(Received for publication, January 4, 1996)
From the
Genetic analyses of Caenorhabditis elegans has
identified three genes that function in the regulation of nematode cell
death. Mammalian homologs of two of these genes, ced-9 and ced-3, have been identified and comprise proteins belonging to
the Bcl-2 and ICE families, respectively. To date, it is unclear where
the negative regulators, ced-9 and bcl-2, function
relative to the death effectors, ced-3 and the mammalian ced-3 homologs, respectively. Here, the molecular order of the
cell death pathway is defined. Our results establish that Bcl-2 and
Bcl-x function upstream of two members of the ICE/CED-3
family of cysteine proteases, Yama (CPP32/apopain) and ICE-LAP3 (Mch3).
Apoptosis, or programmed cell death, is a physiologic process important in the normal development and homeostasis of multicellular organisms(1, 2) . It is encoded by an endogenous program, conserved throughout metazoan evolution, ultimately resulting in cellular suicide. Derangements of apoptosis can have deleterious consequences as exemplified by several human disease states, including acquired immunodeficiency syndrome, neurodegenerative disorders, and cancer (3) .
Despite its paramount importance, the molecular
mechanism of apoptosis is poorly understood. The nematode Caenorhabditis elegans has been a powerful tool in the
identification of critical components of the cell death
machinery(4) . Systematic genetic analyses have elucidated
three genes, ced-3, ced-4, and ced-9, that
are important in the regulation of nematode cell death. Mutations of ced-3 and ced-4 abolish all somatic cell deaths that
normally occur during the development of C. elegans,
suggesting that these genes encode effector components of the
pathway(4) . By contrast, ced-9 encodes a negative
regulator that functions to suppress inappropriate cellular
suicide(5) . Mammalian homologs of ced-9 and ced-3 have been identified and include proteins belonging to the Bcl-2
and ICE ()family, respectively(6, 7) .
However, no homologs of ced-4 have thus far been identified.
Bcl-2 is the prototypic member of a growing family of cell death regulators(8, 9) . First identified for its role in B-cell malignancies, expression of Bcl-2 has been shown to inhibit cell death due to a variety of apoptotic stimuli and in numerous cell types(10) . Bcl-2 can substitute functionally for ced-9, preventing nematode cell death and further emphasizing the highly conserved nature of the cell death pathway(6) .
An important advance came with the discovery that the nematode death
effector ced-3 had significant homology with the mammalian
protease interleukin-1 converting enzyme (ICE)(7) . ICE is
involved in the proteolytic processing of pro-IL-1
to the active
cytokine (11, 12) . Overexpression of ICE or CED-3 in
mammalian cells induces apoptosis, suggesting that ICE, or a related
protease, is an essential component of the mammalian cell death
pathway(13) .
Evidence is growing, however, that ICE itself
may not be the mammalian ced-3 equivalent as: 1) a number of
cell types stably secrete mature IL-1 without undergoing
apoptosis; 2) ICE-deficient mice, although unable to generate active
IL-1
, fail to exhibit a prominent cell death defective
phenotype(14, 15) . Recently, several ICE/CED-3
homologs have been identified which comprise an emerging family of
aspartate-specific cysteine proteases that include
Nedd-2/ICH1(16, 17) ,
Yama/CPP32/apopain(18, 19, 20) , TX/ICH2/ICE
rel-II (21, 22, 23) , ICE-rel
III(21) , Mch2(24) , and
ICE-LAP3/Mch3(25, 26) . Distinct from the prototype
ICE, Yama and ICE-LAP3 cleave the death substrate poly(ADP-ribose)
polymerase (PARP) into signature apoptotic
fragments(18, 19, 26) . Importantly, CED-3
has a similar substrate specificity as it also cleaves PARP at the same
site (27) .
Although important regulators of the cell death
pathway have been identified, little is known about their molecular
order of function. To date, it is unclear whether ced-9 and bcl-2 function upstream or downstream of ced-3 and
the mammalian ced-3 equivalent, respectively. Biochemically
defining the sequence of the pathway is of utmost importance if one is
to gain insight into how the pathway functions and how it is regulated.
Here we report that Yama and ICE-LAP3, the two mammalian proteases most
related to CED-3, are both activated by apoptotic stimuli. Importantly,
we demonstrate order in the pathway by providing biochemical evidence
that Bcl-2 and Bcl-x function upstream of Yama and
ICE-LAP3.
