* Division of Cellular Immunology, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego,
California 92121
We have begun to explore the mechanisms
of apoptosis using a cell-free system based on extracts
from Xenopus eggs. Nuclei assembled or placed in
these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in
apoptosis of proteins containing Src homology 2 (SH2)
domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a
number of signaling proteins, including Lck, Src, and
Abl, inhibited apoptosis when present at concentrations of 10-100 nM. The inhibition was dependent on
specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family
SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined
three phases in the apoptotic process occurring in this
cell-free system. SH2 domains and ceramide act
throughout the first 60-90 min of the process (the "initiation" phase). Next, Bcl-2, interleukin-1 converting
enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring ~90-120
min after the start of incubation (the "sentencing"
phase). In the final phase ("execution"), the process of
active nuclear destruction ensues.
Apoptosis is a normal physiological process of cell
death observed in multicellular organisms (for reviews see 7, 34, 37, 42, 45, 46). The apoptotic death
pathway can be initiated by a variety of stimuli, including
DNA damage, the withdrawal of growth factors, and the
binding of certain ligands to cell surface receptors. Although the death stimulus and cellular context can vary
widely, it is thought that apoptosis involves a conserved
biochemical machinery. Supporting this notion of a common pathway are several observations: first, apoptotic
cells undergo a stereotypical sequence of morphological
changes, including plasma membrane blebbing, chromatin
condensation, and the shrinkage and fragmentation of the
nucleus and cytoplasm, resulting in the formation of cell fragments termed "apoptotic bodies." Second, endonucleases and proteases are usually activated, causing the
fragmentation of genomic DNA and the cleavage of certain proteins such as fodrin (25), nuclear lamins (19, 33),
and poly (ADP-ribose) polymerase (16, 20, 31, 40). Finally, there are a number of proteins whose function in
regulating apoptosis is conserved phylogenetically. For example, the human Bcl-2 protein can inhibit apoptosis
when expressed in the cells of a variety of species, including the nematode, Caenorhabditis elegans (e.g., 15, 32, 37, 44, 45). Bcl-2 belongs to a family of proteins (for reviews
see 32, 37). Some members of this family, e.g., Bcl-2, Bcl-xL,
C. elegans Ced-9, and the adenovirus E1B 19-kD protein,
are able to inhibit apoptosis. Others, such as Bax, Bad,
Bcl-xS, and Bak (1, 9, 17), have the opposite effect of promoting cell death. Thus, cell survival may be regulated in
part by the ratio of Bcl-2-type to Bax-type molecules (38,
47, 49).
Also showing conserved function in apoptosis is a large
family of cysteine proteases related to the C. elegans protein, Ced-3 (50), and the vertebrate protein, interleukin-1 The precise roles of these molecules in the death pathway, as well as the identities of other molecular participants in the process, remain to be elucidated. We recently
described a novel cell-free system that may help to unravel
aspects of apoptotic biochemistry and the function of Bcl-2
(29). This system is based on extracts from eggs of the
South African clawed frog, Xenopus laevis. Xenopus egg
extracts have been an important tool for studying cellular
processes such as nuclear membrane assembly, nuclear
protein import, DNA replication, and cell cycle regulation (for review see 28). We found that it was also possible to
prepare "apoptotic" extracts that mimic the nuclear shrinkage, chromatin condensation, and DNA fragmentation observed in cells dying by apoptosis (29). Apoptotic activity
in these extracts requires the presence of a heavy membrane (HM) fraction consisting mostly of mitochondria and
is inhibited by the addition of baculovirus-expressed Bcl-2
protein. Moreover, we found that apoptosis in this system involves a latent phase, lasting ~90-120 min, that occurs
whether or not nuclei are present. Protection by Bcl-2 occurs only if Bcl-2 is present during this latent period; thus,
it would appear that the antiapoptotic role of Bel-2 is due
primarily to its interaction with cytoplasmic, rather than
nuclear, targets.
