From the Department of Pathology and the Center for
Neurobiology and Behavior and the ¶ Department of Pharmacology,
Columbia University College of Physicians and Surgeons, New York, New
York 10032
Received for publication, October 18, 2000, and in revised form, January 5, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have identified a novel isoform of
rat caspase-9 in which the C terminus of full-length caspase-9 is
replaced with an alternative peptide sequence. Casp-9-CTD (where CTD is
carboxyl-terminal divergent) is expressed in multiple tissues, with the
relative highest expression observed in ovary and heart. Casp-9-CTD was
found primarily in the cytoplasm and was not detected in the nucleus.
Structural predictions suggest that in contrast to full-length
caspase-9, casp-9-CTD will not be processed. Our model is supported by
reduced protease activity of casp-9-CTD preparations in
vitro and by the lack of detectable processing of casp-9-CTD
proenzyme or the induction of cell death following transfection into
cells. Both neuronal and non-neuronal cell types transfected with
casp-9-CTD were resistant to death evoked by trophic factor deprivation
or DNA damage. In addition, cytosolic lysates prepared from cells
permanently expressing exogenous casp-9-CTD were resistant to caspase
induction by cytochrome c in reconstitution assays. Taken
together, our observations indicate that casp-9-CTD acts as a
dominant-negative variant. Its expression in various tissues indicates
a physiological role in regulating cell death.
Apoptosis is a physiological form of cell death that
serves a number of functions, including tissue development and
remodeling, cellular homeostasis, and removal of damaged cells. In
regular animal development, cell death is involved not only in
morphogenesis, but also in optimizing function of the nervous and
immune systems (for review, see Refs. 1 and 2). Apoptosis requires
regulated activation of members of a family of cysteine proteases
(caspases) that cleave cellular substrates at the carboxyl side of Asp
residues, thereby initiating and executing an apoptotic death program.
Caspases are produced in cells as full-length zymogens in a poorly
active or inactive form, but autocleavage and processing by other
proteases yield active enzyme. The processing normally involves the
removal of an N-terminal prodomain and cleavage of the remaining
protein sequence to yield large and small subunits (for review, see
Refs. 2 and 3). Proteolytic cleavage between these protein domains results in caspase activation by formation of heterotetramers composed
of small and large subunits, but it has been shown for caspase-9
activation that caspase activity in vitro is observed also
in the absence of caspase processing (4).
There are presently 14 known caspase family members, and
each has distinguishable enzymatic properties and substrate
specificities (for review, see Ref. 2). Depending on the mode of
activation or downstream effectors, caspase cascades have been
identified that involve the orchestrated activation of downstream
caspases by upstream initiator proteases. Of these, caspase-9 appears
to be one of the major initiators of cell death cascades in diverse apoptotic paradigms, and transgenic as well as null animal models exhibit significant phenotypes that underscore its importance (for
review, see Ref. 5). A wide array of apoptotic stimuli lead to release
of cytochrome c (and in certain cell types, even caspase-9)
from mitochondria, and its presence in the cytosol induces the
formation of a multimeric complex that consists of caspase-9, the
caspase-9/cytochrome c-binding protein Apaf-1
(apoptotic protease-activating
factor-1), and accessory proteins. Caspase-9 is activated
during the formation of the complex and subsequently cleaves and
activates downstream death-promoting executioner caspases such as
caspase-3, -6, and -7 (6-9).
Considering the importance of self-association and complex formation
with downstream substrates for the regulation and consequences of
caspase-9 activity, it is not surprising that catalytically inactive
forms inhibit the activation of downstream executioner caspases and
inhibit cell death. Furthermore, endogenous forms of caspase-2, -8, and
-9 that act as apoptosis inhibitors have been found (10-14).
In this work, we have identified and
characterized a novel isoform of rat caspase-9 that we have designated
casp-9-CTD (where CTD is carboxyl-terminal divergent). This form
differs from previously described isoforms of caspase-9 in that the
small caspase subunit found in full-length caspase-9 is replaced by an
alternative sequence. Here we examined the consequences of the
alternative C terminus on caspase activity, intracellular localization,
and function.
Materials and Cell Culture
RPMI 1640 medium,
DMEM,1 Taq
platinum DNA polymerase, the reverse transcriptase
pre-amplification kit, and LipofectAMINE 2000 were from Life
Technologies, Inc. The Marathon cDNA amplification library kit was
from CLONTECH, and PCR primers were obtained from Integrated DNA Technologies or Life Technologies, Inc. Tissue total RNA
was purchased from Ambion Inc. Hoechst dye 33342 and anti-FLAG antibody
were from Sigma. IPTG was from Life Technologies, Inc.
Escherichia coli BL21(DE3) pLys was from Novagen. Anti-His antibody was from Invitrogen, and anti-caspase-9 antibody was from
Medical and Biological Laboratories, Co., Ltd. Ac-DEVD-pNA and
benzyloxycarbonyl-VAD-fluoromethyl ketone were from Enzyme Systems
Inc., and AMC- and AFC-conjugated substrate peptides were from
Pharmingen or BIOMOL Research Labs Inc. Metal-chelating affinity chromatography was performed on HiTrap chelating columns from Amersham
Pharmacia Biotech.
Cell Culture
PC12 cells were cultured as described (15) in collagen-coated
dishes with RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum. Neuronal differentiation was
induced with 100 ng/ml human recombinant NGF (a kind gift of
Genentech). Rat-1a cells, Rat-1a cells overexpressing
c-myc (a gift from Dr. R. Dalla-Favera, Columbia
University), and 293 cells were grown in DMEM with 10% fetal bovine serum.
Cloning of Rat Full-length Caspase-9 and Casp-9-CTD
To clone rat full-length caspase-9 and casp-9-CTD, primers
5'-TCTATGGCACAGATGGATGCTC-3' and 5'-ATGGATGCTCCGTGTCCATCGA-3'
were used for 3'-RACE and nested 3'-RACE amplification. For 5'-RACE, primer sets 5'-CTCCCAGGCTGGGGCAGCCGG-3' and
5'CAGCCGGTCCCATTGAAGAT-3', 5'-CATGTCTCTCGATCTTCCTGG-3' and
5'-AGTACAGACATCATGAGCTCC-3', and 5'-CTCTAAGCAGGAGATGAAGC-3' and
5'-CTCGAGTCTCAAGATCTATGAC-3' were used in serial PCRs. The open
reading frames of full-length caspase-9 and casp-9-CTD were cloned from
rat brain mRNA or from total RNA isolated from rat tissues. PCR was
conducted with primers 5'-GAATTCCAGCAATCCGCTAGCCATGGAGG-3' and
5'-GAATTCAACTCATGAAGTTTAAAGAACAG-3' for full-length caspase-9 and with
5'-GAATTCGCAGCCAACTCAGATCTTGCTA-3' as the reverse primer for
casp-9-CTD. All PCR products were sequenced.
