(Received for publication, November 8, 1996, and in revised form, February 7, 1997)
From the Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium and the § Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105
Emerging evidence suggests that
multiple aspartate-specific cysteine proteases (caspases (CASPs)) play
a crucial role in programmed cell death. Many cellular proteins have
been identified as their substrates and serve as markers to assay the
activation of CASPs during the death process. However, no substrate has
yet been unambiguously identified as an effector molecule in apoptosis.
PITSLRE kinases are a superfamily of Cdc2-like kinases that have been
implicated in apoptotic signaling and tumorigenesis. In this paper
we report that tumor necrosis factor (TNF)-mediated apoptosis is
associated with a CrmA- and Bcl-2-inhibitable cleavage of PITSLRE
kinases, indicating a role for CASPs. Testing of seven murine CASPs for their ability to cleave p110 PITSLRE kinase 2-1 in vitro
revealed that only CASP-1 (ICE (interleukin-1
-converting enzyme))
and CASP-3 (CPP32) were able to produce the same 43-kDa cleavage
product as observed in cells undergoing TNF-induced apoptosis.
Mutational analysis revealed that cleavage of p110 PITSLRE kinase
2-1 occurred at Asp393 within the sequence YVPDS, which
is similar to that involved in the CASP-1-mediated cleavage of
prointerleukin-1
. TNF-induced proteolysis of PITSLRE kinases was
still observed in fibroblasts from CASP-10/0 mice. These
data implicate CASP-3 as a potentially important CASP family protease
responsible for the cleavage of PITSLRE kinases during TNF-induced
apoptosis.
Apoptosis is a fundamental process for normal development of
multicellular organisms and is involved in the regulation of the immune
system, normal morphogenesis, and maintenance of homeostasis (1).
Aspartate-specific cysteine proteases belonging to the interleukin-1-converting enzyme (ICE)1
family, recently renamed the caspase (CASP) family (2), have been
implicated as principal effectors of apoptosis, presumably by their
proteolytic action on specific targets, including members of the
ICE-related protease family themselves, poly(ADP-ribose) polymerase,
DNA-dependent protein kinase, the 70-kDa small U1 ribonucleoprotein, lamins, protein kinase C
, D4-GDP dissociation inhibitor, and various components of the cytoskeleton (3, 4). Many of
these proteins are likely to be involved in the morphological and
biochemical changes that accompany apoptosis, or in aspects of DNA
damage sensing and repair, and are used as markers to assay the
activation of CASPs during the death process. However, no substrate has
yet been unambiguously identified as a downstream effector molecule in
apoptosis.
PITSLRE kinases are a superfamily of protein kinases related to the master mitotic protein kinase Cdc2 (4-6). Ectopic expression of the smallest member of this superfamily has previously been shown to induce apoptosis (5). In addition, deletion of the PITSLRE gene complex and complete loss of expression of specific isoforms occur in many neuroblastoma cell lines and is frequently observed in human cancers (8-10). Induction of apoptosis via the Fas receptor in human T cells has recently been shown to be correlated with proteolysis and increased activity of PITSLRE kinases (7). Triggering of Fas or the related TNF receptor is known to induce the activation of several CASPs (3, 4). Hence we examined the possibility that PITSLRE kinases are substrates for CASP-1 (ICE) and six other members of the murine CASP family in vitro as well as in vivo and demonstrate that CASP-3 (CPP32) is likely to be a crucial CASP responsible for PITSLRE kinase cleavage during TNF-induced apoptosis.
All cell lines were cultured
in appropriate media using standard tissue culture conditions.
PC60p55p75 is a rodent T cell hybridoma that has been transfected with
the p55 and p75 human TNF receptors (11). HeLaH21 is a human cervix
carcinoma. Transfection, isolation, and characterization of
Bcl-2-expressing PC60p55p75 cells were described previously (11).
HeLaH21 cells expressing CrmA were obtained by transfection with 30 µg of pCAGGS-CrmA and 1 µg of pSV2neo using the calcium
phosphate-DNA co-precipitation method and selecting G418-resistant
transformants by culturing cells in medium containing 500 µg
ml1 G418 for 3 weeks. pCAGGS-CrmA was constructed by
inserting the cowpox virus DNA fragment encoding CrmA, provided by Dr.
J. Pickup (12), into the mammalian expression vector pCAGGS (13). CrmA expression was confirmed by Western blotting. Embryonic fibroblasts were prepared from wild-type and CASP-10/0 mice as
described previously (14).
