(Received for publication, November 11, 1996, and in revised form, December 24, 1996)
From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109
The pivotal discovery that Fas-associated death
domain protein (FADD) interleukin-1-converting enzyme (FLICE)/MACH
was recruited to the CD95 signaling complex by virtue of its ability to
bind the adapter molecule FADD established that this protease has a role in initiating the death pathway (Boldin, M. P., Goncharov, T. M.,
Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815; Muzio, M., Chinnaiyan, A. M., Kischkel, K. C., O'Rourke, K.,
Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M.,
Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827). In this report, we describe
the cloning and characterization of a new member of the caspase family,
a homologue of FLICE/MACH, and Mch4. Since the overall architecture and
function of this molecule is similar to that of FLICE, it has been
designated FLICE2. Importantly, the carboxyl-terminal half of the small
catalytic subunit that includes amino acids predicted to be involved in substrate binding is distinct. We show that the pro-domain of FLICE2
encodes a functional death effector domain that binds to the
corresponding domain in the adapter molecule FADD. Consistent with this
finding, FLICE2 is recruited to both the CD95 and p55 tumor necrosis
factor receptor signaling complexes in a FADD-dependent manner. A functional role for FLICE2 is suggested by the finding that
an active site mutant of FLICE2 inhibits CD95 and tumor necrosis factor
receptor-mediated apoptosis. FLICE2 is therefore involved in CD95 and
p55 signal transduction.
The conserved mechanisms of programmed cell death that play a
fundamentally important role in tissue homeostasis, embryogenesis, and
cellular defense mechanisms have only recently been subject to
molecular analysis (1). Studies in Caenorhabditis elegans were important in providing a molecular framework for the cell death
pathway. In particular, the discovery that the C. elegans death gene, Ced-3, possessed substantial homology to the mammalian interleukin-1-converting enzyme (ICE)1
was a major step forward (2). ICE is an unusual cysteine protease that
processes pro-interleukin-1
to the mature cytokine by cleaving after
Asp residues. Subsequently, several mammalian Ced-3 homologues have
been characterized that are unable to process pro-interleukin-1
but
cleave poly(ADP-ribose) polymerase (PARP), a protein known to be
proteolytically processed early in apoptosis. This and other evidence
suggested that these related family members played a more prominent
role in apoptosis. Recently, this family of cysteine, aspartate-specific proteases has been named the caspase family to
denote cysteine, aspartate-specific proteases (3). Depending upon
susceptibility to tetrapeptide inhibitors, this family of proteases can
be divided into CPP32-like (DEVD-inhibitable) and ICE-like
(YVAD-inhibitable) enzymes (4). Evidence is accumulating for the
existence of a cascade of caspases that can potentially activate each
other, thereby amplifying the death signal leading to precipitous
cleavage of death substrates and rapid demise of the cell (5, 6). The
caspases are activated by a number of physiological and pharmacological
stimuli that induce apoptosis (7).
A number of intriguing questions remain, however, including the identity of the caspases that initiate the cascade, the mechanism of activation, and the exact sequence of events leading to activation of downstream effector caspases.
The identification of FLICE/MACH as a receptor-associated caspase like protease suggested a surprisingly direct mechanism for engagement of the death pathway by the death receptors CD95 and the p55 tumor necrosis factor 1 (8, 9). Upon activation, both these receptors use their death domains to bind the corresponding domain of adapter molecules. Therefore, the death domain appears to represent a protein-protein interaction motif. The death domain containing adapter molecule FADD plays a central role as a conduit for death signals from both the CD95 and p55 receptors (10). Dominant negative versions of FADD that lack the amino-terminal segment (yet retain the death domain) effectively attenuate both CD95- and p55-induced apoptosis (11, 12). Since the amino-terminal domain of FADD appeared necessary to engage the downstream components of the death pathway, it was dubbed the death effector domain (DED) (10). The importance of this domain was underlined by the discovery that a caspase (FLICE/MACH) possessed sequences homologous to the DED within its pro-domain. Biochemical and mutagenesis studies revealed that the DED of FADD, by virtue of its ability to bind to the corresponding sequence motif in the pro-domain, recruited FLICE/MACH to the receptor signaling complex (8). These studies for the first time suggested a homophilic binding mechanism involving DEDs that allowed the death receptors to physically engage the caspases through the adapter molecule FADD.
