From the Hanson Institute, Frome Road,
Adelaide 5000, the § Department of Biochemistry and
Molecular Biology, Monash University, Clayton, Victoria 3800, and the ¶ Department of Medicine, Adelaide University,
Adelaide 5005, Australia
Received for publication, November 12, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Caspase-2 is unique among mammalian caspases
because it localizes to the nucleus in a
prodomain-dependent manner. The caspase-2 prodomain also
regulates caspase-2 activity via a caspase recruitment domain that
mediates oligomerization of procaspase-2 molecules and their subsequent
autoactivation. In this study we sought to map specific functional
regions in the caspase-2 prodomain that regulate its nuclear transport
and also its activation. Our data indicate that caspase-2 contains a
classical nuclear localization signal (NLS) at the C terminus of the
prodomain which is recognized by the importin Caspase activation is a key event in the execution of
apoptosis (reviewed in Refs. 1 and 2). Based on structure and function, caspases are grouped into two main classes (reviewed in Refs.
3 and 4). The initiator caspases, including caspase-2, have a long
prodomain that mediates their autoactivation early during apoptosis.
The effector caspases, including caspase-3, -6, and -7, have short
prodomains and require cleavage by initiator caspases for their
activation. Effector caspases are primarily responsible for the
cleavage of a large number of cellular substrates which leads to the
morphological changes that characterize apoptotic cell death
(1-4).
The long prodomain of caspase-2 contains a caspase recruitment domain
(CARD),1 a six Caspase-2 has been shown recently (17-20) to be the most proximal
caspase in the caspase cascade activated during stress signaling. It
appears to be required for mitochondrial permeabilization and the
release of apoptogenic factors such as cytochrome c and
Diablo (17-20). Although adaptor proteins have been described for most initiator caspases, an in vivo adaptor for caspase-2 has yet
to be identified. Via its CARD, caspase-2 can interact with the
CARD-containing proteins RAIDD (21, 22) and ARC (23), although the
physiological significance of these interactions remains to be
confirmed. Following gel filtration, endogenous caspase-2 elutes in
high molecular weight fractions of cell lysates incubated at 37 °C,
suggesting that caspase-2 has the ability to be recruited to and/or
form a large protein complex in vivo, in a similar manner to
the Apaf-1-mediated caspase-9 apoptosome (24).
One of the most distinguishing features of caspase-2 is its ability to
localize to the nucleus constitutively (7, 25-28), although its role
in this subcellular compartment is not known. A recent study (27)
reports that caspase-2 is retained in the nucleus until the late stages
of apoptosis, suggesting that the nucleus is the site of procaspase-2
activation. Supporting this, another study has shown that in the
absence of cytosolic factors, the nuclear pool of caspase-2 is
sufficient to induce cytochrome c release from mitochondria
(18).
Previously, we have shown that caspase-2 localizes to the nucleus in a
prodomain-dependent manner (7). Deletion of a 44-amino acid
region containing a putative bipartite NLS was sufficient to interrupt
nuclear localization of caspase-2. However, this region alone was not
sufficient to direct nuclear import of GFP, suggesting the existence of
additional or alternative NLSs in the caspase-2 prodomain (7). NLSs
normally mediate nuclear entry by conferring interaction with the
cellular nuclear import machinery (reviewed in Ref. 29). The best
characterized pathway of nuclear protein import involves importin
This study focuses on dissecting the functional regions of the
caspase-2 prodomain that modulate nuclear transport and enzyme activation. By using various caspase-2 mutants, we define a classical NLS in the prodomain of mouse caspase-2 that contains a conserved Lys
residue critical for its function. This newly characterized NLS is
shown to be necessary and sufficient for nuclear transport of
caspase-2. We provide evidence that this NLS strongly interacts with
the importin Expression Constructs for Localization Studies--
WT and
catalytically inactive C320G mutant mouse caspase-2 GFP and C2PD-GFP
constructs have been described previously (7, 15, 26). Caspase-2 GFP
fusions containing various prodomain deletions were generated by PCR
amplification using Pfu polymerase, with either the
wild-type (WT) or the catalytically inactive mutant (C320G) caspase-2
(11, 12, 30) as a template and forward and reverse primers containing
XhoI and BamHI restriction sites, respectively.
