From the University of Michigan Medical School,
Department of Pathology, Ann Arbor, Michigan 48109, ¶ Howard
Hughes Medical Institute, Harvard University, Department of Chemistry
and Chemical Biology, Cambridge, Massachusetts 02138, and the
** Burnham Institute, La Jolla, California 92037
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ABSTRACT |
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The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic. We hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this was provided by (i) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2. Cells transfected with Fpk3FLICE underwent apoptosis after exposure to FK1012H2; (ii) the creation of a nonprocessable zymogen form of FLICE that retained low but detectable protease activity.
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INTRODUCTION |
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Apoptosis, or programmed cell death, is a cell deletion mechanism that is critical to metazoan survival (1, 2). The cell death machinery is conserved throughout evolution and is composed of several distinct parts including effectors, inhibitors, and activators (1, 3).
Mammalian cysteine proteases (designated caspase for cysteine aspartic acid-specific protease) related to the Caenorhabditis elegans cell death gene CED-3 represent the effector components of the apoptotic machinery participating in a regulated proteolytic cascade (4, 5). Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen (6-8).
The assembly of the Fas receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains (9-12). Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits that amplify the apoptotic signal by activating other downstream caspases (6, 8, 13). However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic.
We hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this model was provided by two independent approaches: (i) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2. Cells transfected with Fpk3FLICE underwent apoptosis after exposure to FK1012H2; (ii) the creation of a nonprocessable zymogen form of FLICE that retained low but detectable protease activity.
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MATERIALS AND METHODS |
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Expression Vectors and Recombinant Proteins-- MFpk3Eu vector containing a myristoylation site, three copies of Fpk in tandem and a hemagglutinin epitope tag (HA) was originally made by D. Spencer (Baylor College of Medicine). Fpk is a double mutant of FKBP12 where residues 89 and 90 have been mutated from GI to PK (14). The catalytic domain of FLICE (encoding Ser-217 to Asp-479) was obtained by polymerase chain reaction and sub-cloned in-frame between the last Fpk and the epitope tag at the SalI site in MFpk3Eu. The same catalytic domain of a mutant version of FLICE, in which Cys-360 was replaced by Ser, was similarly cloned. For production of recombinant purified proteins, the catalytic domain of FLICE or mutant versions of FLICE in which the catalytic Cys-360 was replaced by Ser and/or the cleavage sites Asp-374 and Asp-384 were replaced by Ala, were obtained by polymerase chain reaction and sub-cloned into the pET15b expression vector (Novagen). The proteins were expressed in the BL21 pLysS Escherichia coli strain and purified using the QIAexpress Kit (Qiagen) following the manufacturer`s instructions.
Cells and Transfections-- Human embryonic kidney 293 and HeLa cells were cultured as described previously. Cell death assays were performed as described (9). Hela cells were transfected using the lipofectAMINE procedure (Life Technologies, Inc.) according to the manufacturer's instructions. 293 cells were transfected using calcium phosphate precipitation.
Western and Coimmunoprecipitation Analysis-- For immunoblotting analysis, cells (5 × 105) were lysed in 50 µl of buffer (50 mM KCl, 50 mM HEPES, 5 mM EGTA, 2 mM MgCl2, protease inhibitor mixture) followed by three cycles of freeze thaw. The membrane-rich fraction was pelleted, resuspended in 8 M urea and, after boiling in sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with anti-HA antibody. For coimmunoprecipitation analysis, cells (2 × 106) were lysed in 1 ml of buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, 20 mM HEPES, protease inhibitor mixture) and incubated either with anti-HA1 antibodies or anti-FLICE (small subunit-specific) rabbit antiserum. Immune complexes were precipitated by the addition of protein G-Sepharose (Sigma). After extensive washing, the Sepharose beads were boiled in sample buffer, and the eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-HA antibody.
Enzymatic Analysis of Recombinant Mutant Proteins--
The
enzymatic reaction was carried out at 37 °C in 20 mM
PIPES, 0.1 mM substrate, 100 mM NaCl, 10 mM dithiothreitol (fresh), 1 mM EDTA, 0.1%
CHAPS, and 10% sucrose, pH 7.2. The initial rates of hydrolysis were
measured by release of AFC (7-amino-4-methyl cou marin) from the
substrate by the enzymes at appropriate emission and excitation
wavelengths using a Perkin-Elmer LS50B fluorimeter equipped with a
thermostated plate reader. Titrations of wild type and FLICE-DD
[arrow] AA were carried out by preincubating the enzyme with varying
concentrations of Z-DEVD-FMK or CrmA in assay buffer for 30 min at room
temperature. The inhibitor was added at concentrations spanning from 0 to significantly above the concentration of active enzyme. After the
preincubation, Z-DEVD-AFC was added in assay buffer to a final
substrate concentration of 0.1 mM. The relative amount of
uninhibited enzyme was evaluated from the initial rates of hydrolysis
of the substrate as described above, and the concentration of active
enzyme was calculated by extrapolating data points to their
intersection with the x axis. The caspase-8 concentration
used in the titration assays were 0.2 micromolar for the native enzyme
and 20 micromolar for the DD AA mutant.