As part of our continuing effort to characterize additional members of the caspase family, we have identified a new member, designated FLICE2, that is a close structural homologue of FLICE and very similar to Mch4 with certain important exceptions. We demonstrate for the first time that FLICE2 is a Ced-3 homologue capable of interacting with both p55 and CD95 receptors through the adapter molecule FADD. Further, a catalytically inactive form of FLICE2 inhibited both p55- and CD95-induced apoptosis, suggesting that FLICE2 can be recruited to the receptor signaling complex and participate in the propagation of the death signal.
The following oligonucleotides were used:
T96R, GAAAGATGACACAGGTACACG; pCDM8 5F2,
AATGTCGTAACAACTCCGCCCC; FL2R, CCTTTAGAGCACAATGGATCTCGAGGT; pCDM8 3
, CACACCACAGAAGTAAGGTTCCTT; FL#2, ACAACCAGCAAGTCTTGAAGTCTC; T96stop, AAGCCTCTGGAAAGAACTAGGAAACGCTG.
A FLICE2 peptide, ISAQTPRPPMRRWS, that corresponds to amino acids 505-518 of the small catalytic subunit was used to immunize rabbits and obtain polyclonal antiserum.
Cloning of Human FLICE2An EST clone (GenBank accession no.
T96912[GenBank]) was identified as a new caspase family member. This clone
contained a 1.5-kilobase insert encoding sequences corresponding to the
carboxyl-terminal segment of caspases, stop codon, 3-untranslated, and
poly(A)-tail. Full-length sequence was obtained by two rounds of PCR
using gene- and vector-specific primers. Initially, cDNA from a
human K562 library was used as template, T96R as the gene-specific
reverse primer, and pCDM8 5
F2 as the vector-specific forward primer. A
second gene-specific reverse primer, FL2R, was designed, and an
additional upstream sequence was obtained from a melanoma library employing FL2R as the gene-specific primer and pCDM8 3
as the vector-specific primer. Finally, a full-length clone was obtained by
PCR with the two gene-specific primers FL#2 and T96stop using a
thermostable proofreading polymerase (Clontech). The resulting PCR
product was subcloned, sequenced, and used as template for other
constructions.
All eukaryotic expression vectors were constructed in pcDNA3 (Invitrogen) by standard PCR techniques using custom-designed primers encoding epitope tags and appropriate restriction sites. FLICE2 harboring a His6 tag at its carboxyl terminus was constructed in the pET23b vector (Novagen).
Northern Blot AnalysisHuman multiple tissue and human cancer cell line poly(A)+ RNA blots were obtained from Clontech. 293, 293EBNA, Jurkat, U937, MCF7, and THP1 RNA were purified using the RNeasy kit (Qiagen) following the manufacturer's instructions and analyzed by Northern blotting as described previously (13). The 32P-labeled FLICE2 probe encoded amino acids 229-269.
Granzyme B Activation and PARP CleavageHis6-tagged FLICE2 was generated by coupled in vitro transcription/translation using the TNT kit (Promega). The translated protein was purified as described previously (14). In vitro activation of purified FLICE2 by granzyme B (gift from C. J. Froelich, Northwestern University Medical School) was performed essentially as described previously (14). Briefly, 0.3 pmol of granzyme B were used to cleave 50 nmol of FLICE2. The tetrapeptide aldehyde inhibitors, YVAD-CHO and DEVD-CHO (Bachem), were added at a final concentration of 1 µmol following incubation with granzyme B.
In Vitro Binding[35S]Methionine-radiolabeled proteins obtained by coupled in vitro transcription/translation were incubated with bacterially expressed His6-tagged proteins immobilized onto Ni-NTA agarose beads. Binding reactions were performed as described previously (8). Fraction of input radiolabeled protein that bound to the beads was quantitated by phosphorimager analysis.
Co-immunoprecipitation and Western Blot AnalysisTransient
transfections of 293 cells were performed as described previously (15).
A CrmA expression construct was included to suppress apoptosis (Fig. 2,
D and E). Cells were harvested 40 h
following transfection, precleared, immunoprecipitated with the
indicated antibodies, and analyzed by immunoblotting (16).