Apoptosis in the Xenopus system is not affected by inhibitors of tyrosine phosphorylation such as herbimycin A
(29) and genistein (see Results). Thus, protein tyrosine kinases are apparently not involved in the portion of the cell
death pathway at work in the cell-free system. However,
free phosphotyrosine (PTyr; 10-20 mM) was found to
have a marked inhibitory activity (29). This suggested that
protein tyrosine phosphatases or other molecules interacting with tyrosine-phosphorylated proteins might be important for apoptosis in this system. We were thus led to explore the possibility that Src Homology 2 (SH2) domains
are involved in the apoptotic pathway. SH2 domains (for
reviews see 2, 36) are a type of modular sequence element
often found in signaling molecules. These domains are
known to interact with specific protein sequences containing phosphorylated tyrosine residues. The binding of SH2 domains to their ligands provides a means for the cell to
assemble protein complexes in a manner readily controlled by tyrosine kinases and phosphatases. We now
show that the SH2 domains from certain signaling proteins, when added to the Xenopus cell-free system in the
form of glutathione-S-transferase (GST) fusion proteins, can inhibit apoptosis. Moreover, apoptosis in this system is
inhibited by a synthetic peptide known to interact with the
SH2 domains of Src family kinases; this effect is dependent
on tyrosine phosphorylation of the ligand peptide.
The data support a role of SH2-PTyr interactions in the
apoptotic pathway. Moreover, our investigations show that
the events controlled by SH2 domain interactions, ceramide,
Bcl-2, CPP32-like proteases, and the HM fraction define
two distinct temporal phases in the apoptotic process. These
phases precede a third stage, during which the nuclei are
actively destroyed.
The preparation, fractionation, and use of Xenopus egg extracts were as
described (28, 29). In experiments with unfractionated extracts, 50 µl of
extract was mixed with ATP-regenerating system (consisting of 2.5 µl of a
200 mM solution of phosphocreatine, 1 µl of a 100 mM solution of ATP,
pH 7.0, and 1.5 µl of a solution of creatine phosphokinase (5 mg/ml; Sigma
Chemical Co., St. Louis). Then, 1-3 µl of a suspension of rat liver nuclei
(at ~106 nuclei per µl) was added, as well as 1 µl of rhodamine-labeled nuclear import substrate (TRITC-HSA-NLS [a TRITC-labeled conjugate of human serum albumin with a synthetic nuclear localization signal peptide]) to monitor nuclear intactness, as described (28). GST-SH2 domain
fusions and synthetic peptides were then added from stock solutions at
20-50 times the final concentration. HM-depleted extracts were reconstituted from 45 µl of egg cytosol and 5 µl of light membrane suspension obtained from M-phase extracts, as described (28).
The expression and purification of GST fusion proteins were as described (4). Briefly stated, the GST-SH2 domains of Lck were produced
by PCR amplification of the desired region of wild-type (Y192), F192, and
E192 Lck constructs (amino acids 121-224), using the proofreading Vent
DNA polymerase (New England Biolabs, Beverly, MA). The PCR fragments were inserted in frame into the pGEX-3T prokaryotic expression
vector (14), and the resulting GST-SH2 fusion proteins were purified by
glutathione-Sepharose affinity chromatography. The fusion proteins were
dialyzed (using a microdialysis apparatus [Pierce Chemical Co., Rockford,
IL]) against a buffer containing Hepes (20 mM) and KCl (50 mM) before
use. Synthetic peptides were obtained from Research Genetics (Huntsville, AL).
Phosphorylation of the synthetic peptide (RNLDNGGFYIAPR) comprising the site surrounding Y192 in Lck was assayed essentially as described previously (27): peptide (4 mg/ml), GST-SH2 Domain Fusions Specifically Inhibit
Apoptotic Activity in the Cell-free System
To investigate whether SH2-PTyr interactions are involved in the cell-free apoptosis system, we prepared GST
fusion proteins containing the SH2 domains from several
signaling proteins and added them to the extracts. Fig. 1
shows that the SH2 domains from c-Src and c-Abl produced a concentration-dependent inhibition of apoptosis
when present at concentrations of
To determine whether the effects of SH2 domains
added to the cell-free system were based on specific interactions with phosphorylated ligands, we took advantage of
point mutations in the SH2 domain of Lck that disrupt
PTyr-dependent binding. Previous studies (4) had shown
that the function of the Lck SH2 domain can be regulated by modification of a particular amino acid residue, Y192.