Amplified products were subcloned into the GFP N-terminal fusion vector
pEGFP-N1 (Clontech). To generate prodomain deletion mutants PDM Constructs for Expression of Proteins in Escherichia
coli--
Bacterial expression plasmids encoding GFP-caspase-2 fusion
proteins were derived using the GatewayTM technology
(Invitrogen). DNA fragments encoding the caspase-2 prodomain (residues
19-167) or the caspase-2 linker (residues 122-167) were derived by
PCR using either wild-type or mutant (K152A) plasmid DNA templates and
appropriate primers. PCR fragments were gel-purified and then
recombined successively with plasmid pDON201 (BP clonase reaction) and
then pGFP-attC (LR clonase reaction) to yield the expression plasmids:
pGFP-caspase-2 (residues 19-167), pGFP-caspase-2 (residues 19-167,
K152A), pGFP-caspase-2 (residues 122-167), and pGFP-caspase-2
(residues 122-167, K152A). Plasmid pGFP-attC was derived previously
from plasmid pTRCAgfp (31) by restriction digestion and blunt end
ligation of the appropriate cassette and the Gateway Plasmid Conversion
Kit (Invitrogen) to enable an in-frame GFP fusion, containing a
His6 tag at the N terminus.
Plasmid Constructs for Immunoprecipitation
Studies--
HA-tagged full-length caspase-2, PDM Cell Culture and Transient Transfection--
NIH3T3 and COS
cells were maintained in Dulbecco's modified Eagle's medium and
HEK293T cells in 1640 RPMI, each supplemented with 10% fetal bovine
serum. For cell death assays, 2 × 105 NIH3T3 cells
were plated per 35-mm dish the day before transfection. GFP expression
constructs (2 µg of total DNA) were transfected into cells using the
FuGENE 6 reagent (Roche Molecular Biochemicals) according to the
manufacturer's protocol. Cells were scored for apoptotic morphology
24 h after transfection using a fluorescence microscope (Olympus
BH2-RFCA), as described previously (15, 16). Cells transfected with GFP
fusion constructs were fixed in 4% paraformaldehyde for 30 min and
then photographed using a camera mounted on the fluorescence
microscope. A confocal microscope (Bio-Rad) was used to capture images
for localization experiments. For immunoprecipitation experiments,
1.5 × 106 HEK293T cells were plated per 100-mm dish
the day before transfection. Cells were transfected with 4 µg of
total DNA (2 µg of each construct) as described above and harvested
24 h after transfection.
Protein Expression and Purification--
His-tagged GFP fusion
proteins were expressed essentially as described previously (31) under
denaturing conditions (8 M urea) followed by refolding on
the nitrilotriacetic acid-affinity column. Caspase fusion proteins
eluted at 200 mM imidazole. Proteins were subsequently
dialyzed, concentrated using Centricons (30,000 molecular weight
cut-off), and stored in small aliquots at Native Gel Electrophoresis--
Native gel electrophoresis
followed by fluorimaging was performed as described previously (31).
GFP fusion proteins (5-25 pmol), with or without preincubation (15 min, room temperature), with importins ( Immunoprecipitation--
For immunoprecipitation, cells were
lysed on ice in 50 mM Tris-HCl, pH 7.6, 150 mM
NaCl, 0.1% Nonidet P-40, and CompleteTM protease
inhibitors (Roche Molecular Biochemicals). Cellular debris was removed
by centrifugation at 4 °C, and antibody binding was carried out
overnight at 4 °C with 2 µg of an anti-GFP monoclonal antibody
(Roche Molecular Biochemicals). Antibody-protein complexes were
precipitated with protein G-Sepharose (Amersham Biosciences), and
precipitated proteins were resolved by SDS-PAGE and then transferred to
polyvinylidine difluoride membrane. Blots were probed with an anti-HA
monoclonal antibody (Roche Molecular Biochemicals) followed by anti-rat
horseradish peroxidase-coupled secondary antibody (Amersham
Biosciences). Signals were detected using the ECL system (Amersham Biosciences).
Yeast Two-hybrid Methods--
The components of the yeast
two-hybrid system were purchased from Clontech.
Caspase-2 lacking the first 44 amino acids (PDM The Caspase-2 Linker Region of the Prodomain Mediates Nuclear
Localization--
In previous studies (7) we have shown that nuclear
transport of caspase-2 is mediated by the prodomain region of the
molecule. When ectopically expressed, caspase-2 concentrates in the
nucleus of transfected cells in distinct structures resembling dots or filaments. A potential bipartite NLS identified in the N terminus of
the caspase-2 prodomain was shown by us to be necessary but not
sufficient for nuclear transport of caspase-2 (7), suggesting the
presence of an additional element(s) in the caspase-2 prodomain.
