From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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The DNA fragmentation factor (DFF) is composed of
two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and
its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290-345) abrogated the
caspase-3-induced nuclease activity of DFF40/CAD but not its ability to
interact with DFF45/ICAD. Conversely, removal of the regulatory domain
(residues 1-83) yielded a constitutively active DFF40/CAD nuclease
that neither bound to its inhibitor nor required caspase-3 for
activation. Amino acid alignment revealed that the regulatory domain of
DFF40/CAD has homology to the N-terminal region of mammalian and
Drosophila DFF45/ICAD and CIDE-N, a regulatory domain
previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus,
CIDE-N represents a new type of domain that is associated with the
regulation of the apoptosis/DNA fragmentation pathway.
Apoptosis, a morphologically defined form of programmed cell death
plays an essential role in animal development and tissue homeostasis
(1). Apoptotic cells undergo multiple changes including membrane
blebbing, nuclear condensation, and fragmentation of genomic DNA into
nucleosomal fragments. These morphological and biochemical changes are
promoted by caspases, a family of cysteine proteases that are activated
by apoptotic stimuli. Activated caspases can cleave multiple
cytoplasmic and nuclear substrates, a process that appears to play a
pivotal role in the execution phase of apoptosis (2).
DNA fragmentation associated with apoptosis is induced by the DNA
fragmentation factor (DFF)1,
which is activated by caspases, mainly caspase-3 (3-8). DFF is
composed of two protein subunits, a 40-kDa caspase-activated nuclease
(DFF40/CAD), also called CPAN, and its 45-kDa inhibitor (DFF45/ICAD)
(3-8). Cleavage of DFF45/ICAD by caspase-3 releases DFF40/CAD from its
inhibitor leading to the induction of nuclease activity, nuclear
condensation, and DNA fragmentation in vitro (3-8).
However, the molecular basis for DFF40/CAD function and its regulation
by DFF45/ICAD remains poorly understood.
In a previous study, we identified a conserved family of proteins named
CIDEs, that include CIDE-A, CIDE-B, and Fsp27, based on amino acid
homology to the N-terminal region of DFF45/ICAD (9). Unlike DFF45/ICAD,
CIDEs are pro-apoptotic proteins that induce DNA fragmentation as
well as other apoptotic features such as membrane blebbing and nuclear
condensation (9). The activity of CIDEs is inhibited by DFF45/ICAD (9).
Mutational analysis revealed that CIDEs contains two domains, CIDE-N
and CIDE-C (9). The CIDE-C domain is necessary and sufficient for
apoptosis, whereas CIDE-N acts as a regulatory domain required for the
inhibitory activity of DFF45/ICAD (9).
Here we report that DFF40/CAD has two domains with distinct biological
functions. A catalytic domain located in the C-terminal region mediates
nuclease activity, whereas a regulatory domain is located in the
N-terminal half of DFF40/CAD. The regulatory domain of DFF40/CAD has
homology to a conserved CIDE-N domain that was previously identified in
the pro-apoptotic CIDE proteins. These results identify the N-terminal
region of DFF40/CAD as a conserved domain that regulates the activation
and activity of this apoptosis-associated nuclease.
Construction of Expression Plasmids--
The entire cDNA
open reading frame of mouse DFF40/CAD was amplified by polymerase chain
reaction from cDNA synthesized from total RNA from mouse testis and
cloned into the XbaI and EcoRI sites of
pcDNA3-HA and pcDNA-3-Myc (9) to produce expression plasmids
pcDNA3-HA-CAD and pcDNA34-Myc-CAD encoding HA-tagged and Myc-tagged
DFF40/CAD, respectively. pcDNA3-Flag-DFF45,
pcDNA3-caspase-9-HA, pcDNA3-TNFR1-Flag (tumor necrosis factor
receptor-1), and pcDNA3- Transfection, Expression, and Immunodetection of Tagged
Proteins--
5 × 106 human 293T cells were
transfected with expression plasmids by a calcium phosphate method as
described (10). The total amount of transfected plasmid DNA was
adjusted with pcDNA3 plasmid to be the same within individual
experiments. 24 h post-transfection, 293T cells were harvested and
lysed with 0.2% Nonidet P-40 isotonic lysis buffer (12). Total lysates
were subjected to 15% SDS-polyacrylamide electrophoresis and
immunoblotted with mAb to Flag (Kodak).
In Vitro Nuclease Assay and in Vivo DNA Fragmentation
Assay--
The in vitro nuclease activity was assayed with
immunopurified DFF in the presence or absence of recombinant caspase-3.