Constructs
The cysteine residue in the QACGG active-site motif of
full-length caspase-9 was mutated to alanine by overlapping PCR using primers 5'-CATCCAGGCCGCTGGTGGTGAGCAGAAA-3' and
5'-TTTCTGCTCACCACCAGCGGCCTGGATG-3'. Primers
5'-GAATTCCAGCAATCCGCTAGCCATGGAGG-3' and
5'-GAATTCTCATTTGTCATCATCTCCTTGTAGTCCATTCCTGAAGTTTTA-3' were used to
generate the FLAG epitope tag. Casp-9-CTD was FLAG-tagged using the
primer pair 5'-GAATTCCAGCAATCCGCTAGCCATGGAGG-3' and 5'-GAATTCTCATTTGTCATCATCGTCCTTGTAGTCCATCCCGGCCTTTGGTGG-3'. The open
reading frames of full-length caspase-9, caspase-9(C327A)-FLAG, casp-9-CTD, and casp-9-CTD-FLAG were subcloned into the
EcoRI sites of the pCMS-EGFP vector or into pcDNA3.0 for
eukaryotic expression. Human full-length caspase-3 (16), rat caspase-9 and casp-9-CTD, and active-site mutants of caspase-9 enzymes (C327A) were fused with a C-terminal His6 epitope into
pET23b for expression in E. coli BL21(DE3) pLys (8).
For caspase-3, primers 5'-ATGAATTCGGGTGATAAAAATAGA-3' and
5'-ATGAATTCGGGTGATAAAAATAGA-3' were used; for caspase-9, primers 5'-ATCGAATTCGGAGGAGGCTGACCGG-3' and 5'-CTTGTCGACTGAAGTTTTAAAGAA-3' were
used; and for casp-9-CTD, primers 5'-ATCGAATTCGGAGGAGGCTGACCGG-3' and
5'-CTTGTCGACGGCCTTTGGTGGGGA-3' were used. Active-site mutants were
generated in pET23B using the above primers for caspase-9 or primers
5'-TTCTTCATCCAGGCCGCAGGTGGTGGTGAG-3' and
5'-CTCACCACCACCTGCGGCCTGGATGAAGAA-3' for casp-9-CTD.
In Vitro Transcription and Translation
pCMS-EGFP-full-length caspase-9 and pCMS-EGFP-casp-9-CTD vectors
were mixed with single-step T7 promoter-driven TNT-coupled reagents (Promega) and with [35S]methionine (1000 Ci/mmol). The protocol was carried out as recommended by the supplier.
25% of the radiolabeled mixture of full-length caspase-9 and
casp-9-CTD was resolved on 4-20% Novex gradient gels. The gel was
then dried and exposed to x-ray film.
Purification of Recombinant Caspases and Protease Activity
Assays
Following IPTG induction (caspase-3 at 0.2 mM IPTG
for 45 min and caspase-9 isoforms at 0.4 mM IPTG for
1.5 h at 30 °C), caspases were purified from bacterial cell
lysates using standard metal-chelating affinity chromatography and an
imidazole gradient as the eluent as described (17). Following imidazole
elution, purified caspases were dialyzed against caspase buffer (20 mM Hepes, 100 mM KCl, 10 mM
dithiothreitol, 1 mM EDTA, 0.1% Chaps, and 10% sucrose, pH 7.2) and stored as concentrated aliquots in caspase buffer. Enzyme
concentrations were determined by active-site titration using
benzyloxycarbonyl-VAD-fluoromethyl ketone or by comparison with known
amounts of caspase enzymes using Western blotting. Caspase activity
assays were performed at 37 °C in 50-µl reaction volume using 0.2 mM synthetic peptide or caspase-3 as a substrate. For
comparison of different AMC-coupled substrate peptides, an end point
analysis was performed in a Turner TD-700 fluorometer using an
excitation wavelength of 365 nm and an emission wavelength filter of
455-500 nm after a 30-min incubation at 37 °C. The reaction rates
of hydrolysis of Ac-DEVD-pNA or Ac-DEVD-AFC substrate peptides were
determined using a Beckman DU530 spectrophotometer equipped with a
Peltier temperature module in kinetic rate mode. Liberated pNA or AFC
was recorded at A405 nm or
A380 nm in 5-s intervals, respectively, and the
concentrations of pNA and AFC were calculated using a standard
concentration curve. The activity of caspase-3-like enzymes in
reconstituted cell lysates was determined as described previously (18).
In brief, concentrated cytoplasmic protein lysates were obtained by
resuspending PC12 control cells and polyclonal PC12 cell cultures
expressing casp-9-CTD-FLAG at 4 °C in hypotonic cell lysis buffer
(20 mM Pipes, 10 mM KCl, 5 mM EDTA,
2 mM MgCl2, and 1 mM
dithiothreitol, pH 7.4), followed by cell lysis and centrifugation at
13,000 × g.
Transfections and Trophic Factor Deprivation
293 cells were transfected using calcium phosphate as described
(19). For PC12 cells, transfection with 10 µg of plasmid/well was
performed after 2 days of NGF treatment for NGF-differentiated PC12
cells or immediately after plating for naive cells. PC12 and Rat-1a
cells were transfected using LipofectAMINE 2000. After 9 h for
PC12 cells and 5 h for Rat-1a cells, medium with LipofectAMINE 2000 was exchanged for fresh medium with NGF or insulin or with serum
containing DMEM for NGF-treated naive PC12 cells and Rat-1a fibroblasts. On day 3 after transfection for PC12 cells or on day 2 for
Rat-1a fibroblasts, cultures were washed 5-10 times with RPMI 1640 medium for PC12 cells or with DMEM for Rat-1a cells. The cells were
scraped from their dishes and replated onto collagen-coated 15-mm wells
at 2 × 105 cells/well. For PC12 cells, trophic
factor-deprived cultures were maintained in RPMI 1640 medium, and the
control nondeprived cells were grown with NGF or NGF/insulin for
NGF-differentiated or naive cells. Rat-1a and Rat-1a
c-myc-overexpressing fibroblasts remained in DMEM only.
Establishment of Permanent PC12 Cell Lines
PC12 cells (100-mm plates) were transfected with 28 µg of rat
casp-9-CTD-FLAG cloned into the pcDNA3.0 plasmid or with empty pcDNA3.0 plasmid. After 1 week, permanently transfected cells were
selected for neomycin resistance using 500 µg/ml G418 (Cellgro). The
surviving cells were expanded to establish clonal lined or polyclonal
mass cultures. Cells were tested for expression of FLAG-tagged
casp-9-CTD by Western blot analysis with anti-FLAG and anti-caspase-9
antibodies. Survival in the absence of serum was determined by counting
intact nuclei after 2 days (20).
Western Immunoblotting
293 or PC12 cells were harvested in Laemmli sample buffer, and
concentration of proteins was measured by the Bio-Rad method. The proteins were resolved by SDS-polyacrylamide gel electrophoresis using a 4-20% gradient gel and transferred onto nitrocellulose according to standard procedures. The nitrocellulose membrane was
stained with Ponceau S to locate proteins; cut into sections; and
blocked with 5% milk in PBS, pH 7.4, at room temperature for 1 h.
The blot was probed with anti-caspase-9 p20 antiserum (Bur73, a kind
gift from Guy Salvesen, Burnham Institute, San Diego, CA) for 293 cells
or with anti-FLAG antibody for PC12 cells, followed by ECL,
according to the manufacturer's instructions (Amersham Pharmacia Biotech).
Assessment of Cell Survival
Strip Counting--
The numbers of healthy, non-apoptotic,
EGFP-positive cells in a defined strip across the culture were assessed
by phase and fluorescence microscopy. The same strip was scored each
day during the time course, and the percentage of surviving cells was
calculated relative to the number present in the same well at 1.5 h after plating (20, 21).