Cell Death Assays
MCF7 cells were transfected by the
lipofectamine procedure (Life Technologies, Inc.) with 0.5 µg of
FLICE2 and 1.5 µg of p35 or CrmA expression constructs (17). Mutant
green fluorescent protein, pEGFP-N1 (50 ng), was included as a
transfection marker (Clontech). Cells were fixed 30 h after
transfection in 4% formaldehyde, permeabilized and stained with 0.1 µg/ml 4,6-diamidino-2-phenylindole dissolved in phosphate-buffered
saline plus 1% Triton X-100, and nuclear morphology of transfected
cells evaluated by fluorescence microscopy. Nuclei with chromatin
margination and condensation were scored as apoptotic. Human embryonic
kidney cells, 293 and 293EBNA, were transfected by CaHPO4
using a 1:10 ratio of pCMV-
GAL and the appropriate death-inducing
construct. Cells were fixed and stained after 24-40 h (17). The
percentage of apoptotic cells was determined by calculating the
fraction of membrane blebbed blue as a function of total blue cells.
All assays were evaluated in triplicate, and the mean and the standard
deviation were calculated.
A search of the GenBank EST data base
revealed a clone T96912[GenBank] derived from a human fetal spleen library with
high homology to the conserved GSW sequence contained within the small catalytic subunit of all caspases. Sequencing of the EST clone revealed
a downstream stop codon as well as 1.3 kilobases of 3-untranslated region followed by a poly(A) tail. Full-length sequence was obtained by
two rounds of PCR extension employing vector- and gene-specific primers. The derived open reading frame encoded a protein of 521 amino
acids with a molecular mass of 59 kDa. Because of its significant homology over its entire sequence (28% identity) to FLICE/MACH (8, 9),
it was designated FLICE2. Recently, however, a third FLICE homologue,
Mch4, has been described (18). The alignment in Fig.
1A shows that FLICE2 and Mch4 have a high
degree of identity (90%). This identity extends to the nucleotide
level and includes 200 base pairs of 5
-untranslated sequence.
Therefore, it is likely that these two proteins are encoded by the same
gene and represent alternatively spliced products. The two sequences
differ completely, however, in two coding segments (highlighted in
blue). First, FLICE2 has a 50-amino acid insert at the carboxyl
terminus of the pro-domain. PCR analysis using primers flanking this
divergent sequence revealed sequence length polymorphism, with FLICE2
being the longest transcript (data not shown). Thus, the insert
probably arises from differential splicing. Second, the 48 carboxyl-terminal amino acids of FLICE2 are distinct and possess only a
low level of homology to Mch4. This sequence unique to FLICE2 was,
however, present in all PCR products tested and was, in fact, encoded
by the original EST (T96912[GenBank]) as indicated in Fig. 1A.
Significantly, the enzymatic activity of FLICE2 is expected to be
substantially different from Mch4 since this divergent region encodes
for half of the small catalytic subunit.
A, alignment of the deduced amino acid
sequence of FLICE2 with Mch4 and FLICE. Differences between FLICE2 and
Mch4 are highlighted in blue. The open reading frame
corresponding to EST T96912[GenBank] is indicated by a black line.
Amino acids boxed in red identify death effector domain
residues in FADD that are conserved with the two DEDs in FLICE and
FLICE2. The conserved pentapeptide QACQG is boxed in green. An asterisk indicates the cleavage
site between the large and small subunits of the catalytic domain.
Based on the CPP32 crystal structure, the symbols above the
alignment indicate residues involved in contacting the substrate. The
corresponding amino acids in CPP32 are also indicated above the
symbols. +, active site cysteine; , contacts with P1 residue;
,
second tier hydrogen bonds with P1; ×, contacts with P4 where the
intensity of the violet shading indicates the level of sequence
divergence from CPP32. B, PARP cleavage of Granzyme
B-processed FLICE2. In vitro translated His6
FLICE2 was purified and activated with granz yme B as described under
"Materials and Methods." PARP cleavage was performed in the absence
or presence of the indicated tetrapeptide aldehyde inhibitors (1 µm).
C, multiple human tissue and cell line mRNA blots were
probed with a 32P-labeled probe specific for FLICE2.