Affinity Labeling of Recombinant Mutant Proteins-- 100 ng of purified proteins were incubated with or without N-(biotinyl-Asp-Glu-Val-Asp-[(2,6-dimethylbenzoyl)oxy]methyl ketone (BIO-DEVD-AMK) (15) at a concentration of 0.5 µM in a final volume of 50 µl of buffer (0.1% CHAPS, 10 mM dithiothreitol, 10 mM Tris, pH 7.5) for 15 min at 25 °C. For competition experiments, increasing amounts of a nonbiotinylated version of the DEVD tetrapeptide (DEVD-CMK; Bachem) were added to the reaction. Samples were boiled in sample buffer in the absence of reductant, resolved by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, blotted onto nitrocellulose, blocked in phosphate-buffered saline containing 3% bovine serum albumin and 0.1% Tween 20, incubated with streptavidin-conjugated horseradish peroxidase (ICN) in phosphate-buffered saline, 0.1% bovine serum albumin, 0.1% Tween, washed with 50 mM Tris, pH 7.5, 0.25% gelatin, 0.05% Tween 20, 150 mM NaCl, 5 mM EDTA, and developed by ECL. Alternatively, the membrane was analyzed by immunoblotting with anti-FLICE antiserum specific for the small catalytic subunit.
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RESULTS AND DISCUSSION |
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To determine if FLICE oligomerization in vivo could result in activation, chimeric FpkFLICE expression constructs were engineered. Fpk (molecular mass 12 kDa), a double mutant of FKBP (FK binding protein FK506) (16) contains a single binding site for the cell-permeable immunosuppressive drug FK506. The FKBP-FK506 complex is a potent inhibitor of calcineurin, a protein phosphatase that plays a key role in signaling. An FK506 dimer (FK1012H2) was previously synthesized by introducing a cross-linker into the domain of FK506 necessary for inhibition of calcineurin. FK1012H2, although unable to bind calcineurin, still retains its ability to bind and dimerize Fpk polypeptides given its bivalent nature (17-19) (Fig. 1a).
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To mimic recruitment of FLICE to its receptor signaling complex (11,
12), the prodomain of FLICE was substituted with a myristoylation
signal followed by three tandem repeats of Fpk, (MFpk3FLICE). A catalytically inactive version was
constructed by mutating the active-site Cys-360 to Ser
(MFpk3FLICE(C S)). An additional control was the
construct MFpk3Eu that encoded only the
myristoylation signal, three Fpk repeats in tandem, and an HA epitope tag (Fig. 1b). To confirm the ability of FK1012H2
to induce oligomerization of Fpk3-containing proteins,
human 293 cells were transiently transfected with the
MFpk3Eu construct and the catalytically
inactive chimera MFpk3FLICE(C
S). The MFpk3FLICE immunoprecipitates contained associating
MFpk3Eu only in the presence of FK1012H2 (Fig.
1c), confirming the validity of the dimerization
approach.
Ectopic expression of the catalytically active chimera MFpk3FLICE resulted in the production of a protein of predicted molecular mass (69 kDa) that was membrane-associated. The addition of the synthetic ligand FK1012H2 induced rapid disappearance of the catalytically competent MFpk3FLICE chimera. Emergence of the active subunits, however, was not detectable under these experimental conditions. As predicted, the catalytically inactive derivative was efficiently expressed, but on exposure to FK1012H2, did not undergo processing even after prolonged incubation (Fig. 2a).
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We next observed if MFpk3FLICE oligomerization resulted
in the expected apoptotic demise of transfected cells. Human 293 and HeLa cells were transiently transfected with
MFpk3FLICE(C S) or MFpk3FLICE
expression constructs together with a reporter plasmid encoding
-galactosidase. Thirty six h after transfection, cells were left
untreated or treated with 250 nM FK1012H2, stained, and
analyzed by phase contrast microscopy. As shown in Fig. 2b, oligomerization of MFpk3FLICE but not
MFpk3FLICE(C
S) induced phenotypic alterations
characteristic of apoptosis. Cells shrank, displayed membrane blebbing,
and detached from the dish. Additionally, the apoptotic substrate
poly(ADP-ribose) polymerase was cleaved to its signature 85-kDa form,
and genomic DNA was cleaved into characteristic internucleosomal size
fragments (data not shown), both biochemical hallmarks of apoptosis.