Phosphorylation at this site, which is located next to the
EF loop of the SH2 domain, markedly lowers the affinity
of the SH2 domain for phosphotyrosine-containing protein ligands. Mutants in which this Tyr residue was changed to Phe (F192) and Glu (E192) had been found to mimic
the unphosphorylated and phosphorylated states, respectively, of Y192. We added the wild-type and mutant Lck
SH2s to the Xenopus cell-free system. As shown in Fig. 2,
the SH2 domain from the constitutively inactive Lck-E192
mutant was unable to inhibit apoptosis. On the other hand,
the Lck-F192 mutant SH2, which binds constitutively to
phosphotyrosine-containing ligands, was as potent an inhibitor as the Src and Abl SH2 domains.
The wild-type Lck SH2 domain, like the nonbinding
E192 mutant, was unable to inhibit apoptosis. This result
could be explained if we supposed that the wild-type Lck
SH2 domain became phosphorylated on Y192 by a kinase
present in the extract and consequently had a lowered affinity for the tyrosine phosphorylated ligand, similar to the
E192 mutant. This hypothesis predicted that if phosphorylation on Y192 were prevented, the wild-type SH2 domain
would function like Lck-F192; i.e., it would inhibit apoptosis in the cell-free system. To test this prediction, we added
the tyrosine kinase inhibitor, genistein, either separately
or with the wild-type Lck SH2 domain. Genistein, added
by itself, had only a slight effect on the kinetics of nuclear
destruction in the apoptotic cell-free system (Figs. 3 and
4 B; this suggests that any critical tyrosine phosphorylation
events involving proteins endogenous to the extract had
already occurred before the addition of genistein). However, the coaddition of genistein and the wild-type Lck SH2 domain produced a marked inhibitory effect, similar to that
of the F192 mutant (Fig. 3 A). Therefore, the Lck SH2 domain is likely to be phosphorylated on Y192 by an endogenous protein tyrosine kinase in the Xenopus egg extracts,
and thus inactivated. To examine this more directly, we assayed the phosphorylation of a synthetic peptide corresponding to the sequence surrounding Y192. Fig. 3 B
shows that this peptide is phosphorylated by the extract in a time-dependent manner; the specific activity was estimated at 7.7 ± 0.2 pmol per min per mg of extract protein.
Thus, a kinase is present in the extract that is able to phosphorylate this site in the Lck SH2 domain. We conclude
from the experiments shown in Figs. 1-3 that the effects of
the exogenous SH2 domains are dependent on specific interactions with endogenous tyrosine-phosphorylated ligands.
To confirm that PTyr-SH2 domain interactions are important for apoptosis in the cell-free system, we examined
the effects of adding an exogenous synthetic phosphopeptide, EPQY*EEIPIYLK (abbreviated Y*EEI; Y* represents PTyr), known to interact with Src family SH2 domains (41). Fig. 4 A shows that this peptide was able to
inhibit apoptosis even at concentrations as low as 1 nM. In
control experiments (Fig. 4 B), we found that a nonphosphorylatable mutant peptide, EPQFEEIPIYLK (FEEI),
was ineffective, arguing again that the inhibitory effect is
dependent on the specific interaction of SH2 domains with
sequences containing PTyr residues. The effect of adding
the unphosphorylated peptide (YEEI) was similar to that
seen with Y*EEI, suggesting that YEEI can be phosphorylated by kinases endogenous to the extract (Fig. 4 B). In
support of this idea, we found that the protein tyrosine
kinase inhibitor, genistein, was able to reverse the inhibitory effect of YEEI (Fig. 4 B). To probe the specificity
of these effects further, we tested another synthetic phosphopeptide, corresponding to the SH2 domain-binding region surrounding residue Y1021 of phospholipase-C SH2 Domains Interact in the Early Phase of the
Apoptotic Process
To determine when in the apoptotic process PTyr-SH2 interactions are important, we performed experiments in
which the Src SH2 domain-GST fusion was added to the
cell-free system at various times after the start of incubation (Fig. 5). As before (Fig. 1), the Src SH2 domain
slowed the kinetics of nuclear destruction markedly when
added at the start of incubation. However, when the addition of this SH2 domain was delayed by intervals ranging from 30-120 min, its inhibitory activity was gradually reduced. These results demonstrate that the SH2 domain interactions are important throughout the initial portion of
the apoptotic process.