These were performed
essentially as described previously (6, 7). PITSLRE kinases were
detected using antibody P2N100 directed against the first 50 amino
acids of the p110 isoforms or antibody GN1 directed against the first
72 amino acids of p58 PITSLRE kinase 1 (6). ECL (Amersham Life
Science, Amersham, United Kingdom) was used for visualization.
p110 PITSLRE kinase 2-1 and proIL-1
cDNAs were subcloned into the pGEM11Zf(+) plasmid (Promega,
Madison, WI) behind the T7 and SP6 promoter, respectively, using
routine recombinant DNA techniques. 35S-Labeled proIL-1
and p110 PITSLRE kinase
2-1 were prepared by the in vitro
coupled transcription-translation reticulocyte lysate system (Promega)
with SP6 and T7 polymerase, respectively, and incubated for 70 min at
30 °C with either purified recombinant human CASP-1 (a gift from
Drs. N. Thornberry (Merck Research Laboratories) and R. Talanian (BASF
Bioresearch Corp., Worcester, MA)), murine CASP-1 or CASP-3, or crude
cell extracts from Escherichia coli transformed with
different murine CASP expression plasmids, in a total volume of 25 µl
of CASP reaction buffer containing 10 mM HEPES, pH 7.5, 2 mM EDTA, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg ml
1
leupeptin, and 10 µg ml
1 aprotinin. In some
experiments, p110 PITSLRE kinase was first purified from the
reticulocyte lysate by immunoprecipitation with antibody GN1 as
described (6). Reactions were stopped by the addition of SDS-PAGE
sample buffer. Cleavage products were analyzed by SDS-PAGE and
fluorography.
The p110 PITSLRE kinase 2-1
D393A mutant was generated by PCR methodology using the mutagenic
oligonucleotide primer TTCGACCGATCCGGGGA and a kit from
CLONTECH. The presence of the introduced mutation (underlined) and fidelity of PCR replication were confirmed by sequence
analysis.
Cell death was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (15), or in the case of PC60 cells, by propidium iodide uptake, as described previously (11).
Bacterial Expression and Purification of Murine CASPsThe full-length cDNA clones of murine, CASP-1, CASP-2, CASP-3, CASP-6, CASP-7, CASP-11, and CASP-12 (16), were used as a template for classical PCR amplification with Vent polymerase and primers designed to generate CASPs lacking the N-terminal prodomain and appending a 10-amino acid Strep-tag to the C terminus. The resulting products were cloned into the bacterial expression vector pLT10TH downstream of the His tag and transformed in E. coli strain MC1061pT7POL23 (17). Empty pLT10TH was used as a negative control. Exponentially growing E. coli at 28 °C were induced by a temperature shift to 42 °C. Bacterial extracts were prepared by sonication in CASP reaction buffer and either used as active enzyme preparations for in vitro cleavage experiments or further purified by TALON immobilized metal affinity chromatography (CLONTECH) and elution with 20 mM Tris-HCl, pH 7.5, 100 mM imidazole, 50 mM NaCl, 10% glycerol, and 1 mM glutathione. All CASPs were expressed at approximately equal levels, except for CASP-7 which was about 5-fold higher.
We first studied the cleavage of PITSLRE kinases and the role of
CASPs in the killing of a rodent PC60p55p75 T cell hybridoma by human
TNF. Expression and proteolysis of PITSLRE kinases were examined by
Western blotting using PITSLRE kinase-specific antibodies P2N100 and
GN1 (6, 7). In the cells studied, the former recognized the p110
PITSLRE kinase 2 and
2 isoforms (Fig.