Apoptotic Nuclei--
At various time points, PC12 or Rat-1a
cells were fixed in 4% formaldehyde for 10 min and then, after washing
with PBS, exposed to 10% nonimmune goat serum and 0.2% Triton X-100
in PBS for 30 min at room temperature (20). Staining for FLAG fusion
protein was carried out for 1-2 h at room temperature with 20 µg/ml
anti-Flag antibody in PBS containing 10% nonimmune goat serum and
0.2% Triton X-100, followed by a 1-h exposure to rhodamine-labeled
secondary antibody (1:100 dilution). To visualize nuclear DNA, the
fixed cells were incubated with Hoechst dye 33342 at 1 µg/ml in PBS and 0.08% Triton X-100 for 15 min at room temperature. Cells
possessing condensed nuclei and fragmented chromatin were scored as
apoptotic. Data are presented as the proportion of either EGFP- or
FLAG-expressing cells with apoptotic nuclei.
Cloning of a Rat Carboxyl-terminal Divergent Caspase-9
(Casp-9-CTD)--
Efforts aimed at the cloning of a full-length
cDNA of rat caspase-9 by 5'- and 3'-RACE/PCR led to the discovery
of a rat caspase-9 variant cDNA that encodes an alternative
C-terminal sequence immediately following the QACGG active site
(GenBankTM/EBI accession number AY008275) (Fig.
1A, lower
sequence). This alternative C terminus (amino acids 327-383)
shows no homology to other reported sequences, including those
previously reported for full-length caspase-9 or other short forms of
caspase-9. From the N-terminal methionine through QACGG, the predicted
protein sequence is identical to rat full-length caspase-9
(GenBankTM/EBI accession number AF308469) (Fig.
1A, upper sequence). The variant was
independently confirmed by 3'-RACE/PCR from rat PC12 cells and by PCR
from rat brains, in each case using multiple alternative primer pairs
(data not shown). RT-PCR and subsequent sequencing also detected the
variant in several different rat tissues, including adult liver and
ovary, and in whole rat embryos (see also Fig. 3). We have designated
the novel variant as casp-9-CTD for caspase-9 carboxyl-terminal
divergent. A portion of the casp-9-CDT 5'-UTR (16 base pairs) was
sequenced, and this showed complete identity to the corresponding
sequence of full-length caspase-9 (Fig. 1A). In contrast,
the 3'-UTR of casp-9-CTD contains no similarity to the 3'-UTR of the
full-length form, thus suggesting that the variant C terminus of
casp-9-CTD is derived from an alternative exon of the rat caspase-9
gene. Furthermore, Southern blots of rat genomic DNA showed a single
distinct band when hybridized with a probe shared by sequences of both
forms of caspase-9 (Fig. 1A). The failure to detect any
additional hybridizing bands using these enzymes and the fact that the
5'-UTR sequences of both cDNAs are identical suggest the presence
of a single caspase-9 gene in the rat genome that gives rise to both
full-length caspase-9 and casp-9-CTD by alternative splicing.
Structural Analysis of Casp-9-CTD Suggests an Inactive
Isoform--
Alternative short isoforms of caspase-9 are catalytically
inactive and act as endogenous inhibitors of caspase-9 activation. These isoforms include a human isoform (caspase-9S, caspase-9b, CASP9,
or caspase-9
A closer examination of the predicted amino acid sequence of rat
casp-9-CTD reveals that the alternative carboxyl terminus does not
contain the two aspartic acid residues that are present in the
full-length forms of caspase-9 (Fig. 1A). These residues are
required for the processing of the zymogen into large (p37) and small
(p12) subunits that are necessary for the formation of a fully
activated enzyme (4). Using the Smith-Waterman algorithm for sequence
alignment (22), we detected similarities of 51.7 and 53.7% between the
predicted protein sequence of human caspase-3 (GenBankTM/EBI accession number U26493) and those of rat
caspase-9 and casp-9-CTD, respectively. Using PrISM
(protein informatics system for
modeling) (23), we were able to unambiguously compute a model for the protease domain of both caspase-9 isoforms using the
known structures of human caspase-3 (24, 25) as a template. A further
analysis of casp-9-CTD using PrISM did not predict any alternative
secondary structure for the divergent C-terminal domain in the context
of the casp-9-CTD protein. Thus, it is unlikely that the alternative C
terminus in casp-9-CTD protein will contribute to the formation of a
small subunit or any other predictable secondary structure (Fig.
2).
RT-PCR Analysis of Casp-9-CTD Reveals Expression in Multiple Rat
Tissues--
To compare the tissue distributions of rat full-length
caspase-9 and casp-9-CTD, we conducted semiquantitative RT-PCR using primers that span the open reading frames of each form using total RNA
from various tissues (Fig.
3A). Products corresponding to the predicted size were detected in all tissues examined. The identity
of the 1188-base pair casp-9-CTD product was confirmed by sequence
analysis. A fast migrating DNA product was also detected using these
primer pairs, but it encoded an unrelated DNA product. Expression of
casp-9-CTD was consistently highest in ovary, whereas the relative
ratio of casp-9-CTD to full-length caspase-9 was highest in heart and
ovary. The presence of casp-9-CTD transcripts in PC12 cells was
confirmed, but we did not find regulation by NGF (Fig. 3A).
5'- and 3'-RACE/PCR as well as Western blotting confirmed the presence
of full-length caspase-9 in PC12 cells (data not shown).
Expression Analysis of Casp-9-CTD Reveals a Molecular Mass of 45 kDa--
The molecular masses of full-length caspase-9 and casp-9-CTD
were predicted to be 50.4 and 42.3 kDa, respectively, using PeptideMass (26). In vitro transcription and translation followed by
SDS-polyacrylamide gel electrophoresis analysis revealed apparent
molecular masses of ~45 kDa for rat casp-9-CTD and ~55 kDa for
full-length caspase-9 (Fig. 3B). Similar results were
obtained with protein expressed in E. coli or after
transient transfection of casp-9-CTD into 293 cells (Fig.
3B). In transient transfections, however, we detected only
cleavage of full-length caspase-9, possibly because of autoprocessing (data not shown).
Casp-9-CTD Expression Is Limited to the Cytoplasm--
To examine
the subcellular distribution of casp-9-CTD, a C-terminal FLAG construct
was transiently transfected into PC12 (Fig. 4A) and Rat-1a (data not
shown) cells and visualized by staining with an anti-FLAG antibody. To
facilitate comparison with full-length caspase-9, we used a
full-length, FLAG-tagged form of rat caspase-9 that is rendered
catalytically inactive by mutation of the cysteine residue within the
QACGG active site (C327A) and that will not induce cell death (see also
Figs. 6 and 7). Confocal fluorescence microscopy revealed that
casp-9-CTD was localized to the cytoplasm and excluded from the
nucleus, whereas full-length caspase-9(C327A) was detected in both the
cytoplasm and the nucleus. These observations indicate that full-length
caspase-9 and casp-9-CTD differ in their subcellular distributions and
underscore the potential contribution of the C terminus in regulating
nuclear localization. Similar results were obtained following
subcellular fractionation of lysates from transiently transfected
cells, followed by Western blot analysis (data not shown).
Overexpression of Casp-9-CTD Does Not Cause Cell Death--
Our
structural considerations suggested that casp-9-CTD is catalytically
compromised, but possibly not inactive. Because expression of exogenous
full-length caspase-9 leads to activation, processing, and consequent
cell death in many cell types, we compared the effects of full-length
caspase-9 and casp-9-CTD on cell survival. 293 cells were transiently
transfected with either full-length caspase-9 or casp-9-CTD.
Transfection of cells was confirmed by examining the expression of EGFP
encoded by the same plasmid. Counts of healthy, EGFP-expressing cells
at 1 and 2 days revealed nearly comparable numbers in cultures
transfected with vector alone or with casp-9-CTD, but many fewer in
cultures transfected with full-length caspase-9 (Fig. 4B).