Bcl-2 and the Heavy Membrane Fraction Act Later
Than SH2 Domains in the Apoptotic Process
Previously, we showed that Bcl-2 can inhibit apoptosis by
acting during the early cytoplasmic phase of the apoptotic
process (29). To define more precisely the period during
which Bcl-2 acts, we performed experiments similar to
those described above. Bcl-2 was added at various intervals after the start of incubation, and the time course of
nuclear survival was determined. Unlike the SH2 domains,
Bcl-2 inhibited apoptosis effectively even if its addition
was delayed up to ~1.5 h (Fig. 6). Thus, it would appear
that the events controlled by Bcl-2 occur later in the apoptotic process than those regulated by SH2 domain-PTyr
interactions.
Our previous studies also identified a requirement for
an HM fraction that was enriched in mitochondria but also
contained other membrane material (29). Extracts depleted of heavy membranes were found to lack apoptotic
activity, and the addition of HMs restored this activity in a
dose-dependent manner. To determine when the HM fraction is required during the time course of the apoptotic
process occurring in the cell-free system, we performed an
experiment in which the HM fraction was added at various
times after the start of incubation. Fig. 7 shows that delaying the addition of the HM fraction by as much as 1.5 h
had essentially no effect on the kinetics of nuclear destruction in the apoptotic extract. Only when the HM fraction
was added at 2 h after the start of incubation was the time
course of nuclear destruction delayed. We conclude that,
like Bcl-2, the heavy membranes are required at a relatively late stage in the apoptotic process.
We also showed previously that a soluble ceramide analog could substitute for the HM fraction in the cell-free
apoptotic system (24) This finding raised the possibility
that the function of HMs was simply to produce ceramide.
However, as Fig. 8 shows, ceramide acts earlier in the apoptotic process than the HMs. Like the GST-SH2 domain
fusions, ceramide was most active when added at the start
of incubation, and its potency declined gradually as the
time of its addition was delayed. Thus, it is unlikely that
the function of HMs is merely to produce ceramide.
Previous studies from many laboratories have demonstrated the involvement of cysteine proteases from the
Ced-3/ICE family in the apoptotic process (see Introduction). To determine whether such proteases are involved
in the Xenopus system, we tested the effects of aldehydebased tetrapeptide derivatives that have been shown to be
specific inhibitors of ICE family proteases. The compound Ac-DEVD-CHO (DEVD) has been shown to inhibit
CPP32-like proteases preferentially, while Ac-YVAD-CHO
(YVAD) is more selective for ICE and its close relatives
(30). Fig. 9 A shows that micromolar concentrations of
DEVD block apoptosis in the Xenopus system. YVAD also
inhibited apoptosis in this system, but concentrations ~500-fold higher were required. These results are similar
to those described by Nicholson et al. (1995) (30) for mammalian cells, and argue that a CPP32-like, rather than an
ICE-like, protease activity is required for apoptosis in the
Xenopus cell-free system. However, because these compounds can inhibit multiple caspases, it is possible that several such enzymes are involved in this system. After this
manuscript was submitted, other studies showed directly
that CPP32-like caspases are activated in Xenopus egg extracts (3, 18).
Using the same approach as described above, we investigated the effect of delaying the addition of DEVD. The results (Fig. 9 B) showed that the protease activity was required relatively late in the apoptotic process, near the end
of the 1.5-2-h latent phase. In this experiment, when AcDEVD-CHO was added as late as 1 h after the start of the
incubation, almost no reduction in its inhibitory activity
was seen, and only a partial loss of activity was observed
when the inhibitor was added after 1.5 h of incubation. In
contrast, the inhibitor had no activity when added after 2 h.
A Role for SH2 Domains in Apoptosis
In this report, we have shown that apoptosis in the Xenopus cell-free system is inhibited by the addition of SH2 domains from several signaling proteins (Figs. 1-3 and 5).