Briefly, 5 × 106 293T cells were transfected with
plasmids as indicated in the figure legends. 24 h
post-transfection, cells were lysed with Nonidet P-40 lysis buffer, and
tagged DFF40/CAD proteins were immunoprecipitated with anti-HA or
anti-Myc polyclonal antibody and protein-A-Sepharose, washed five times
with Nonidet P-40 lysis buffer, and then with CPAN buffer (10 mM HEPES, 1 mM EGTA, 5 mM MgCl2, 50 mM NaCl, 1 mg/ml bovine serum
albumin, pH 7.5) (6). The in vitro nuclease activity of the
immunoprecipitated protein was assayed by incubation in CPAN buffer
with or without 50 units of recombinant caspase-3 (a gift from
Margarita Garcia-Calvo, Merck) and 1 µg of plasmid DNA substrate and
subsequent analysis of plasmid DNA degradation by agarose gel
electrophoresis, as described (6). For assay of in vivo DNA
fragmentation, genomic DNA was extracted from 2 × 104
293T cells transfected with pcDNA3, pcDNA3-caspase-9-HA,
pcDNA3-TNFR1-Flag, or DFF40/CAD expression plasmids as indicated in
the figure legends, 24 h post-transfection as described (8). The
same amount of each DNA was subjected to 1% agarose gel
electrophoresis. The amount of DNA loaded was 10-fold lower than
previously described (9). The cell death assay of 293T was performed as
reported (9).
The N-terminal Half of DFF40/CAD Mediates Binding to DFF45/ICAD
whereas the C-terminal Region Is Required for Nuclease
Activity--
To identify regions of DFF40/CAD that are important for
nuclease activity, binding, and sensitivity to DFF45/ICAD, we
constructed a series of plasmids to express deletion mutants of
DFF40/CAD (Fig. 1A). We
transfected these expression plasmids encoding HA-tagged or Myc-tagged
wild type and mutant DFF40/CAD proteins into 293T embryonic kidney
cells and purified the proteins by affinity chromatography.
HA-tagged wild type and C-terminal deletion mutant DFF40/CAD proteins
were co-expressed with Flag-tagged DFF45/ICAD and immunoprecipitated by
anti-HA antibody. In vitro nuclease activities of these
immunoprecipitated proteins were measured by their ability to degrade
plasmid DNA substrate in the presence or absence of caspase-3. This
in vitro assay revealed that the DFF40/CAD deletion mutants
We tested next the ability of WT and mutant DFF40/CAD to promote
genomic DNA fragmentation in vivo. In these experiments, we
transiently transfected constructs producing WT, The N-terminal Region of DFF40/CAD Has Homology to the N Termini of
DFF45/ICAD and CIDEs--
DFF40/CAD exhibits no significant amino acid
or structural homology to other known nuclease families (4). Close
inspection of the DFF40/CAD amino acid sequence by the BLAST2 program,
however, revealed significant amino acid homology between the
regulatory domain of DFF40/CAD and the CIDE-N domain previously
identified in DFF45/ICAD and CIDE proteins (Fig.
2). The regulatory (CIDE-N) domain of
DFF40/CAD showed 49 to 55% similarity with those of CIDE-A, CIDE-B,
Fsp27, DFF45/ICAD, and Drosophila DREP-1, respectively (p = 10 Mutant Forms with Substitution of Conserved Residues in the CIDE-N
Domain of DFF40/CAD Exhibit Diminished Nuclease Activity--
To
determine whether the conserved residues in the CIDE-N domain play a
role in the regulation of DFF40/CAD activity, we introduced site-directed mutations at five conserved residues of the CIDE-N domain
of DFF40/CAD (Fig. 2). Anti-HA immunoprecipitation showed that all five
of these point mutants still bound to DFF45/ICAD (Fig.
3A). However, the G55I, F63S,
and W81C mutants of DFF40/CAD exhibited reduced nuclease activity
in vitro (Fig. 3B) and in vivo (Fig.
3D), whereas the K35A and T56L mutants did not.
In vitro nuclease analysis of 50-fold dilutions of the
immunoprecipitated DFF40/CAD proteins from Fig. 3B confirm
these differences (Fig. 3C). Immunoblotting revealed similar
levels of expression of the mutants, indicating that reduced nuclease
activity was not because of diminished expression (Fig. 3A).
Under our experimental conditions using the in vitro assay,
DFF45/ICAD bound to either WT DFF40/CAD or any of the mutants was
completely cleaved by
caspase-3.2 In addition,
neither WT DFF40/CAD nor any of the mutants bound to DFF45/ICAD protein
cleaved by caspase-3.2 These data indicate that reduced
nuclease activity of the mutants is not because of caspase-3
insensitivity of DFF45/ICAD bound to mutant proteins or inhibition of
DFF40/CAD by an interaction with cleaved DFF45/ICAD fragments.