Furthermore, cultures transfected with full-length caspase-9 exhibited
many fragmented and shrunken fluorescent cells, whereas few, if any,
such cells were present in cultures transfected with vector alone or
casp-9-CTD. Similar results were achieved in cultures of PC12 and Rat-1
cells (data not shown). These findings indicate that in contrast to
full-length caspase-9, which evokes death, overexpression of casp-9-CTD
does not compromise cell survival.
Casp-9-CTD Is a Poorly Active Protease Enzyme--
To examine the
activity of rat caspase-9 and casp-9-CTD in vitro, we
expressed both isoforms and human caspase-3 in E. coli BL21(DE3) pLys using the pET/IPTG prokaryotic expression system (17).
Recombinant proteins were purified using standard metal-chelating affinity chromatography, and the concentration of caspase-3 and full-length caspase-9 was determined by active-site titration using
benzyloxycarbonyl-VAD-fluoromethyl ketone (17). For casp-9-CTD, protein concentrations were determined by quantitative Western blot
analysis against known protein standards. Following IPTG induction, we
were able to purify caspase-3 in its full-length, zymogen form.
In contrast, both full-length caspase-9 and casp-9-CTD were partially
processed, and the extent of cleavage was dependent upon the time of
induction. For full-length caspase-9, two major bands (12 and 43 kDa)
were observed, most likely as a result of cleavage at amino acids 167 and 367. A smaller proportion remained uncleaved or was fully processed
into the p35/p12 enzyme (data not shown). In contrast, the majority of
casp-9-CTD protein migrated at the expected size of 45 kDa, with
smaller fragments at 35 kDa depending on the induction time (data not
shown). These smaller fragments were likely to be caused by degradation
and not by autoprocessing since they were also present in preparations
of mutated casp-9-CTD (C327A).
We first examined whether purified full-length caspase-9 or casp-9-CTD
preparations exerted any activity against the caspase-9-specific substrate Ac-LEHD-AMC or other synthetic peptide substrates that are
used to detect caspase activity in vitro (27). For these experiments, we used the caspase-9 isoforms at 10 nM, a
concentration that is similar to the concentration of rat caspase-9
that we have observed in cytosolic cell lysates from PC12 cells
(concentration of total protein, ~12 mg/ml). This concentration
resembles the concentration of human caspase-9 (20 nM)
that has been observed in concentrated cytosolic lysates derived from
293 cells (4). Whereas 10 nM purified rat full-length
caspase-9 efficiently cleaved the Ac-LEHD-AMC synthetic substrate
peptide and, to a much reduced extent, also Ac-VEID-AMC > Ac-IETD-AMC > Ac-DEVD-AMC > Ac-YVAD-AMC substrate peptides,
no protease activity above background was detected with any of these
substrates when using 10 nM casp-9-CTD (Fig.
5A). These data suggest either
that casp-9-CTD is catalytically impaired or, alternatively, that
casp-9-CTD possesses distinct catalytic properties that are not
detected in our assay.
We then determined the ability of purified caspase-9 and casp-9-CTD to
cleave recombinant caspase-3 in vitro. Whereas caspase-3 (150 nM or 1 µM) was processed when incubated
with 10 nM full-length caspase-9 (Fig. 5B),
casp-9-CTD did not induce caspase-3 processing at 20 nM
when using 150 nM caspase-3 or even at 200 nM
when using 1 µM caspase-3 as a substrate (Fig.
5B). The reduced activity of casp-9-CTD against caspase-3
that we established by Western blot analysis (Fig. 5B) was
further confirmed after examining the kinetic rate of conversion of the
caspase-3 peptide substrate Ac-DEVD-AFC in a coupled protease assay
(8). In this assay, the activity of purified caspase-3 against
substrate peptide is an indication of the ability of initiator protease
to cleave caspase-3 and to induce caspase-3 activity. Using a ratio of
10 nM initiator protease to 100 nM caspase-3,
which resembles the ratio of caspase-9 to caspase-3 in cell lysates (4,
18), we observed significant caspase-3 activity following incubation
with full-length caspase-9 (Fig. 5C). 10 nM
granzyme B also activated caspase-3 in our experiments, but no
significant caspase-3 activation was observed after incubation of
caspase-3 with casp-9-CTD (Fig. 5C). The residual hydrolysis of the Ac-DEVD-AFC substrate that was observed when caspase-3 was
incubated with casp-9-CTD was similar to that of Ac-DEVD-AFC hydrolysis
by casp-9-CTD alone and possibly due to protease activity against this
substrate (see also Table I).
Using a similar approach, we determined the reaction rates of
Ac-DEVD-pNA hydrolysis using initiator proteases alone or with caspase-3 in a coupled caspase assay at a molar ratio of 200 nM caspase-9/casp-9-CTD to 100 nM caspase-3
(Table I). In these experiments, 200 nM full-length caspase-9 was able to induce a significant
activation of 100 nM caspase-3 in a coupled enzyme assay.
Casp-9-CTD at 200 nM was not able to induce significant Ac-DEVD-pNA hydrolysis through secondary activation of caspase-3, but
showed residual proteolytic activity against the peptide substrate. This activity exceeded that of caspase-3 or caspase-9 alone. Taken together, these findings indicate that casp-9-CTD is an inactive caspase enzyme with respect to its ability to cleave caspase-3, a
physiological substrate of full-length caspase-9. However, at high
concentrations, the intact catalytic site in casp-9-CTD may contribute
to proteolysis of certain substrate peptides.
Casp-9-CTD Protects Neuronal and Non-Neuronal Cells from
Apoptosis--
The findings that casp-9-CTD is catalytically
compromised and does not induce death raised the possibility that it
could, in turn, act to suppress apoptosis. To test this, several
different cell lines were subjected to different death paradigms after
transient transfection with casp-9-CTD (with or without a C-terminal
FLAG tag) cloned into the pCMS-EGFP vector. A FLAG-tagged construct verified expression of casp-9-CTD. For comparison, cells were also
transfected with the FLAG-tagged inactive construct of full-length caspase-9(C327A).
We first tested a paradigm in which naive PC12 cells undergo apoptotic
death by serum deprivation. Cultures were fixed and stained with
Hoechst dye number 33342 to visualize nuclei. Counterstaining with anti-FLAG antibody identified the cells expressing the desired proteins, and transfected cells were then scored for the presence of
apoptotic nuclei. As shown in Fig.
6A, ~30% of the transfected cells deprived of support by serum had apoptotic (condensed) nuclei when vector alone was present compared with <3% of cells that expressed casp-9-CTD or inactive full-length caspase-9(C327A) or that
were treated with NGF (a trophic factor for PC12 cells). A parallel
experiment performed on neuronally differentiated PC12 cells evoked to
die by removal of both serum and NGF also showed excellent protection
by casp-9-CTD as well as inactive full-length caspase-9(C327A) (Fig.
6B).
We also determined whether expression of casp-9-CTD would protect
neuronal PC12 cells from apoptosis evoked by DNA damage. Cultures were
transfected and then exposed to the topoisomerase I inhibitor
camptothecin (28). Both casp-9-CTD and inactive full-length
caspase-9(C327A) protected cells from DNA damage-induced apoptosis for
at least 3 days (Fig. 6C). To evaluate the possibility that
the protective properties of casp-9-CTD were affected by the presence
of the FLAG tag, we carried out parallel experiments without a
FLAG tag version. The total numbers of surviving transfected (EGFP-positive) cells were assessed at various times after trophic deprivation. As shown in Fig. 6D, the version without the
tag also proved to be highly protective from death.