Our results with the Lck SH2 domain (Figs. 2 and 3) demonstrate that the effect is specifically due to interaction
with tyrosine-phosphorylated ligands, in that it is abrogated by phosphorylation on residue Y192 of Lck; phosphorylation at this site was shown elsewhere to disrupt binding
of the SH2 domain to tyrosine-phosphorylated ligands (4). Similarly, the inhibition produced by the ligand peptide,
YEEI, was dependent on phosphorylation of the tyrosine
residue required for interaction with SH2 domains. Thus,
our results show specificity in the sense that they reflect the
PTyr-dependent binding of SH2 domains and their ligands.
On the other hand, the exogenous SH2 domains appear to
be acting in a generic manner, inasmuch as the SH2 domains from a number of proteins can block apoptosis in
this system when present at concentrations of at least 10 nM.
Our results therefore do not identify which particular
SH2-containing protein(s) and corresponding tyrosinephosphorylated ligand(s) are involved in the cell-free apoptosis system.
The synthetic phosphopeptide Y*EEI inhibited apoptosis in the extract at lower concentrations than those required for the GST-SH2 domain fusions. The reason for
this is unclear. Possibly these reagents act in different
ways, e.g., by interacting in a regulatory manner with specific molecules. However, we consider it likely that these
molecules act by disrupting PTyr-dependent interactions
between proteins endogenous to the extract. What could
be the function of these endogenous proteins? SH2 domains are frequently found in signaling molecules that regulate cell proliferation. Such molecules include tyrosine kinases, tyrosine phosphatases, and the adapter molecules
(e.g., Grb2) that link these enzymes to other members of
signaling cascades. Grb3-3, a naturally occurring variant of
Grb2 that contains a truncated, nonfunctional SH2 domain, was found to promote apoptosis, presumably by acting as a dominant negative inhibitor of a cell survival pathway (10). Our results (Figs. 1-5) are just the opposite: i.e., they suggest that some SH2 domains can be involved in
promoting cell death, not survival. We suggest that the fate
of the cell may rest in the balance between apoptotic and
survival pathways, both controlled in part by SH2 domain
interactions. After this manuscript was submitted, it was
reported that c-Crk, an adapter protein containing an SH2
domain, is required for apoptosis in Xenopus egg extracts,
and that the SH2 domain of Crk inhibits apoptosis potently in this system (8). The activity of Crk could account
for our results; however, an involvement of other proteins
containing SH2 domains cannot be excluded at present.
In view of our findings that suggest a role for PTyr-SH2
domain interactions in apoptosis, it may seem surprising
that genistein, an inhibitor of multiple tyrosine kinases,
has little or no effect on apoptosis in the Xenopus system.
This paradox can be resolved if we suppose either that the
critical phosphorylation events have already taken place
before the preparation of the extracts, or that the kinases
responsible for these critical phosphorylation events are
insensitive to genistein. We have also observed that pervanadate (1-10 mM), an inhibitor of protein tyrosine phosphatases, inhibits apoptosis effectively in the Xenopus system (unpublished data; this inhibition is not merely due to
the presence of peroxide in the pervanadate preparation,
as control experiments with added H2O2 [10 mM] showed
no effect on the kinetics of apoptosis). This result may suggest that tyrosine dephosphorylation of specific proteins is
required for the apoptotic process in the Xenopus system.
However, it is also possible that pervanadate blocks apoptosis by inhibiting activities other than phosphatases.
The Relationship of Ceramide and the Heavy
Membrane Fraction
The role of the heavy membrane fraction is still under investigation. Previously we reported that a soluble ceramide
analog can induce apoptosis in HM-depleted extracts (24).
Further experiments have shown that this effect is apparently due to the synergistic effect of ceramide and a small
amount of contaminating heavy membranes still remaining
in the HM-depleted extracts (Farschon, D.M., and D.D.