Comparison between wild-type, site-directed mutants of DFF40/CAD and an
N-terminal deletion mutant, A Regulatory Domain Located in the N-terminal Half of
DFF40/CAD--
The results of the experiments shown in Fig. 1
suggested that the nuclease activity of DFF40/CAD resides in the
C-terminal half of the protein. To test this hypothesis, we assessed
the nuclease activity of the N-terminal deletion mutant,
Fig. 4D is a model depicting the mechanism by which
DFF40/CAD nuclease activity may be regulated during apoptosis. When
DFF40/CAD is bound to DFF45/ICAD via its N-terminal region, its
nuclease activity is suppressed by this interaction. Once DFF45/ICAD is cleaved by caspase-3, the N-terminal region of DFF40/CAD is released, a
process that enables the N-terminal region of DFF40/CAD to then activate the C-terminal nuclease domain. Cleavage of DFF45/ICAD by
caspase-3 downstream of Asp-117, which is adjacent to its CIDE-N domain, results in the release of DFF45/ICAD fragments from DFF40/CAD (3, 5), supporting an important role for the CIDE-N domain in
DFF45/ICAD function. The regulation of the CIDE-N domain of DFF40/CAD
is reminiscent of that of the recently described CIDE-N domain of
CIDE-A (8). In this protein, the CIDE-N domain is required for
DFF45/ICAD inhibition of CIDE-A-mediated apoptosis (8). In addition,
deletion of the CIDE-N domain results in enhanced apoptosis, supporting
its role as a regulatory domain. Thus the CIDE-N domain represents a
novel class of regulatory domains that can control the function of
adjacent domains. In the case of DFF40/CAD, its N-CIDE domain appears
to play an important role in the activation and regulation of nuclease activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
MATERIALS AND METHODS
-gal have been previously described
(9-11). The expression plasmids encoding deletion mutants of
HA-DFF40/CAD, pcDNA3-HA-CAD
290-345, pcDNA3-HA-CAD
162-345,
and pcDNA3-Myc-CAD-
1-83 were constructed by polymerase chain
reaction methods. The authenticity of all constructs was confirmed by
dideoxy sequencing.
RESULTS AND DISCUSSION
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Fig. 1.
C-terminal deletion mutants of DFF40/CAD lose
nuclease activity but not the ability to bind DFF45/ICAD.
A, schematic structure of WT DFF40/CAD and deletion mutants.
Conserved CIDE-N sequence is indicated by a closed box (see
Fig. 3) B[en]C, 293T cells were transfected
with pcDNA3-HA-CAD, pcDNA3-HA-CAD 290-345, or
pcDNA3-HA-CAD
162-345 in the absence or presence of
pcDNA3-Flag-DFF45. 24 h post-transfection, DFF40/CAD proteins were
immunoprecipitated by anti-HA mAb. The in vitro nuclease
activities of immunoprecipitated DFF40/CAD proteins were assayed with
or without caspase-3 treatment, as described under "Materials and
Methods." Activity was measured as degradation of added plasmid DNA
substrate, visualized by agarose gel electrophoresis (B,
top panel). m, DNA size marker fragments. In
total lysates (C) and anti-HA immunoprecipitates
(B, middle and bottom panels),
DFF40/CAD and DFF45/ICAD proteins were detected first with anti-HA
polyclonal Ab and then subsequently with anti-Flag polyclonal Ab. The
intact 45-kDa DFF45/ICAD (B, middle panel) and
14-kDa fragments of DFF45/ICAD digested by caspase-3 (B, bottom
panel) were detected by 5- and 60-s exposures of x-ray film,
respectively. D, 293T cells in 4 wells of 12-well plates
were co-transfected with pcDNA3-HA-CAD (WT),
pcDNA3-HA-CAD
290-345 (
290-345), or
pcDNA3-HA-CAD
162-345 (
162-345) and
pcDNA3-TNFR1-Flag (TNFR1) or pcDNA3-caspase-9-HA
(Casp. 9) plus pcDNA3-
-gal. To assay for in
vivo DNA fragmentation, genomic DNA was extracted from 1 well of
transfected cells and subjected to 1% agarose electrophoresis as
described under "Materials and Methods." The percentage of
apoptotic cells (apop.) was determined by the
-galactosidase assay using transfected cells in 3 wells as described
under "Materials and Methods." Standard deviation of each
percentage was less than 10%.