To assess the protective action of casp-9-CTD in a non-neuronal cell
type, wild-type Rat-1a cells and Rat-1a cells constitutively overexpressing c-myc (Rat-1a-myc) were
transiently transfected with the above constructs and deprived of
serum. Serum deprivation causes apoptosis of both cell types, but cell
death is greatly accelerated in Rat-1a-myc cells (29). The
data in Fig. 7 demonstrate that for both
Rat-1a and Rat-1a-myc cells, casp-9-CTD provided protection
from serum deprivation similar to or even greater than that observed
after transient expression of inactive full-length caspase-9(C327A).
Cells with Permanent Overexpression of Casp-9-CTD Are Resistant to
Apoptosis--
To characterize the underlying deficiency of apoptosis
induction in cells overexpressing casp-9-CTD, we generated PC12 cells lines that permanently expressed FLAG epitope-tagged casp-9-CTD or
control vector. Cells were selected for neomycin resistance and
examined by Western blot analysis using anti-FLAG and anti-caspase-9 antibodies. Several cell lines as well as polyclonal cultures were
generated that expressed levels of casp-9-CTD below or equal to the
levels of endogenous caspase-9 (data not shown). Following trophic
factor deprivation, polyclonal cultures and cell lines with detectable
amounts of casp-9-CTD (CTD1-3, -7, and -10) were resistant to apoptosis
induction, whereas control or nonexpressing cells (CTD1-1) were not
(Fig. 8). Examination of cytosolic
cytochrome c levels using subcellular fractionation revealed
that these cells responded comparably to control cells, with release of
mitochondrial cytochrome c during trophic factor
deprivation and etoposide treatment (data not shown). These data
indicate that the release of cytochrome c from mitochondria
was not affected in cells expressing casp-9-CTD.
Exogenous Casp-9-CTD Inhibits Caspase-3 Activation by Cytochrome
c--
To test the caspase activation cascade downstream of
mitochondrial damage responses in vitro, we tested cytosolic
extracts from control cells or polyclonal cultures expressing
casp-9-CTD protein for caspase-3-like activity following cytochrome
c addition. The activity of endogenous caspase-3-like
enzymes was determined using the fluorogenic substrate Ac-DEVD-AFC
under continuous reading conditions. Cytosolic lysates from these cells
did not exert any caspase-3-like activity before addition of cytochrome
c (Fig. 9). Treatment of
cytosolic lysates prepared from control and casp-9-CTD cells with 20 nM purified granzyme B resulted in hydrolysis of 6.7 µM Ac-DEVD-AFC substrate
peptide·s Our findings indicate that both casp-9-CTD and full-length
caspase-9 are derived from the same rat gene. The caspase recruitment and p20 domains of both forms are identical, and their identity continues through the active-site sequence QACGG. The 5'-UTR sequence thus far determined for the carboxyl-terminal divergent form is also
identical to that of the full-length form. It is only 3' of the active
site that the two forms diverge. In addition, results from Southern
blot experiments are consistent with the assumption of only a single
gene that gives rise to both caspase-9 isoforms. These observations are
similar to those reported for the short form of caspase-2 (Ich-1 short)
that is an alternative splice product diverging from the full-length
form beginning with sequences after the catalytic core and that acts as
a dominant-negative inhibitor of apoptosis (10).
The isoform casp-9-CTD that we have identified in rat is distinct from
other reported variant forms of caspase-9. A human form lacks a caspase
catalytic domain, and its overexpression in cells inhibits apoptosis,
at least in part, by means of caspase recruitment domain-mediated
interaction with Apaf-1 (12, 13). A short variant of mouse caspase-9
has also been reported that is not processed under conditions that lead
to cleavage of the full-length enzyme and that does not significantly
increase overall caspase-9 activity when overexpressed (11). However,
transcripts encoding this mutant were not detected in mouse embryos or
tissues, suggesting a cell line-specific expression that resembles that of mutant forms of caspase-3 in the human breast cancer cell line MCF-7
(30). In contrast, casp-9-CTD was detected in various rat tissues, cell
lines, and whole rat embryos, indicating a potential role in apoptosis regulation.
The lack of a defined secondary structure prediction for the
alternative C-terminal sequence of casp-9-CTD could be relevant when
considering the contribution of the Structural consideration led us to predict that casp-9-CTD does not
exert significant protease activity against physiological caspase-9
substrates such as caspase-3. Our prediction was confirmed by examining
the catalytic activity of casp-9-CTD in vitro and using
cytosolic lysates. The observation that the overexpression of
casp-9-CTD in different cell types does not induce apoptosis is
consistent with the likelihood that this form is functionally inactive
within living cells. Because casp-9-CTD shares many sequences with full-length caspase-9, including caspase recruitment and catalytic
core domains, we further postulated that this caspase-9 isoform should
act as a dominant-negative for full-length caspase-9. There are
several nonexclusive mechanisms by which casp-9-CTD might function as a
dominant-negative mutant. One is that it binds to the Apaf-1 apoptosome
complex via its caspase recruitment domain and thereby competitively
prevents binding and activation of full-length caspase-9. A second is
that it forms oligodimers with full-length caspase-9, thereby
interfering with the formation of an active heterodimeric caspase-9
enzyme. A third is that it competitively binds to downstream protein
complexes that include caspase-3. In line with its in vitro
anti-apoptotic activity, we have shown that transiently expressed
casp-9-CTD effectively protects PC12 cells and Rat-1a fibroblasts from
trophic deprivation and DNA damage and that cell lines with permanent
expression of this isoform are protected from cell death evoked by
serum deprivation. Past studies have implicated caspase-2 as a required
element in the mechanism by which trophic factor-deprived PC12 cells
undergo apoptosis (21). Our findings that casp-9-CTD and
caspase-9(C327A) overexpression protects PC12 cells from death evoked
by serum and NGF deprivation implicate caspase-9 in the apoptotic process.
We observed that casp-9-CTD was primarily cytoplasmic and excluded from
the nucleus, whereas inactive full-length caspase-9 was present in both
the cytoplasm and nucleus. The intracellular localization of casp-9-CTD
clearly contrasts with that of full-length caspase-9, which was
previously found in the nuclei of cultured mammary epithelial
cells, where it is activated in response to apoptotic signals (31).
Faleiro and Lazebnik (32) have recently presented additional evidence
that caspase-9 re-localization to the nucleus is important in nuclear
disassembly. Thus, it will be important to determine the role of
nuclear exclusion of casp-9-CTD in the induction of nuclear breakdown.
At this point, our observations indicate that in the paradigms we
studied, the step(s) in the death pathway suppressed by casp-9-CTD
occur outside the nucleus and that any independent nuclear events
involving full-length caspase-9 are not sufficient to evoke death. An
additional point raised by our findings is that the C-terminal
sequences regulate the nuclear localization of caspase-9 isoforms. It
will be of interest to determine whether the alternative C-terminal
domain of the caspase-9-CTD form promotes specific interactions with other proteins and/or subcellular compartments.