Comparison of the crystal structure of CPP32 and the sequence of FLICE2 reveals complete conservation of substrate contacts for the P1 aspartate residue (19). Surprisingly, the amino acids that contact the P4 residue in the CPP32 crystal structure and the homologous residues in FLICE2 are divergent. Of the seven amino acids contacting the P4 residue, only one is conserved (Trp-457). Three are conserved substitutions (Phe-449, Glu-454, Trp-491). The remaining are predicted to significantly change the properties of the P4 pocket (His-451, Val-452, Glu-453). Glu-453, in particular, represents the loss of two positive charges as it is equivalent to Lys-210 in CPP32. Therefore, FLICE2 is likely to have a unique substrate and inhibitor specificity with respect to the P4 position. The first indication that these structural correlates have functional consequences comes from inhibitor studies using CrmA, a pox virus-encoded serpin. CrmA is a poor inhibitor of CPP32 but a strong inhibitor of FLICE (20). The P4 position in CrmA is a leucine. The corresponding P4 binding pocket in FLICE contains an alanine at the position equivalent to Lys-210 in the P4 binding pocket of CPP32. Therefore, the loss of a positive charge in the P4 pocket leads to an active site that can readily accommodate P4 substrate residues that are not negatively charged. Given that the P4 binding pocket in FLICE2 has a negatively charged residue (Glu-453) in place of Lys-210 in CPP32, it is conceivable that FLICE2 may accept positively charged P4 residues.
FLICE2 Cleaves PARPAll mammalian caspases are synthesized as zymogens that need to be proteolytically processed at internal Asp residues to produce the active dimeric species (21). Granzyme B, an aspartate-specific protease from cytotoxic T cells granules, is capable of activating CPP32-like caspase zymogens in vitro (8, 14, 17, 18, 22, 23). His6-tagged FLICE2 was obtained by coupled in vitro transcription/translation, purified, and activated by granzyme B. Residual granzyme B activity was neutralized by addition of anti-GraB, a specific inhibitor of granzyme B (14). FLICE2 enzymatic activity was assessed by the addition of the substrate PARP. Immunoblot analysis revealed FLICE2 to be a competent Ced-3-like protease that was capable of cleaving PARP to its signature 85-kDa apoptotic form (Fig. 1B). Additionally, PARP cleavage was inhibited by the tetrapeptide inhibitor DEVD-CHO, but not by YVAD-CHO, consistent with FLICE2 being a CPP32-like but not ICE-like protease.
FLICE2 ExpressionHuman tissue and cell line RNA blots were probed with a 32P-labeled cDNA specific for FLICE2 and not contained within Mch4 (Fig. 1C). A transcript of 4.4 kilobases was detected and is consistent with the size of the cloned cDNA. The tissue distribution was strikingly similar to that of FLICE (8) In particular, tissues enriched in lymphoid cells expressed a substantial amount of FLICE2 transcript. Embryonic expression was high in all tissues with the exception of the brain. A variety of transformed cell lines expressed low levels of FLICE2. K562, a chronic myelogenous leukemia line, displayed significant expression. Importantly, the cell lines used for transfections in this study including 293, 293EBNA, and MCF7 did not express detectable levels of endogenous FLICE2 transcript.
While the mRNA expression patterns are consistent with FLICE-related proteins being involved in the maturation of the lymphoid system, additional functions are likely as suggested by the high level of expression of MCH4 and FLICE in the heart (9, 18). FADD and FLICE2 mRNA expression patterns are not identical, suggesting that situations may exist where the two function independently of each other.
FLICE2 Binds the Death Adapter Molecule FADDDeath effector
domains have been shown to be the protein interaction motifs that
mediate the binding of FLICE to FADD (8). FLICE2, like FLICE, contains
two DEDs, with the first being more conserved. To establish the
in vivo function of the DEDs, FLICE2/FADD binding
experiments were undertaken (Fig. 2A).
Co-immunoprecipitation analysis clearly revealed the ability of FLICE2
to specifically bind full-length FADD but not FADD, which lacks a
functional DED due to truncation of the first 18 amino-terminal amino
acids. Conversely, FLICE2 lacking the DEDs (encoding only the catalytic subunits) did not coprecipitate with FADD. Indeed, the DED containing pro-domain of FLICE2 by itself was fully capable of binding FADD (Fig.
2B). This interaction was specific since the pro-domain of
FLICE2 did not bind to FADD with a disrupted DED (
FADD). The unrelated caspase Mch2 served as a negative control. Notably, the
catalytically inactive cysteine mutant C401S FLICE2 retained its
ability to bind FADD, suggesting potential for use as a dominant negative inhibitor.
Fig. 2C shows the result of analogous binding experiments performed in vitro using purified recombinant FADD and FADD-DN that lacks the DED. Again, FLICE2 specifically bound the DED of FADD through its pro-domain. This interaction as assessed by binding of input radiolabeled protein was equivalent for both FLICE and FLICE2. The ability to reconstitute FLICE2-FADD binding in vitro using purified molecules suggested that the interaction was direct and not mediated by an intermediary molecule.