Newmeyer, unpublished results). We note that both the
production of ceramide and the molecules that respond to
ceramide are reported to reside in the plasma membrane
(e.g., 22). If so, to explain the activity of ceramide in HMdepleted extracts, we would need to postulate that some
amount of contaminating plasma membrane-derived material is present. The HM fraction could also contain a certain amount of such membranes. Thus, we considered the
possibility that the function of our HM preparation was
simply to increase the amount of plasma membrane-producing ceramide. However, as Figs. 7 and 8 show, time-
dependent changes occur in the cell-free system that at
first diminish its responsiveness to ceramide and later
make it responsive to the HM fraction. Thus, it is unlikely that the function of HMs is merely to produce ceramide.
Recent studies have shown that, during apoptosis, mitochondria release cytochrome c into the cytosol, leading to
the activation of CPP32-like proteases. Bcl-2, through its
interactions with the outer mitochondrial membrane,
blocks the efflux of cytochrome c (18a, 18, 48). Whether
ceramide can directly or indirectly modulate the release of
cytochrome c from mitochondria is still unknown.
Temporal Stages in Apoptosis
Apoptosis could be envisioned as occurring in three
stages. The first of these, "initiation," would refer to the
events that cause entry into the common death pathway.
The second stage, which might be termed "sentencing,"
encompasses the intracellular events that commit the cell
irreversibly to the death process. Finally, the cell enters
the "execution" stage, in which effector molecules, such as
particular nucleases and proteases, accomplish the overt changes associated with apoptotic cell death. Each of these
hypothetical stages could itself involve a series of events.
To understand the apoptosis machinery, it will be necessary to know both the identities of the molecular participants in the process and whether these molecules act in sequence or in parallel.
In this report we have investigated the function of several components of the apoptotic machinery: SH2 domains, the HM fraction, ceramide, ICE family proteases,
and Bcl-2, particularly with regard to the time intervals
during which these reagents could affect the kinetics of nuclear destruction in the cell-free system. The events we examined fell into three main time windows, as depicted in
Fig. 10. We propose that these experimentally defined
time periods correspond to the three conceptual phases of
apoptosis listed above.
The earliest distinguishable phase in the cell-free system, which we call "initiation," occurs in the first ~60-90
min of incubation. (Events occurring before lysis of the
eggs may of course also be important for apoptosis initiation, but are outside the scope of our investigations.) The
importance of SH2 domains and ceramide in this period
(Figs. 5 and 8) is consistent with the expectation that these
molecules would function in signal transduction. The second period, which occurs ~90-120 min after the start of incubation, is the time when Bcl-2 protein, the heavy membrane fraction, and the CPP32-related protease(s) act
(Figs. 6, 7, and 9). We propose that this represents a "sentencing" phase, during which the system becomes committed irreversibly to the apoptotic pathway. Finally comes
the third, or "execution" phase, beginning after ~1.5-2 h
of incubation. This is the period when overt changes occur
in the cell nuclei, culminating in extensive DNA fragmentation and nuclear destruction. During this phase, we also
observed the cleavage of two polypeptides whose proteolysis is characteristic of apoptotic cell death (25, 33): fodrin, a cytoskeletal protein endogenous to the extract, and
lamin B, present in the rat liver nuclei added to the system
(18a). Our kinetics data suggest that the cleavage of fodrin
and lamin B occur later than the proteolytic events inhibited by DEVD. Thus, this system appears to involve at
least two distinct protease activities, which may belong to
an enzymatic cascade.
It is important to note that the timing of apoptotic
events in the Xenopus system is somewhat variable from
extract to extract. This may be due, for example, to variation between animals in the proportion of eggs that are undergoing apoptosis. In extreme cases, the onset of nuclear
destruction in a given extract can be as early as 45 min
or as late as 4 h after incubation has begun. However, in
the majority of experiments, the various events occurred within ± 30 min of the times indicated in Fig. 10. This
means that, while the data in this report show that Bcl-2,
the HM fraction, and DEVD-inhibitable protease activity
all act in roughly the same time period, other approaches
have been required to determine the sequence in which
they act. Studies have now shown that mitochondria release cytochrome c into the cytosol, and that this event is blocked by Bcl-2 (18, 48); cytochrome c, when present in
the cytosol, causes the activation of CPP32-related caspases (23, 18a).