Figure 1:
The mammalian CED-3 homologs, Yama
and ICE-LAP3, are activated by apoptotic stimuli. A, schematic
representation of the active and inactive forms of ICE, Yama, and
ICE-LAP3. B, activation of Yama and ICE-LAP3 by Fas/APO-1 and
staurosporine. Jurkat cells were either left untreated or treated with
2 µM staurosporine or 200 ng/ml anti-APO-1 antibody for 3
h. Cytosolic extracts from 10 10
Jurkat cells were
analyzed by SDS-PAGE and subsequently immunoblotted with antibodies
directed against the p17 and p12 subunits of Yama or an antibody
against the p20 subunit of ICE-LAP3. Similar results were obtained
using BJAB or CEM cells (data not shown). Intermediate forms of the p17
subunit of Yama were also observed (specific only to antibody directed
against the p17 subunit) likely containing the prodomain. Upon
activation, a 30-kDa species of ICE-LAP3 was often observed and
corresponds to a form lacking the
prodomain.
A related member of the ICE/CED-3 family was recently cloned and designated ICE-LAP3/Mch3 ((25, 26) Fig. 1A). Of the homologs thus far identified, ICE-LAP3 is the most closely related to Yama, sharing 58% sequence identity and 75% similarity(25) . More importantly, endogenous pro-ICE-LAP3 (p35) is also activated by Fas/APO-1 (25) and staurosporine (Fig. 1B), generating p20 and p12 subunits. A 30-kDa form of ICE-LAP3 (p30) was often observed upon activation and likely represents a prodomain-less species.
Taken together, these results show that Yama and ICE-LAP3, the two ICE-like proteases sharing the most sequence homology and substrate specificity with CED-3(18, 19, 25, 26, 27) , are activated by an apoptotic signal. In contrast to C. elegans, this suggests that the mammalian cell death pathway may require more than one ICE/CED-3 family member to execute the cell death program.
Figure 2:
Bcl-2 and Bcl-x function
upstream of Yama and ICE-LAP3. A, expression of Bcl-2 and
Bcl-x
in Jurkat cell lines. Jurkat cells were transfected
with control vector, pEBs-bcl-x
, or pEBs-bcl-2 and selected
with hygromycin. SDS-PAGE and immunoblotting were done as described
under ``Materials and Methods.'' B, Bcl-2 and
Bcl-x
prevent staurosporine-induced cell death but not
Fas/APO-1-induced cell death. Jurkat cells (2
10
)
were left untreated, treated with staurosporine (2 µM) for
18 h, or treated with anti-APO-1 antibody (200 ng/ml) for 6 h. Cells
were then analyzed for DNA fragmentation as described under
``Materials and Methods.'' Additionally, nuclear morphology
assessed by acridine orange staining yielded similar results (data not
shown). C, staurosporine-induced cleavage of the death
substrate, poly(ADP-ribose) polymerase, is blocked by Bcl-2 and
Bcl-x
. Jurkat cells were left untreated, treated with 2
µM staurosporine, or treated with 200 ng/ml anti-APO-1
antibody for 6 h and analyzed for PARP cleavage as described
previously(18) . D, Bcl-2 and Bcl-x
block
staurosporine-induced Yama and ICE-LAP3 activation. Jurkat cells were
left untreated, treated with 2 µM staurosporine for 3 h,
or treated with 200 ng/ml anti-APO-1 antibody for 1.5 h and analyzed as
in Fig. 1B. See Fig. 1B for a
description of the different subunits
observed.
To provide biochemical proof of whether
Bcl-2 and Bcl-x function upstream or downstream of the
apoptotic ICE/CED-3 cysteine proteases, we directly assessed Yama and
ICE-LAP3 activation under various conditions. Overexpression of Bcl-2
or Bcl-x
abrogated staurosporine-induced generation of
active Yama and ICE-LAP3 (Fig. 2D). Similar results
were obtained using another Bcl-2/Bcl-x
-inhibitable death
stimulus, thapsigargin (data not shown), which is a specific inhibitor
of the endoplasmic reticulum Ca
-ATPase(37) .
The inhibitory action of Bcl-2 and Bcl-x
could be
``bypassed'' by treatment with anti-APO-1 antibody, resulting
in activation of the apoptotic proteases (Fig. 2D). Our
conclusions are not a peculiarity of Jurkat cells since similar results
were obtained using a well characterized Bcl-2-expressing CEM cell line
( (35) and data not shown).