1A), while the latter reacted with a p90
PITSLRE kinase isoform, which has been tentatively identified as an
alternatively spliced PITSLRE kinase isoform lacking much of exon 2, but containing all other exons (6),2 and a
170-kDa protein, which is presumably another unknown PITSLRE kinase
isoform (Fig. 1B). The PITSLRE kinase
1 (p65) and
1
(p58) isoforms could not be detected and are presumably not present in
the cells used. Following treatment of PC60p55p75 cells with TNF, the
p110 PITSLRE kinase isoform was cleaved to fragments of 60 and 43 kDa
and a minor 52-kDa product (Fig. 1A). These processing products seemed to be very unstable, because they were only detectable when p110 cleavage was still incomplete and after long exposure times
of the blots. In parallel, the p170 isoform was cleaved to a 130-kDa
protein, which accumulated with increasing TNF exposure times, while
the p90 isoform remained unaffected (Fig. 1B). Proteolysis of PITSLRE kinases was already detectable after 2-h TNF treatment, slightly preceding the onset of TNF-induced apoptosis. Similar results
were obtained in HeLa cells that had been treated with anti-Fas
antibody or TNF (data not shown). Nonspecific proteolysis could be
excluded by the fact that several other proteins were not degraded
(data not shown). As mentioned above, the p90 PITSLRE kinase isoform
lacks exon 2, corresponding to amino acids 375-415 of the PITSLRE
kinase
2-1 110-kDa isoform. This region of the protein contains a
putative cleavage site for proteases of the CASP family
(YVPD393
S). The observation that the p90 PITSLRE kinase
isoform, in contrast to the p110 and p170 PITSLRE kinase isoforms, was
not cleaved after TNF treatment, suggests a possible role for CASPs in
TNF-induced cleavage of the p110 and p170 PITSLRE kinase isoforms. The
latter hypothesis was further substantiated by investigating the effect of TNF in cells stably transfected with either the human
bcl-2 or cowpox virus crmA gene, which encode
proteins known to act as upstream (18) or direct (12) inhibitors,
respectively, of at least some CASPs. Overexpression of these genes has
been shown previously to protect cells against TNF-induced apoptosis (11, 19, 20). Both CrmA and Bcl-2 expression completely inhibited the
TNF-induced cleavage of PITSLRE kinases as well as cell death (Fig.
2). Similarly, pretreatment of cells with the
CASP-specific inhibitor
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Ref. 4; Enzyme
Systems Products, Dublin, CA), in contrast with inhibitors of granzyme
B, cathepsin B, serine, and cysteine proteases, completely protected
cells from TNF-induced apoptosis and PITSLRE kinase cleavage (data not
shown). These results further indicate a role for CASPs in the cleavage
of PITSLRE kinases during the TNF-induced cell death pathway.
In the human system, the CASP family comprises seven different members
that share a conserved QACRG pentapeptide containing the active site
cysteine (2-4). More recently, three additional CASPs have been
described, viz. CASP-8 (MACH = FLICE = Mch5;
Refs. 21-23), CASP-9 (ICE-LAP6; Ref. 24), and CASP-10 (Mch4; Ref. 21). These differ from the other CASPs by a nonconservative substitution in
the active site QACRG pentapeptide, and the presence of a FADD-like effector domain in the case of CASP-8 and CASP-10. In mice, only the
cloning of CASP-1 (ICE), CASP-2 (NEDD-2 = human Ich1) and CASP-11
(Ich3) has been reported so far (2-4, 25). We have recently isolated
and characterized seven murine CASPs containing the conserved QACRG box
(16)3: CASP-1 (ICE), CASP-2 (Ich1), CASP-3 (CPP32, Yama, or
apopain), CASP-6 (Mch2), CASP-7 (Mch3, ICE-LAP3, or CMH-1), CASP-11
(presumably the murine homolog of human CASP-4, TX, Ich2, or
ICErel-II), and CASP-12 (related but presumably not
homologous to human CASP-5 or ICErelIII). To analyze
whether one of these CASPs can directly cleave PITSLRE kinases, we
treated in vitro translated p110 PITSLRE kinase 2-1 with
crude cell extracts of bacteria expressing the specific murine CASPs.
Interestingly, CASP-1 and CASP-3 were the only CASPs that could cleave
p110 PITSLRE kinase to a 43-kDa processed product (Fig.
3). In some experiments, weak 60- and 80-kDa cleavage products could also be observed (see also Fig. 4). The
size of the 43- and 60-kDa PITSLRE kinase cleavage products corresponds to the size of the protein bands that were observed after TNF-induced cleavage of the p110 PITSLRE kinase in vivo (Fig.
1A), further suggesting that the latter is mediated by
CASP-1 or CASP-3. None of the other CASPs displayed any effect on the
PITSLRE kinase, although they were all found, except CASP-12 (which was
nevertheless able to induce apoptosis after transfection; Ref.