The occurrence of kinetically distinguishable phases in
apoptotic extracts now serves to underscore the value of
this cell-free system for dissecting the crucial intracellular
events involved in apoptosis. In particular, we expect this
system to be useful in uncovering both the activation
mechanisms and the critical substrates of the CPP32/Ced-3
family proteases.
converting enzyme (ICE)1 (26). Most recently, a particular
subfamily of these enzymes, consisting of Ced-3, CPP-32/
Yama/Apopain (5, 11, 30, 39, 43), MCH2 (12), and ICELAP3/CMH-1/MCH3 (6, 13, 21), has been suggested to
play a central part in the apoptotic process.
Materials and Methods
-[32P]ATP (1 mCi/ml), and
Xenopus egg extract (7.4% vol/vol, corresponding to a protein concentration of ~2.6 mg/ml) were incubated at 22°C for the times indicated, in 25 µl of
a buffer containing 50 mM Hepes/K, pH 7.4, 10 mM MgCl2, 5 mM MnCl2,
and 1.2 mM Na3VO4. The reaction was stopped with the addition of 155 µl
of 3.2% TCA and 20 µl of a solution of BSA (10 mg/ml). After 30 min on
ice, the samples were centrifuged, and 100 µl of each supernatant was applied to a 2.5-cm circle of phosphocellulose membrane (Whatman P-81). The filters were washed seven to eight times in 75 mM o-phosphoric acid
in 50-ml conical tubes. After drying, the filters were placed in 4 ml of
scintillation fluid and the radioactivity was counted. All the results presented here were qualitatively reproducible. However, the time at which
nuclear destruction began to occur varied from extract to extract, making
it impossible to average the results from separate experiments in a meaningful way. Each figure is representative of at least four experiments giving similar results.
Results
10 nM. A similar effect was seen when fusions of GST with the SH2 domains
of Grb-2 and Stat1 were added (not shown).
Fig. 1.
SH2 domains from Abl and Src inhibit apoptosis in the
cell-free system. GST fusions of the SH2 domains of c-Abl and
c-Src were added, at the indicated concentrations, to an apoptotic
egg extract containing sperm chromatin. The fraction of intact
nuclei was assayed at various times thereafter, as previously described (29).
[View Larger Version of this Image (25K GIF file)]
Fig. 2.
Effect of wild-type and mutant SH2 domains from Lck.
The experiment was performed as in Fig. 1. GST-SH2 domain fusions were added at 100 nM. Note that the wild-type (Y192) Lck
SH2 domain is unable to inhibit apoptosis, as is the E192 mutant,
which mimics the phosphorylated form of Y192. However, the
F192 mutant, which mimics the unphosphorylated form of Y192,
inhibits apoptosis in the extract.
[View Larger Version of this Image (19K GIF file)]
Fig. 3.
The wild-type Lck SH2 domain is inactive in the extract
because it becomes phosphorylated. (A) Genistein, an inhibitor
of tyrosine kinases, restored the ability of the wild-type Lck SH2 domain to inhibit apoptosis. The experiment was done as in Fig. 2, except that genistein (10 µM) was added to the indicated samples either separately or along with the wild-type (WT) Lck SH2
domain-GST fusion (100 nM). (B) A kinase in the Xenopus egg
extract can phosphorylate a synthetic peptide corresponding to
the site surrounding Y192 in Lck. The peptide (RNLDNGGFYIAPR) was incubated in a buffer containing -[32P]ATP and Xenopus egg extract (diluted as described in Materials and Methods)
for the times indicated, and the incorporated radioactivity was assayed (see Materials and Methods). Two separate experiments are shown.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
A synthetic peptide ligand for Src family SH2 domains,
EPQY*EEIPIYLK (Y*EEI), inhibits apoptosis in the Xenopus
cell-free system in a PTyr-dependent manner. (A) The indicated
concentrations of phosphopeptide were added at the start of incubation. (B) (Top) The phosphopeptide (denoted by Y*EEI)
was added to the extract at a final concentration of 5 nM, in the
presence or absence of genistein. Genistein has no effect on its
activity. (Bottom) The unphosphorylated form of this peptide
(YEEI) and an unphosphorylatable mutant peptide (FEEI) were
added at 5 nM. Note that the FEEI peptide has no inhibitory activity. The unphosphorylated peptide (YEEI) inhibits apoptosis
nearly as well as the Y*EEI, but this inhibition is reversed in the
presence of genistein. Thus, the peptide is apparently phosphorylated by a kinase present in the extract.