Figure 3:
The CrmA target is upstream of Yama and
ICE-LAP3. A, expression of CrmA in Jurkat cell lines. Jurkat
cells were transfected with control vector or pZEM-CrmA and selected
with neomycin. Seven anti-APO-1-resistant clones were identified and
pooled. SDS-PAGE and immunoblotting were done as described under
``Materials and Methods.'' The BJAB CrmA cell line was
described previously(28) . B, CrmA prevents
Fas/APO-1-induced cell death but not staurosporine-induced cell death.
Jurkat cells (2 10
) were left untreated, treated
with staurosporine (2 µM) for 18 h, or treated with
anti-APO-1 antibody (200 ng/ml) for 6 h. Cells were then analyzed for
DNA fragmentation as described under ``Materials and
Methods.'' Additionally, nuclear morphology assessed by acridine
orange staining yielded similar results (data not shown). C,
Fas/APO-1-, but not staurosporine-, induced cleavage of PARP is blocked
by CrmA. Jurkat cells were left untreated, treated with 2 µM staurosporine, or treated with 200 ng/ml anti-APO-1 antibody for 6
h and analyzed for PARP cleavage as described previously(28) . D, CrmA blocks Fas/APO-1-induced Yama and ICE-LAP3 activation.
Jurkat cells were left untreated, treated with 2 µM staurosporine for 3 h, or treated with 200 ng/ml anti-APO-1
antibody for 1.5 h and analyzed as in Fig. 1B. See Fig. 1B for a description of the different subunits
observed.
To determine whether CrmA directly inhibited Yama and/or ICE-LAP3 in vivo, activation of these apoptotic proteases was directly
assessed. Consistent with DNA fragmentation and PARP analysis (Fig. 3, B and C), staurosporine-induced Yama
and ICE-LAP3 activation were not blocked by CrmA (Fig. 3D). Interestingly, Fas/APO-1-induced generation
of active Yama and ICE-LAP3 was potently abrogated by CrmA, suggesting
the existence of a CrmA-inhibitable ICE-like protease upstream of Yama
and ICE-LAP3. The fact that staurosporine-induced apoptosis is
prevented by Bcl-2/Bcl-x and not by CrmA, coupled
with the observation that Fas/APO-1-induced cell death is inhibited by
CrmA but not by Bcl-2/Bcl-x
, argues for the
existence of distinct apoptosis signaling pathways.
Figure 4:
Molecular ordering of the cell death
pathway. Three signals for cell death are illustrated: 1) a
Bcl-2-inhibitable pathway in which Bcl-2 and Bcl-x function
upstream of the CED-3-like apoptotic proteases, 2) granzyme B-mediated
direct activation of the apoptotic proteases, 3) Fas/APO-1 activation
of a proteolytic cascade.
In our Jurkat cell system,
Fas/APO-1-induced cell death was not blocked by Bcl-2 or Bcl-x (Fig. 2B). Thus, it was not surprising that Bcl-2
or Bcl-x
failed to inhibit Fas/APO-1-induced PARP cleavage
and Yama/ICE-LAP3 activation (Fig. 2, C and D). CrmA, on the other hand, potently abrogated
Fas/APO-1-induced cell death, PARP cleavage, and Yama/ICE-LAP3
activation (Fig. 3). Interestingly, CrmA failed to inhibit the
death pathway triggered by staurosporine (Fig. 3). Taken
together, our data suggest the existence of distinct apoptosis
signaling pathways displaying differential sensitivity to
Bcl-2/Bcl-x
and CrmA (Fig. 4).
In vitro, CrmA interacts only with the active forms of ICE or Yama(18) . However, both Yama and ICE-LAP3 remained as proenzymes in anti-APO-1-treated CrmA expressing cells, suggesting that CrmA inhibits an ICE-like protease upstream of Yama and ICE-LAP3. This hypothesis is especially attractive considering that granzyme B-induced cell death, which is not Bcl-2-inhibitable, likely occurs via direct activation of Yama (and/or Yama-related proteases)(41, 42) . Therefore, we postulate that Fas/APO-1 signals the activation of a proteolytic cascade, analogous to the coagulation or complement systems, and in this case, comprised of related ICE/CED-3 family members (Fig. 4).