16)3, to hydrolyze the synthetic peptide substrates
acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC) and/or
acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin (YVAD-AMC): CASP-1, CASP-2,
CASP-6, CASP-7, and CASP-11 were about 2,000, 700, 4,000, 100, and
2,000 times, respectively, less active than CASP-3 on DEVD-AMC; CASP-1
and CASP-3 were the only enzymes found to be active on YVAD-AMC, CASP-3
being about 25 times less active than CASP-1 (data not shown). Similar
results were obtained when purified, in vitro translated
p110 PITSLRE kinase and purified recombinant CASPs were used, excluding
any contribution of reticulocyte or bacterial proteases that may become
activated when CASPs are present (see also Fig. 4).
Examination of the amino acid sequence of the p110 PITSLRE kinase
revealed that the 43-kDa protein generated upon incubation with CASP-1
or CASP-3 could be generated by cleavage at the sequence YVPD393S, which is very similar to the YVHD
A cleavage
site for CASP-1 in proIL-1
(26) and which conforms to the specific
hydrolysis of YVAD-AMC by CASP-1 and CASP-3 as described above. Indeed,
in vitro translated proIL-1
and p110 PITSLRE kinase
2
were cleaved with similar efficiency by purified CASP-1, requiring a
minimal CASP-1 concentration of only 0.25 ng (Fig. 4, A and
B). PITSLRE kinase cleavage by CASP-3 required about 25 times higher concentrations than CASP-1 (Fig. 4C). Moreover,
a p110 PITSLRE kinase, in which Asp393 in the sequence
YVPD
S had been mutated to Ala, was no longer cleaved by CASP-1 or
CASP-3 (Fig. 4, B and 4C), although some residual
nonspecific cleavage resulting in a 43-kDa degradation product could
still be observed at microgram concentrations of CASP-3 (data not
shown). These data, together with the observation that a p90 PITSLRE
kinase isoform lacking a region containing the YVPD
S sequence is not
cleaved in cells undergoing TNF-induced apoptosis (see above and Fig.
1A), demonstrate that p110 PITSLRE kinase is efficiently
cleaved by CASP-1 and CASP-3 at position D393.
We finally investigated whether in vivo CASP-1 itself is
required for TNF-induced cleavage of PITSLRE kinases; thus we analyzed the effect of TNF in embryonic fibroblasts obtained from
CASP-10/0 mice (27, 28). As shown in Fig. 5,
TNF-induced cell death and PITSLRE kinase cleavage was similar in
wild-type and CASP-1-deficient fibroblasts. Taking into account that
CASP-3 was the only other CASP that was found to cleave p110 PITSLRE
kinase in vitro, the above results suggest a crucial role
for CASP-3-mediated cleavage of PITSLRE kinases in the TNF-induced cell
death pathway. We cannot, however, exclude an involvement of other
newly identified (CASP-8, CASP-9, CASP-10; Refs. 21-24) or still
unidentified CASPs (although the inhibition by CrmA limits possible
candidates), or a more complex scenario involving two or more proteases
which function redundantly or concomitantly, such that elimination of
CASP-1 alone has no effect. The biological implication for
CASP-3-mediated cleavage of PITSLRE kinases in TNF-induced apoptosis is
still unclear. To date, no specific substrates for PITSLRE kinases have been identified. Cleavage by CASP-1 and CASP-3 occurs at a site that
separates the C-terminal kinase domain from the N-terminal part that
harbors two nuclear localization signals as well as a region that has
been shown to bind SH2 domains (29). Therefore, CASP-3-mediated
cleavage of PITSLRE kinases might be a mechanism to modulate their
localization or interaction with other proteins. It should also be
mentioned that in addition to PITSLRE kinases, Cdc2 itself and other
Cdc2-like enzymes have been shown to play a role in apoptosis induced
by TNF and granzyme B (30, 31).
In conclusion, we have shown that TNF-induced apoptosis is associated with CASP-3-specific proteolysis of PITSLRE kinases. This processing does not occur in cells in which the apoptotic pathway is blocked by Bcl-2 or by CrmA expression. Since the PITSLRE kinases represent the first CASP substrates for which a direct apoptotic function and a role in tumorigenesis have been described (5-10), our findings might have important implications for understanding the mechanism of action of TNF, as well as the regulation of apoptosis in human cancer and other diseases.
We thank Drs. N. Thornberry and R. Talanian for recombinant human ICE, Dr. D. Pickup for crmA cDNA, as well as Dr. R. Kamen and Dr. T. Seshadri for ICE0/0 mice and Ilse Van den brande for expression and purification of CASPs.