FLICE2 Is Recruited to the Death Receptors CD95 and p55The FLICE2-FADD interaction raised the possibility that FLICE2, like FLICE, could be recruited to the CD95 or p55 signaling complexes in a FADD-dependent manner. To directly assess if FLICE2 could be recruited to the CD95 or tumor necrosis factor receptors, FLICE2 was cotransfected with FLAG-tagged p55 or CD95 receptors (Fig. 2, panels D and E). As shown, FLICE2 bound both death receptors, and a substantial increase in binding was observed when FADD was included in the transfections (Fig. 2, panels D and E, lanes 1 and 2). This was consistent with initial binding being mediated by endogenous FADD and being enhanced by the expression of exogenous FADD. Confirming this was the finding that expression of FADD-DN, which lacks a DED and is therefore unable to bind FLICE or FLICE2, attenuated the association of FLICE2 with the death receptors (Fig. 2, panels D and E, lane 3).
Overexpression of FLICE2 Induces ApoptosisThe homology of
FLICE2 with other members of the caspase family suggests that it is a
protease involved in apoptosis. MCF7 or 293EBNA cells were transiently
transfected with FLICE2, and recipient cells underwent morphological
changes including nuclear condensation, cellular shrinkage, and
membrane blebbing, all of which are hallmarks of apoptosis (Fig.
3A). The induction of apoptosis could be
efficiently blocked in both cell lines by the well characterized viral
inhibitors of caspases, CrmA and p35 (20, 24-26). Importantly, the
active site cysteine mutant (C401S FLICE2) inhibited killing by native
FLICE2 in 293EBNA cells. This inhibition was probably due to the
formation of inactive heterodimers composed of wild type and
catalytically inactive molecules as suggested by the crystal structures
of ICE and CPP32 (19, 27, 28).
Inhibition of CD95 and p55-induced Cell Death by the FLICE2 Active Site Mutant
293EBNA cells underwent apoptosis when transiently transfected with CD95 receptor (Fig. 3B). This autoactivation on overexpression occurred in a dose-dependent manner (lanes 1 and 3) and has been reported previously (9). Cotransfection with the active site FLICE2 cysteine mutant effectively inhibited the induction of apoptosis to the same extent as CrmA or p35 (lanes 6 and 7). Similarly, transfected cells overexpressing the p55 receptor underwent an apoptotic demise by 24 h. Again, expression of the FLICE2 active site mutant inhibited apoptosis to the same extent as p35, CrmA, and the active site mutant of FLICE (C360S FLICE). Taken together, these results are in keeping with the involvement of FLICE2 in the death pathway engaged by both CD95 and p55 (Fig. 3C). Additionally, these results are consistent with FLICE2 operating at the apex of the caspase cascade.
FLICE, the first caspase shown to be associated with CD95 and p55 receptors, has similar properties. This is predictable given the conservation of functional domains between the two molecules. Both have functional death effector domains in their pro-sequences that can bind FADD, and the signature sequence surrounding the catalytic cysteine is QACQG and not QACRG as it is in the other mammalian caspases. The amino acids that are predicted to contact the P4 site, however, diverge significantly (Fig. 1A). Therefore, FLICE and FLICE2 probably have different substrate specificities. An attractive hypothesis is the notion that receptor oligomerization activates the caspase cascade by approximating the two FLICEs such that they act as substrates for each other. FLICE activation involves two aspartate specific cleavages: Asp-374 between the large and small catalytic subunits and Asp-216 between the pro-domain and large catalytic subunit. The P4 amino acids are Ile and Arg, respectively. A positively charged residue in position 4 is intriguing, given the glutamate substitution (Glu-453) in the P4 binding pocket of FLICE2 and suggests that FLICE2 may be capable of cleaving the pro-domain of FLICE.
In summary, we have shown that FLICE2 is a signaling caspase able to interact with the death receptors p55 and CD95 through the adapter molecule FADD. A dominant negative version of FLICE2 effectively inhibited apoptosis, establishing a role for this molecule in signaling from the death receptors. Future studies will investigate the possibility of whether FLICE/FLICE2 transactivation is responsible for initiating the caspase cascade.
We thank Chris Froelich for the granzyme B, Arul Chinnaiyan, Marta Muzio, Kim Orth, and Karen O'Rourke for reagents and protocols, and Ian M. Jones for expertise in making the figures.
Caspase-10/b is the name assigned to FLICE2 according to the reorganized ICE/Ced-3 protease nomenclature.