290-345 and
162-345 lost their caspase-activated nuclease
activity when compared with control wild type (WT) DFF40/CAD (Fig.
1B, top panel). In contrast, both DFF40/CAD
mutants retained their ability to bind to DFF45/ICAD (Fig.
1B, middle panel). Loss of catalytic activity was
not due to differences in protein levels between wild type and mutant
proteins because comparable levels of both were detected in total
lysates (Fig. 1C) and immunoprecipitates (Fig.
1B, middle panel). In addition, the sensitivity
of DFF45/ICAD to caspase-3-mediated cleavage was not affected by these
mutations, as DFF45/ICAD bound to either mutant was cleaved by
caspase-3 (Fig. 1B, middle and lower
panels).
290-345,
162-345, or control plasmid into 293T cells. Because DFF40/CAD requires an apoptotic stimulus to be activated, the cells were also
transfected with plasmids producing TNFR1 or caspase-9, both of which
are known to activate apoptotic cell death in 293T cells (13). Although
both caspase-9 and TNFR1 could cause DNA fragmentation, we loaded a
reduced amount of genomic DNA in the in vivo DNA
fragmentation assay to visualize the enhancement of DNA fragmentation
by DFF40/CAD. In the absence of a death stimulus, neither WT DFF40/CAD
nor mutant DFF40/CAD induced DNA fragmentation (Fig. 1D).
However, when the cells were co-transfected with plasmids producing
TNFR1 or caspase-9, WT DFF40/CAD did promote DNA fragmentation (Fig.
1D). In contrast to WT DFF40/CAD, the
290-345 and
162-345 mutants did not induce DNA fragmentation (Fig.
1D). Thus, the C-terminal region of DFF40/CAD is required
for catalytic activity in vitro and in vivo but
not for binding to DFF45/ICAD.
2-10
5). Several
amino acids in the CIDE-N domain of DFF40/CAD including Lys-35, Asp-54,
Gly-55, Thr-56, Phe-63, and Trp-81 are conserved in the CIDE-N domains
of CIDE-A, CIDE-B, Fsp27, and DREP-1 (Fig. 2), suggesting that they are
important for the function of these proteins.
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Fig. 2.
Homology of CIDE-N domains from DFF40/CAD,
CIDE-A, CIDE-B, Fsp27, and DFF45/ICAD. Amino acid sequence and
alignments of N-terminal regions of human and mouse DFF40/CAD (4, 6),
human and mouse DFF45/ICAD (3, 4), Drosophila melanogaster
DREP-1, mouse Fsp27, human and mouse CIDE-A, and mouse CIDE-B (8). The
completely conserved residues and partial conserved residues are
indicated by black and gray
highlights, respectively. Mutations used in the experiments
of Fig. 3 are also shown.
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Fig. 3.
Mutation of conserved residues of CIDE-N
domain result in diminished nuclease activity of DFF40/CAD. 293T
cells were co-transfected with 4 µg of pcDNA3 (CTRL or
), pcDNA3-HA-CAD (WT), pcDNA3-HA-CAD-K35A,
pcDNA3-HA-CAD-G55I, pcDNA3-HA-CAD-T56L, pcDNA3-HA-CAD-F63S, or
pcDNA3-HA-CAD-W81C in the presence or absence of
pcDNA3-Flag-DFF45. To compare the expression levels and the
nuclease activities of WT and mutant DFF40/CAD proteins, 293T cells
were also transfected with 0.01 or 0.1 µg of pcDNA3-HA-CAD
(WT) in the presence of pcDNA3-Flag-DFF45. A,
mutation of the conserved residues does not affect interaction between
DFF40/CAD and DFF45/ICAD. HA-DFF40/CAD and Flag-DFF45/ICAD proteins
were detected with anti-HA and anti-Flag mAb in total lysate
(bottom panel) and anti-HA immunoprecipitates (top
panel). B-C, the in vitro nuclease
activities of WT and mutant DFF40/CAD immunopurified with anti-HA mAb
were measured as described under "Materials and Methods" and Fig.
1. Panel C depicts the relative in vitro nuclease
activities obtained using 50-fold dilutions of the immunoprecipitated
proteins from panel B. D, to compare in
vivo genomic DNA fragmentation activities of the WT and mutant CAD
proteins, 293T cells in 4 wells of 12-well plates were co-transfected
with plasmid expressing WT or mutant DFF40/CAD and
pcDNA3-TNFR1-Flag (TNFR1) or pcDNA3-caspase-9-HA
(Casp. 9). Genomic DNA was analyzed as in Fig. 1.