[View Larger Version of this Image (18K GIF file)]
1
(DNDY*IIPLPDPK; 35). This peptide had no significant
effect on the apoptotic process in the cell-free system, even
at concentrations as high as 1 µM (see Fig. A1). This is additional evidence that interactions involving specific SH2
domains are important in the Xenopus system.
Fig. 5.
The Src SH2 domain inhibits an activity important in
the early portion of the apoptotic process. The experiment was
performed as above, except that the GST-Src SH2 fusion was
added (final concentration 100 nM) at the indicated times after
the start of incubation.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
The Bcl-2 protein acts later in the apoptotic process, after ~1.5 h of incubation. Lysates from Sf9 cells (0.01 vol, as described; 29) infected with Bcl-2 baculovirus or, as a control, -galactosidase baculovirus were added at the indicated times after the
start of incubation. Note that Bcl-2 is much less effective when its addition is delayed at least ~2 h after the start of incubation.
[View Larger Version of this Image (18K GIF file)]
Fig. 7.
The heavy membrane (HM) fraction is required relatively late in the apoptotic process. Percoll-enriched HMs (29)
were added at the indicated times. Note that only when the HMs
were added at least 2 h after the start of incubation was there any
significant delay in the kinetics of nuclear destruction.
[View Larger Version of this Image (21K GIF file)]
Fig. 8.
Ceramide acts early in the cell-free apoptotic process.
A progressive loss of activity occurs when the addition of ceramide (150 µM) is delayed 0.5-1 h.
[View Larger Version of this Image (21K GIF file)]
Fig. 9.
CPP32-like protease
activity is required for apoptosis in the Xenopus system.
The crucial proteolytic event
occurs ~1.5-2 h after the
start of incubation. (A) The
tetrapeptide inhibitors, AcDEVD-CHO (DEVD) and
Ac-YVAD-CHO (YVAD),
were added to the extract from the start of incubation
at the concentrations indicated. Nuclear survival was
quantitated at various times.
(B) The experiment was performed as above, except that
DEVD (100 µM) was added
at the indicated times. Note
that the inhibitor lost its effect only when added after
~2 h of incubation. (C and
D) Fluorescence photomicrographs of rat liver nuclei
placed in apoptotic extract,
incubated for 3 h in the presence (C) or absence (D) of
10 µM DEVD. Bar, 10 µM.
[View Larger Versions of these Images (29 + 15K GIF file)]
Discussion
Fig. 10.
Summary of the approximate time intervals during
which various events occur in the cell-free system.
[View Larger Version of this Image (22K GIF file)]
Fig. A1.
Comparison of SH2 ligand peptides with regard to
their effects on the Xenopus cell-free apoptosis system. The experiment was done as in Fig. 4, except that either the YEEI peptide,
or a synthetic peptide corresponding to the SH2 ligand domain of
phospholipase C (PLC)-1 surrounding Y1021 (PLC peptide,
DNDY*IIPLPDPK; 35) was added to the system at the indicated
concentrations before incubation. Note that the PLC-
1 peptide
had no significant inhibitory activity in this system.
[View Larger Version of this Image (18K GIF file)]
Received for publication 4 March 1996 and in revised form 25 March 1997.
1. Abbreviations used in this paper: GST, glutathione-S-transferase; HM, heavy membrane; ICE, interleukin-1We are indebted to Rajasekaran Baskaran and Jean Wang for helpful advice and gifts of Abl, Src, and Stat1 SH2 domain GST fusions, as well as to David Jones for a GST fusion of the SH2 domain of Grb-2. Gratitude is expressed to our colleagues Ruth Kluck and Doug Green for advice and comments on the manuscript.
This work is supported by grants to D.D. Newmeyer from the American Cancer Society (DB-97) and National Institutes of Health (NIH) (GM50284) and to T. Mustelin from NIH (GM48960 and AI35603).