Our work has shown that casp-9-CTD mRNA transcripts are expressed
in a variety of tissues. This suggests that this form plays a role in
regulating apoptotic death in the intact organism. Two types of roles
can be foreseen. One is that the casp-9-CTD form acts as a constitutive
inhibitor to protect cells from low-level accidental activation of
full-length caspase-9 following mitochondrial cytochrome c
release. The second is that casp-9-CTD protects cells by interfering
with activation of full-length caspase-9 in the presence of otherwise
apoptotic stimuli. As such, casp-9-CTD is among a growing number of
isoforms of apoptosis-related proteins that have the potential to
regulate cell death and that result from alternative splicing. As
reviewed above, several such alternative forms of caspase-9 have been
recognized. Catalytically compromised forms of caspase-2 and caspase-8
have also been found (10, 33, 34). It is of note that these caspases
share the potential to be initiators of apoptotic cascades that
catalyze activation of downstream caspases. Thus, cells may have
evolved alternative forms with the potential to delay or suppress the
untimely onset of apoptotic caspase cascades. It is intriguing
that the ratios of casp-9-CTD to caspase-9 are high in ovary, brain,
and heart. In the case of the ovary, one may speculate that casp-9-CTD
plays a protective role during the portion of the ovarian cycle not associated with cell death. Likewise, the high levels of casp-9-CTD in
heart and brain may serve to protect non-replaceable cells.
It is also remarkable that for caspase-9, multiple complementary
isoforms have been reported in humans and rodents. To date, we have not
found sequences homologous to the alternative C terminus of rat
casp-9-CTD in data bases of human expressed sequence tags and genomic
sequences (data not shown). This could reflect the absence of this form
in humans, the presence of a different alternative C terminus in a
human homolog of casp-9-CTD, or the incomplete state of presently
available data bases. It remains to be determined if the existence of
distinct isoforms of inhibitory variants of caspase-9 in different
species is, in turn, an indication of an ongoing evolutionary process
directed at the optimization of inhibitory caspase proteins or is only
a matter of incomplete sequence information.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
[in a new window]
Fig. 1.
Sequence comparison of rat caspase-9 and
casp-9-CTD. A, the nucleotide and predicted amino acid
sequences of rat full-length caspase-9 are depicted. The predicted
5'-Met residue indicating the translation start site, the translation
stop site, and Asp residues preceding potential cleavage sites between
the N terminus and large and small subunits are indicated in
boldface. The caspase active-site signature motif QACGG is
shaded. Boldface lettering marks differences (at
positions 322 and 754) from known sequences for rat caspase-9. The
alternative C-terminal nucleotide sequences of rat casp-9-CTD are shown
where different from full-length caspase-9, and an amino acid
translation is provided. B, the short isoforms of caspase-9
in mouse (GenBankTM/EBI accession number AB019601) and
human (accession numbers AF110376, AF093130, AB020979, and AB015653)
are shown together with rat full-length caspase-9 and casp-9-CTD. Only
casp-9-CTD contains peptide sequences derived from an alternative
cDNA sequence (striped box). CARD, caspase
recruitment domain.
; GenBankTM/EBI accession numbers AF110376,
AF093130, AB020979, and AB015653, respectively) that is most likely
derived from human full-length caspase-9 by omission of exons 3-6
(Fig. 1B). The ability of the human short isoform to act as
an endogenous inhibitor is not surprising considering that the omission
of these exons will result in the deletion of its active site. A short variant of mouse caspase-9 has been reported (Fig. 1B) that
appears to be generated by deletion of 107 nucleotides and a resulting frameshift within sequences encoding the small caspase subunit (GenBankTM/EBI accession number AB019601). This mutant
form, however, differs from the variant that we have identified in that
a transcript is not normally seen in mouse embryos or tissues (11). In
contrast, the variant of rat caspase-9 that we have observed was
present at varying expression levels in various rat tissues and cell
lines, and it was also detected at different stages in rat embryos (see also Fig. 3A).
View larger version (43K):
[in a new window]
Fig. 2.
Structure modeling of rat caspase-9 and
casp-9-CTD. Structure models for processed rat caspase-9
(residues 192-454) and casp-9-CTD (residues 192-331) were predicted
by PrISM software and are depicted using the InSightII 98 software
package on an O2 R12000 workstation (Silicon Graphics). Side chains for
the active-site sequence QACGG are shown, and the catalytic
Cys327 residue is colored in pink. The N
terminus is marked, and -helixes 1-5 and
-sheets 1-6 are
labeled where applicable. As indicated under "Results," no
unambiguous predictions could be made for the alternative C-terminal
sequences in casp-9-CTD.
View larger version (28K):
[in a new window]
Fig. 3.
Tissue expression and characterization of
casp-9-CTD protein. A: expression of rat full-length
caspase-9 and casp-9-CTD was determined by RT-PCR using total RNA from
the indicated rat tissues and PC12 cells, either without treatment
(naive) or following 1 week of exposure to NGF. RT-PCR using
-actin-specific primers is shown as a standard. B:
left panel, full-length caspase-9 and casp-9-CTD were
labeled with [35S]methionine following in
vitro transcription and translation. Labeled proteins were
resolved on a 4-20% gradient gel and detected by autoradiography.
Right panel, control vector and casp-9-CTD were transiently
transfected into 293 cells. After 24 h, cells were lysed, and cell
lysates were analyzed by Western blotting with anti-caspase-9 antiserum
and ECL.
View larger version (11K):
[in a new window]
Fig. 4.
Casp-9-CTD is a cytoplasmic protein that
fails to induce cell death. A, PC12 cells were
transiently transfected with pCMS-EGFP directing the expression of
casp-9-CTD-FLAG (panels a and b)
or caspase-9(C327A)-FLAG (panels c and
d). On day 3, the cells were fixed and stained
with anti-FLAG antibody, followed by detection with
rhodamine-conjugated secondary antibody. Panels a and
c show autofluorescence of EGFP (green).
Immunofluorescence was used to examine the expression of FLAG-tagged
caspases (red). Panels b and d are
composite images from confocal microscopy. The bar
represents 10 µm. B, 293 cells were transfected with
vector, full-length caspase-9, or casp-9-CTD and assessed for cell
viability at 22 and 48 h after transfection. Values represent the
numbers of non-apoptotic EGFP-positive cells in the same strip at each
time point. Similar results were obtained independently.
View larger version (20K):
[in a new window]
Fig. 5.
Recombinant casp-9-CTD exerts reduced
catalytic activity. A: AMC-conjugated peptide
substrates (0.2 mM) were incubated with buffer only,
purified caspase-9 (10 nM), or casp-9-CTD (10 nM) for 30 min at 37 °C. Following incubation, the
concentration of liberated AMC was determined using standard
fluorometry. Data of three independent samples are summarized and
presented after subtraction of substrate hydrolysis by buffer only.
Residual activities for casp-9-CTD are within the experimental error
range. B: left panels, recombinant
His6-tagged caspase-3 (150 nM) was incubated
with granzyme B (GraB; 10 µM), rat full-length
caspase-9 (Casp-9; 10 or 20 nM), or casp-9-CTD
(C9-CTD; 10 or 20 nM). NT indicates
nontreated. After 30 min at 37 °C, proteins were separated by
4-20% SDS-polyacrylamide gel electrophoresis and blotted onto
nitrocellulose membrane. Proteins were detected using
anti-His6 (upper left panel) or anti-caspase-9
(lower left panel) antibody, followed by ECL. No cleavage
products of caspase-3 were detected when using casp-9-CTD. Right
panels, His6-tagged caspase-3 protein (1 µM) was incubated with rat full-length caspase-9 (200 nM) or casp-9-CTD (200 nM) at 37 °C for the
indicated times. Proteins were separated by 4-20% SDS-polyacrylamide
gel electrophoresis, blotted onto nitrocellulose membrane, and detected
using anti-His6 (upper right panel) or
anti-caspase-9 (lower right panel) antibody, followed by
ECL. No cleavage products of caspase-3 were detected following
incubation with casp-9-CTD. C: caspase-3 activation by
upstream proteases (caspase-9, casp-9-CTD, and granzyme B) was examined
by determining AFC release in a coupled enzyme assay. Caspase-3 does
not exert proteolytic activity on the Ac-DEVD-AFC substrate unless
activated. Thus, AFC release is an indicator of the ability of upstream
proteases to activate caspase-3. Here, 100 nM caspase-3 was
incubated with a 10 nM concentration of each protease using
0.2 mM Ac-DEVD-AFC substrate at 37 °C. The concentration
of AFC was then determined at A380 nm in 5-s
intervals. Substrate autoproteolysis at different time intervals was
substracted before plotting.