N, that expresses residues 84-345 of
DFF40/CAD (see below) revealed that the G55I, F63S, and W81C mutants
and the
N mutant exhibit in vitro nuclease activities that are reduced when compared with WT DFF40/CAD (Fig. 3, B
and C and Fig. 4A).
Thus, amino acids Gly-55, Phe-63, and Trp-81 appear to enhance the
nuclease activity of DFF40/CAD after cleavage of DFF45/ICAD by
caspase-3 but do not play an essential role in the catalysis reaction.
These results suggest that the CIDE-N regulatory domain of CAD/ICAD not
only mediates binding to DFF45/ICAD but also promotes the activity of
the catalytic nuclease domain located in the C-terminal region of the
protein.
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Fig. 4.
N-terminal deletion mutant of DFF40/CAD
retains nuclease activity but not binding to DFF45/ICAD or sensitivity
to DFF45/ICAD mediated inhibition. A, 293T cells were
transfected with pcDNA3 ( ), pcDNA3-Myc-CAD (WT),
or pcDNA3-Myc-CAD
1-83 (
N) in the absence or
presence of pcDNA3-Flag-DFF45. 24 h post-transfection,
DFF40/CAD proteins were immunoprecipitated by anti-Myc mAb. The
in vitro nuclease activities of immunoprecipitated DFF40/CAD
proteins with or without caspase-3 treatment were measured as described
under "Materials and Methods." To compare in vitro
nuclease activity between WT and mutant DFF40/CAD proteins,
immunopurified wild type protein was diluted 1-, 6-, 36-, 216-, or
648-fold, and the nuclease activities were measured. m, DNA
size marker fragments. B, in total lysates (bottom
panel) and anti-Myc immunoprecipitates (top panel),
DFF40/CAD and DFF45/ICAD proteins were detected with anti-Myc
polyclonal and anti-Flag mAb, respectively. C, 293T were
transfected with pcDNA3 (
), 0.01, 0.1, or 1 µg of
pcDNA3-Myc-CAD or 4 µg of pcDNA3-Myc-CAD
1-83
(
N). 24 h post-transfection, transfected cells were
harvested and lysed with Nonidet P-40 buffer. Myc-tagged DFF40/CAD and
Flag-tagged DFF45/ICAD were subsequently immunodetected with anti-Myc
and anti-Flag polyclonal Abs, respectively. Flag-DFF45/ICAD was
co-immunoprecipitated with anti-Myc polyclonal Ab and detected with
anti-Flag mAb (top panel).
N. The
mutant protein retained in vitro nuclease activity, although
its activity was reduced compared with that of WT DFF40/CAD (Fig.
4A). In contrast to the WT protein, the
N mutant of
DFF40/CAD did not bind to DFF45/ICAD (Fig. 4, B and
C), indicating that the N-terminal region of DFF40/CAD is
required for binding to DFF45/ICAD. Significantly, the
N mutant had
constitutive nuclease activity in that it did not require caspase-3 for
enzymatic activity (Fig. 4A). These results indicate that
the N-terminal region of DFF40/CAD, although dispensable for in
vitro nuclease activity, has a regulatory role, as it is required
both for the interaction of DFF40/CAD with its inhibitor DFF45/ICAD and
for caspase-3-dependent activation. In addition, the
N-terminal domain of DFF40/CAD is required for optimal nuclease
activity, suggesting that this domain regulates the activity of the
catalytic C-terminal domain.
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ACKNOWLEDGEMENTS |
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We are grateful to M. Garcia-Calvo and N. Thorberry of Merck for recombinant caspase-3; V. M. Dixit for pcDNA3-TNFR1-Flag; and L. del Peso, V. Gonzalez, D. Ekhterae, and Y. Hu for critical review of the manuscript.
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FOOTNOTES |
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* This research was supported in part by Grant CA-64556 from the National Institutes of Health (to G. N.).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 Fellowship from the Japan Science and Technology Corporation.
§ Supported by a Fellowship from the United States Army Medical Research and Material Command.
¶ Recipient of Research Career Development Award CA-64421 from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109. Tel.: 734-764-8514; Fax: 313-647-9654; E-mail: Gabriel.Nunez{at}umich.edu.
2 N.Inohara, and G. Núñez, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DFF, DNA fragmentation factor; HA, hemaglutinin; CAD, caspase-3-activated DNase; CIDE, cell death-inducing DFF45-like effector; ICAD, inhibitor of caspase-3-activated DNase; mAb, monoclonal antibody; WT, wild type; TNFR1, tumor necrosis factor receptor-1..
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REFERENCES |
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