB cells. KB cells were preincubated
with 50 µM Z-DEVD-FK for 1 h and then treated with anti-Fas
mAb (CH-11, 200 ng/ml) for 4 h. As a control, cells were exposed
to 0.7 M NaCl for 30 min (NaCl). The activities of endogenous
JNK/SAPK and endogenous p38 were determined by immunecomplex kinase assays. Cycloheximide (50 µg/ml) was added in
all samples. (C) Dominant negative effect of KL-MKK7 on the
Fas-induced activation of JNK/SAPK in KB cells. KB cells were
transiently cotransfected with HA-tagged SAPK/JNK and KL-MKK7 (a kinase-negative mutant of MKK7) (KLMKK7) or a vacant plasmid (vector), and then treated with anti-Fas mAb (CH-11,
250 ng/ml). After 2 h, the HA-tagged SAPK/JNK was precipitated by anti-HA antibody and assayed for the kinase activity
(top). The immunoprecipitated HA-SAPK/JNK was analyzed by
immunoblotting with anti-SAPK antibody (bottom). Cycloheximide (50 µg/ml) was added in all samples.
[View Larger Versions of these Images (16 + 27 + 23K GIF file)]
Fig. 7.
Inhibition of CPP32-like proteases did not block Fas-induced morphological changes in
KB cells. (A) KB cells were preincubated for 1 h with or without ICE-family protease inhibitors;
Z-DEVD-FK (50 µM) or Z-VAD-FK (50 µM) in the presence of 50 µg/ml cycloheximide. The
cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 4 and 8 h. Phase contrast images
are shown. (B) KB cells were treated as in A. After incubation for 8 h, the cells were fixed and
stained with DAPI and phalloidin. (C) KB cells were preincubated for 1 h with or without
Z-DEVD-FK (50 µM) in the presence of 50 µg/ml cycloheximide. The cells were then treated
with anti-Fas mAb (CH-11, 150 ng/ml) for 8 h, and collected and lysed. A portion of the cell lysate
was analyzed by immunoblotting with anti-actin mAb (A-4700; Sigma) that recognizes the
COOH-terminal region of actin. An arrow and an arrowhead indicate an intact actin molecule and a 15-kD fragment of actin, respectively.
[View Larger Versions of these Images (126 + 55 + 34K GIF file)]
; McCarthy et al., 1997
). To examine whether the
Fas-induced disruption of actin network is associated with
the cleavage of actin molecule, the cell lysate obtained
from the anti-Fas-treated KB cells in the presence or absence of Z-DEVD-FK was subjected to immunoblotting with anti-actin mAb (A-4700) that recognizes the COOH-terminal region of actin. In the absence of Z-DEVD-FK,
15-kD COOH-terminal fragment of actin appeared after
Fas stimulation (Fig. 7 C, middle lane), but in the presence
of Z-DEVD-FK, this cleavage was not seen (Fig. 7 C, right
lane). As the disruption of actin network occurs even in
the presence of Z-DEVD-FK, the Fas-induced morphological changes may be independent of the cleavage of actin molecule.
Discussion
; Juo et al., 1997
; Lenczowski et al.,
1997
) and p38 (Juo et al., 1997
). Rather surprisingly, the
activation of both kinase cascades was not suppressed by
specific inhibitors for CPP32-like proteases which were
able to block almost completely the Fas-induced chromatin condensation and DNA fragmentation, indicating that
the signaling pathway of the protein kinase cascades is independent, or lying upstream, of the activation of CPP32-like proteases. Since expression of a dominant negative
form of JNK/SAPK activator that blocked the Fas-induced
activation of JNK/SAPK had no effect on Fas-mediated DNA fragmentation and chromatin condensation (Lenczowski et al., 1997
and our unpublished data), the JNK/
SAPK activation pathway may not lie upstream of the activation of CPP32-like proteases that is essential for DNA
fragmentation. Therefore, in Fas signaling, the activation
of ICE-like proteases is followed by at least two separate
pathways; one is the activation of CPP32-like proteases followed by CPP32-like proteases-dependent processes,
and the other is independent of CPP32-like proteases,
such as the activation of the MAP kinase superfamily molecules. Moreover, the above data clearly indicated that the
MAP kinase superfamily molecules are not involved in the
Fas-mediated chromatin condensation and DNA fragmentation.
). Moreover, a recent report of McCarthy et al. (1997)
has shown that in the apoptosis induced by
c-myc, etoposide, and Bak, the DNA fragmentation but
not the cell shrinkage and membrane blebbing is blocked
by the ICE-family protease inhibitor. It is interesting to
speculate that in the apoptotic signaling the chromatin
condensation and DNA fragmentation, one of the most characteristic events in the apoptotic nuclei, and the morphological change, a phenomenon outside the nucleus, are
separately directed by different pathways. Most recently,
Rudel and Bokoch (1997)
reported that PAK2, a member
of the p21 activated protein kinase family, is cleaved to be
activated by Fas in a CPP32-like protease(s)-dependent manner. They have also shown that expression of dominant-negative PAK inhibits formation of apoptotic bodies
during Fas-induced apoptosis but has no effect on Fas-induced DNA fragmentation. Our observations here showed
that in the presence of the inhibitor for CPP32-like proteases, neither DNA fragmentation nor formation of apoptotic bodies occurred in Fas-treated Jurkat or KB cells (Fig. 3 g, and data not shown). So, not only DNA fragmentation but also formation of apoptotic bodies may lie
downstream of a CPP32-like protease(s), which is not involved in Fas-induced membrane blebbing and cell shrinkage. It is interesting to examine whether Fas-induced cell
shrinkage and membrane blebbing can occur in those cells
expressing dominant-negative PAK. As some members of
PAK family have been shown to activate JNK/SAPK signaling pathways, Fas-induced activation of PAK2 might lead
to activation of JNK/SAPK. However, our observation
clearly indicates that the activation of JNK/SAPK by Fas is
independent of CPP32-like proteases. Additional experiments are necessary to elucidate the Fas-induced signaling pathway that leads to activation of JNK/SAPK and p38.
Fig. 8.
A proposed model for Fas-induced signaling pathways.
[View Larger Version of this Image (21K GIF file)]
Address correspondence to Eisuke Nishida, Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-01, Japan. Tel.: (81) 75-753-4230. Fax: (81) 75-753-4235.
Received for publication 16 May 1997 and in revised form 6 August 1997.
1. Abbreviation used in this paper: ICE, IL-1This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to E. Nishida.
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