Significantly, treatment of Fpk or Fpk-FLICE(C
S)-transfected cells
with higher doses of FK1012H2 for an extended period of time did not
induce apoptosis (16, 17, 19, 20) (data not shown). After 3 h of
exposure to the synthetic ligand, 50-80%
MFpk3FLICE-transfected cells started blebbing, condensing,
and detaching from the dish. This was completely abrogated by the
broad-spectrum caspase inhibitor Z-VAD-FMK that has previously been
shown to inhibit FLICE-induced apoptosis (Fig. 2, c and
d) (12). Importantly, the monomeric form of the ligand, FK506M, did not induce apoptosis, confirming that the results observed
were oligomerization-dependent (Fig. 2, c and
d).
These data demonstrate that specific clustering of FLICE zymogen causes
apoptosis through caspase activation. We hypothesized that this
activation occurs through self-processing of the clustered FLICE due to
an intrinsic proteolytic activity of the zymogen. To further address
this hypothesis, different recombinant versions of FLICE were generated
(Fig. 3a); as shown previously
(6, 8, 11), overexpression of catalytically competent FLICE constructs in E. coli generated an active heterodimeric enzyme composed
of large and small catalytic subunits. Enzymatically inactive FLICE (catalytic Cys-360 mutated to Ser; FLICE(C S)) did not undergo processing, consistent with a requirement for intrinsic enzymatic activity. Importantly, an altered form of FLICE that retained the
catalytic cysteine but was mutated at the internal cleavage sites
(Asp-374 and Asp-384; FLICE(DD
AA)) also did not undergo auto-processing and was expressed as a single polypeptide zymogen as
confirmed by protein staining and anti-FLICE immunoblot analysis (data
not shown and Fig. 3c). The processing mutant FLICE in which the catalytic cysteine was additionally inactivated (FLICE (DDCAAS)) was also expressed as a single polypeptide zymogen. These recombinant forms of FLICE were used to establish whether the zymogen did indeed
possess enzymatic activity.
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The activity of the cleavage site mutant version FLICE(DD AA) was
determined by its ability to hydrolyze the tetrapeptide caspase
substrate Z-DEVD-AFC (21), and the portion of active material was
determined by titration with the protein inhibitor CrmA (22, 23) and
the covalent peptide based inhibitor, Z-DEVD-FMK (21). A 100-fold more
FLICE(DD
AA) was required on a protein basis to give the same rates
of substrate hydrolysis as wild type FLICE (Fig. 3b). Thus,
from these results it is apparent that the bacterially expressed
FLICE(DD
AA) mutant equivalent to the zymogen form has
approximately 1% of the activity of the wild type enzyme. As expected,
the catalytic mutant FLICE(C
S) possessed no enzymatic activity
(data not shown).
To interpret the enzymatic analysis, we tested the ability of the
different versions of FLICE to bind the biotinylated irreversible inhibitor BIO-DEVD-AMK, which covalently binds to the active-site cysteine (15). As expected, the large subunit of processed native FLICE
that contains the catalytic cysteine bound BIO-DEVD-AMK (Fig.
3c). Additionally, the unprocessed cleavage site mutant (FLICE(DD AA)) also bound BIO-DEVD-AMK, indicating that the 1%
activity resides in the single chain. FLICE(DDC
AAS), as anticipated, did not show any specific binding to BIO-DEVD-AMK. The
specificity of the bands indicated was confirmed by competing the
binding with a nonbiotinylated version of the DEVD tetrapeptide (Fig.
3d).
Taken together, these data are consistent with a model wherein FLICE zymogen possesses intrinsic low level caspase activity that upon approximation mediated by the adapter molecule Fas-associated death domain (FADD) attains a sufficient concentration to activate the apoptosis pathway. This study provides a remarkably simple solution to the chicken and egg conundrum of how the initiating caspase (FLICE) is proteolytically processed.
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ACKNOWLEDGEMENTS |
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We are grateful to S. L. Schreiber for helpful suggestions and discussions. We thank David Spencer for MFpk3Eu, Linda Clayton for BIO-DEVD, Ian Jones for his expertise in preparing the figures, and the following members of the Dixit lab for encouragement and discussions: Divya Chaudhary, Arul Chinnaiyan, Hangjun Duan, Shimin Hu, Eric Humke, Justin McCarthy, Karen O`Rourke, James Pan, and Claudius Vincenz.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Human Frontier Science Program Organization (HFSPO) fellowship.
A Howard Hughes Medical Institute predoctoral fellow supported
in part by NIGMS, National Institutes of Health, Grant GM-52067 (to
S. L. Schreiber).
Present address and to whom correspondence should be addressed:
Genentech Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.:
415-225-1000; Fax: 415-225-6000; E-mail: dixit{at}gene.com.
1 The abbreviations used are: HA, hemagglutinin; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BIO-DEVD-AMK, N-(biotinyl-Asp-Glu-Val-Asp-[(2,6-dimethylbenzoyl)oxy]methyl ketone; -CMK, chloromethyl ketone; -FMK, fluoromethyl ketone; -AFC, 7-amino-4-trifluoromethyl coumarin; Z-, carbobenoxy.
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REFERENCES |
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