Reaction rates for Ac-DEVD-pNA substrate hydrolysis
View larger version (17K):
[in a new window]
Fig. 6.
Casp-9-CTD expression protects neuronal
cells from apoptosis. A, replicate cultures of PC12
cells were transfected with the indicated FLAG-tagged constructs
(caspase-9(C327A) and casp-9-CTD in the pCMS-EGFP vector) and
maintained with insulin in serum-free medium. After 72 h, half the
cultures were deprived of insulin, and half were treated with
insulin/NGF. Cells were then evaluated for the expression of EGFP and
the FLAG tag and the presence of apoptotic nuclei. Values represent
percentages of EGFP-expressing or FLAG-tagged protease-expressing cells
with apoptotic nuclei. Comparable results were achieved in an
independent experiment. B, replicate PC12 cell cultures were
exposed to NGF for 2 days in serum-free medium, transfected as
described for A, and maintained for an additional 3 days in
serum-free medium with NGF. The cells were extensively washed free of
NGF and serum and replated with or without NGF. Cultures were evaluated
as described for A for percentages of transfected cells
showing apoptotic nuclei. Comparable results were achieved in an
independent experiment. C, PC12 cells were treated with NGF
for 2 days in medium containing 1% horse serum and then transfected
with pCMS-EGFP (Vector), pCMS-EGFP-casp-9-CTD-FLAG
(Casp-9CTD), or pCMS-EGFP-caspase-9(C327A)
(Caspase-9(C327A)). After 3 days, the cultures were exposed
to 10 µM camptothecin (indicated as time 0) and scored
for the relative percentage of transfected cells that exhibited
apoptotic nuclei and expressed either EGFP- or FLAG-tagged constructs.
The numbers of cells scored per point ranged from 320 to 800. D, PC12 cells were transfected with either vector
(pCMS-EGFP) or pCMS-EGFP-casp-9-CTD in serum-free medium supplemented
with insulin to provide trophic support. On day 3, insulin was
withdrawn, and the numbers of surviving EGFP-positive cells in the same
strip were counted at the indicated times. Data were normalized to
numbers of living transfected cells per strip (ranging from 58 to 99)
scored at the time of insulin withdrawal. Values represent means ± S.E. (n = 3). Comparable findings were obtained in
three independent experiments.
View larger version (15K):
[in a new window]
Fig. 7.
Casp-9-CTD protects fibroblasts from
apoptosis following serum withdrawal. Rat-1a and
Rat-1a-Myc cells were transfected with pCMS-EGFP
(Vector), pCMS-EGFP-casp-9-CTD-FLAG (Casp-9-CTD),
or pCMS-EGFP-caspase-9(C327A)-FLAG (Caspase-9(C327A)). After
2 days (time 0), serum was withdrawn, and cultures were scored for the
percentage of transfected cells that expressed either EGFP- or
FLAG-tagged constructs and exhibited apoptotic nuclei. For Rat-1a
cultures, 300-500 cells were scored per time point; for
Rat-1a-Myc cultures, 60-100 cells were scored per time
point. Comparable results were obtained in an independent
experiment.
View larger version (10K):
[in a new window]
Fig. 8.
Cells with constitutive overexpression of
casp-9-CTD are resistant to apoptosis. Neomycin-resistant PC12
cells transfected with the pcDNA3.0 plasmid expressing
casp-9-CTD-FLAG (clones CTD1-1, -3, -10, -7, and polyclonal mass
cultures) or the pcDNA3.0 empty vector (Control) were
washed four times and then maintained in serum-free RPMI 1640 medium
for 2 days. Expression of casp-9-CTD-FLAG was verified by Western
blotting of cell lysates with anti-FLAG and anti-caspase-9 antibodies.
The control and CTD1-1 lines had no detectable expression of
casp-9-CTD. The percentages of apoptotic PC12 cells were
determined as described under "Experimental Procedures." Values
represent means ± S.E. (n = 3).
1·mg
1 of total protein for
both types of lysates (data not shown). These results suggested that
the levels of activable caspase-3 were not altered in cells expressing
casp-9-CTD. We then examined the ability of exogenous cytochrome
c to induce caspase-3-like activity in our in
vitro reconstitution model. At low concentrations of cytochrome
c (0.1 µM), no significant caspase-3-like
activity was detected in lysates prepared from either cell type,
possibly because the rate of enzymatic conversion was below detectable levels (Fig. 9). However, cytosolic lysates prepared from control cells
and cells expressing casp-9-CTD differed significantly in the extent of
caspase-3 activation when 1 µM cytochrome c
was added (Fig. 9). Significant activity was present with control cell
lysates, whereas no detectable peptide proteolysis was observed in
cytosolic lysates from cells expressing casp-9-CTD (Fig. 9). These
findings indicate that the presence of casp-9-CTD in cell lysates
inhibits the cytochrome c-dependent induction of
caspase-3-like enzyme activity.
View larger version (17K):
[in a new window]
Fig. 9.
Cell lysates from cells overexpressing
casp-9-CTD are resistant to cytochrome c.
Cytosolic extracts were prepared from PC12 control cells and
polyclonal mass cultures expressing casp-9-CTD. dATP (1 mM
final concentration) and buffer or cytochrome c
(CytoC; 0.1 or 1 µM final concentration) were
added to the lysates (10 mg/ml of protein) at 37 °C. Proteolysis of
Ac-DEVD-AFC was recorded at A380 nm in 5-s
intervals. The depicted data were repeated in multiple independent
experiments. NT indicates nontreated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helixes 4 and 5 encoded by the
small subunit to the overall structural integrity of full-length caspases (24, 25). The importance of these helixes is further underscored by their presence in other proteases, where they are thought to be involved in forming the backbone for substrate
recognition and processing in the context of an antiparallel
-sheet
conformation (24, 25). Residues in the small subunit of full-length
caspase-9 that are present in the known structures of caspase-1 and
caspase-3 and that form the binding sites P3-P5 and, to a lesser
extent, are involved in the formation of P1 are also absent in
casp-9-CTD. These residues are especially important in facilitating the
correct relative orientation of the Asp residue N-terminal to the
P4-P5 peptide bond. We have observed a residual proteolytic activity of casp-9-CTD when using the peptide substrates Ac-DEVD-AFC and Ac-DEVD-pNA. Since Ac-DEVD-AFC is not efficiently cleaved by
full-length caspase-9, our data also indicate that casp-9-CTD exerts
either an altered or more promiscuous enzyme specificity compared with full-length caspase-9.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Henning Stennicke and Guy Salvesen for helpful discussions and anti-caspase-9 antibody Bur73, Chris Froehlich (Northwestern University, Chicago) for purified granzyme B, and Andy Marks (Columbia University) for critical comments. We also thank Claudine Bitel for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants NS16036 and NS33689 and the Blanchette Rockefeller Foundation (to L. A. G.), American Heart Association Grant 9951052T, and Department of Defense Grants DAMD 17-99-1-9153 and DAMD 17-00-1-0214 (to T. F. F.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF308469 and AY008275.
§ To whom correspondence should be addressed: Dept. of Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th St., P&S15-401, New York, NY 10032. Tel.: 212-305-6370; Fax: 212-305-5498; E-mail: jma14@columbia.edu.
To whom reprint requests should be addressed. E-mail:
JMA14@columbia.edu or TFF5{at}columbia.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M009523200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
DMEM, Dulbecco's
modified Eagle's medium;
PCR, polymerase chain reaction;
RT-PCR, reverse transcriptase-polymerase chain reaction;
IPTG, isopropyl--D-thiogalactopyranoside;
pNA, p-nitroanilide;
AMC, 7-amino-4-methylcoumarin;
AFC, 7-aminotrifluoromethylcoumarin;
NGF, nerve growth factor;
ECL, enhanced
chemiluminescent;
RACE, rapid amplification of cDNA ends;
Pipes, 1,4-piperazinediethanesulfonic acid;
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PBS, phosphate-buffered saline;
EGFP, enhanced green fluorescent protein;
UTR, untranslated region.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Green, D. R.,
and Reed, J. C.
(1998)
Science
281,
1309-1312 |
2. |
Thornberry, N. A.,
and Lazebnik, Y.
(1998)
Science
281,
1312-1316 |
3. |
Salvesen, G. S.,
and Dixit, V. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10964-10967 |
4. |
Stennicke, H. R.,
Deveraux, Q. L.,
Humke, E. W.,
Reed, J. C.,
Dixit, V. M.,
and Salvesen, G. S.
(1999)
J. Biol. Chem.
274,
8359-8362 |
5. | Zheng, T. S., Hunot, S., Kuida, K., and Flavell, R. A. (1999) Cell Death Differ. 6, 1043-1053[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Slee, E. A.,
Harte, M. T.,
Kluck, R. M.,
Wolf, B. B.,
Casiano, C. A.,
Newmeyer, D. D.,
Wang, H. G.,
Reed, J. C.,
Nicholson, D. W.,
Alnemri, E. S.,
Green, D. R.,
and Martin, S. J.
(1999)
J. Cell Biol.
144,
281-292 |
7. | Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E. S. (1998) Mol. Cell 1, 949-957[Medline] [Order article via Infotrieve] |
8. |
Stennicke, H. R.,
and Salvesen, G. S.
(1997)
J. Biol. Chem.
272,
25719-25723 |
9. |
Rodriguez, J.,
and Lazebnik, Y.
(1999)
Genes Dev.
13,
3179-3184 |
10. | Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, J. (1994) Cell 78, 739-750[Medline] [Order article via Infotrieve] |
11. | Fujita, E., Jinbo, A., Matuzaki, H., Konishi, H., Kikkawa, U., and Momoi, T. (1999) Biochem. Biophys. Res. Commun. 264, 550-555[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Seol, D. W.,
and Billiar, T. R.
(1999)
J. Biol. Chem.
274,
2072-2076 |
13. |
Srinivasula, S. M.,
Ahmad, M.,
Guo, Y.,
Zhan, Y.,
Lazebnik, Y.,
Fernandes-Alnemri, T.,
and Alnemri, E. S.
(1999)
Cancer Res.
59,
999-1002 |
14. |
Hu, S.,
Vincenz, C.,
Ni, J.,
Gentz, R.,
and Dixit, V. M.
(1997)
J. Biol. Chem.
272,
17255-17257 |
15. | Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2424-2428[Abstract] |
16. | Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801-809[Medline] [Order article via Infotrieve] |
17. | Stennicke, H. R., and Salvesen, G. S. (1999) Methods 17, 313-319[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Stennicke, H. R.,
Jurgensmeier, J. M.,
Shin, H.,
Deveraux, Q.,
Wolf, B. B.,
Yang, X.,
Zhou, Q.,
Ellerby, H. M.,
Ellerby, L. M.,
Bredesen, D.,
Green, D. R.,
Reed, J. C.,
Froelich, C. J.,
and Salvesen, G. S.
(1998)
J. Biol. Chem.
273,
27084-27090 |
19. | Angelastro, J. M., Ho, C. L., Frappier, T., Liem, R. K., and Greene, L. A. (1998) J. Neurochem. 70, 540-549[Medline] [Order article via Infotrieve] |
20. | Greene, L. A., Farinelli, S. E., Cunningham, M. E., and Park, D. S. (1998) in Culturing Nerve Cells (Banker, G. G. K., ed) , pp. 161-187, MIT Press, Cambridge, MA |
21. |
Troy, C. M.,
Stefanis, L.,
Greene, L. A.,
and Shelanski, M. L.
(1997)
J. Neurosci.
17,
1911-1918 |
22. | Smith, T. F., and Waterman, M. S. (1981) J. Mol. Biol. 147, 195-197[Medline] [Order article via Infotrieve] |
23. | Yang, A.-S., and Honig, B. (1999) Proteins Suppl. 3, 66-72 |
24. | Rotonda, J., Nicholson, D. W., Fazil, K. M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E. P., Rasper, D. M., Ruel, R., Vaillancourt, J. P., Thornberry, N. A., and Becker, J. W. (1996) Nat. Struct. Biol. 3, 619-625[Medline] [Order article via Infotrieve] |
25. |
Mittl, P. R.,
Di Marco, S.,
Krebs, J. F.,
Bai, X.,
Karanewsky, D. S.,
Priestle, J. P.,
Tomaselli, K. J.,
and Grutter, M. G.
(1997)
J. Biol. Chem.
272,
6539-6547 |
26. | Wilkins, M. R., Lindskog, I., Gasteiger, E., Bairoch, A., Sanchez, J. C., Hochstrasser, D. F., and Appel, R. D. (1997) Electrophoresis 18, 403-408[Medline] [Order article via Infotrieve] |
27. |
Thornberry, N. A.,
Rano, T. A.,
Peterson, E. P.,
Rasper, D. M.,
Timkey, T.,
Garcia-Calvo, M.,
Houtzager, V. M.,
Nordstrom, P. A.,
Roy, S.,
Vaillancourt, J. P.,
Chapman, K. T.,
and Nicholson, D. W.
(1997)
J. Biol. Chem.
272,
17907-17911 |
28. |
Stefanis, L.,
Park, D. S.,
Friedman, W. J.,
and Greene, L. A.
(1999)
J. Neurosci.
19,
6235-6247 |
29. |
Juin, P.,
Hueber, A. O.,
Littlewood, T.,
and Evan, G.
(1999)
Genes Dev.
13,
1367-1381 |
30. |
Janicke, R. U.,
Sprengart, M. L.,
Wati, M. R.,
and Porter, A. G.
(1998)
J. Biol. Chem.
273,
9357-9360 |
31. | Ritter, P. M., Marti, A., Blanc, C., Baltzer, A., Krajewski, S., Reed, J. C., and Jaggi, R. (2000) Eur. J. Cell Biol. 79, 358-364[Medline] [Order article via Infotrieve] |
32. |
Faleiro, L.,
and Lazebnik, Y.
(2000)
J. Cell Biol.
151,
951-960 |
33. |
Alnemri, E. S.,
Fernandes-Alnemri, T.,
and Litwack, G.
(1995)
J. Biol. Chem.
270,
4312-4317 |
34. | Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. (1997) Nature 388, 190-195[CrossRef][Medline] [Order article via Infotrieve] |