The caspase-2 prodomain is 169 amino acids in length and consists of a
34-residue N-terminal region (Met1-Pro34), a
CARD (Asp35-Thr121), and a 48-amino acid
region (Leu122-Asp169) linking the CARD to the
enzymatic domain encompassing the p18 and p14 subunits (Fig.
1A). Because the first 34 amino acids in the prodomain do not appear to be involved in any
specific function (7, 12, 13), we have divided the caspase-2 prodomain
into two regions consisting of the CARD
(Met1-Thr121) at the N terminus and the linker
region at the C terminus (Leu122-Asp169). For
localization studies, we generated a series of prodomain mutants
containing a GFP tag at the C terminus (Fig. 1B).
To test whether the caspase-2 CARD alone is capable of mediating
nuclear transport, we fused the mouse caspase-2 CARD directly to GFP
and assessed the localization of the fusion protein in transfected COS
cells. Unlike the caspase-2 prodomain-GFP fusion that forms elaborate
filaments in the nucleus (Fig.
2A), the CARD-GFP construct is
predominantly localized as perinuclear filamentous structures (Fig.
2B). Thus the CARD alone can not localize efficiently to the
nucleus in the absence of the linker region, despite the fact that it
contains the bipartite NLS. Indeed, when the CARD is fused directly to
the caspase domains, deleting the linker region (PDM
To test further the ability of the linker region to direct nuclear
transport, we fused the mouse caspase-2 linker region to caspase-3.
Addition of the caspase-2 linker region to caspase-3 was sufficient to
drive transport of the usually cytoplasmic caspase-3-GFP fusion protein
wholly to the nucleus (Fig. 2, E and F),
confirming that this region directs nuclear localization. Collectively,
our localization data point to the linker region of mouse caspase-2 as
containing an element(s) that is required for nuclear transport of
caspase-2.
The Caspase-2 Prodomain Contains a Functional NLS in the Linker
Region--
Our deletion mutant analysis implicated a role for the
linker region of the caspase-2 prodomain in mediating nuclear
transport. An examination of the sequence of the caspase-2 prodomain
linker region identified a putative NLS spanning residues 149-156 that is similar to the NLS of the c-Myc protein (29). This 8-amino acid
region contains conserved Pro, Lys, and Leu residues
(PPHKQLRL) that correspond to
identical residues in c-Myc and that are conserved in human, rat, and
mouse caspase-2 (Fig. 3A).
In order to determine whether this putative NLS is functional, we
carried out site-directed mutagenesis studies. By analogy with the well
characterized c-Myc NLS, we predicted that Lys152 would be
a critical residue. Mouse caspase-2 mutants were generated in which
this Lys152 residue was substituted with an Ala (K152A). In
all cases, catalytically inactive forms of each fusion protein were
used for localization studies to prevent cell killing. As shown in Fig.
3, K152A mutation resulted in a strikingly altered caspase-2
localization. Whereas caspase-2-GFP characteristically accumulates in
the nucleus forming dots or filaments (Fig. 3B), the mutant
caspase-2-GFP (K152A) was mostly localized outside and exclusive of the
nucleus, forming dot-like aggregates in the cytoplasm (Fig.
3C). Interestingly, the K152A mutant retains the ability to
form aggregates despite being restricted to the cytoplasm, suggesting
that nuclear localization is not necessary for caspase-2 to form
aggregates when overexpressed.
To determine whether mutation of Lys152 also affects
nuclear localization of the caspase-2 prodomain, we generated a mutant
caspase-2 prodomain-GFP (K152A) construct and transfected it into COS
cells. As mentioned before, caspase-2 prodomain-GFP forms elaborate
filament-like structures in the nucleus of transfected COS cells (Fig.
3D). Similar to the data obtained for the full-length
molecule, K152A mutation resulted in caspase-2 prodomain-GFP localizing
outside of the nucleus (Fig. 3E). The pattern of
localization of the prodomain (K152A) mutant resembled that of CARD-GFP
(compare Fig. 3E to Fig. 2B) with many cells
exhibiting filamentous structures that lie outside and often around the
nucleus. Thus as for the full-length mutant, caspase-2 prodomain-GFP
(K152A) is still able to form aggregates, in this case filament-like
structures, despite its impaired nuclear localization. We also
constructed a K152A mutant of the PDM The Caspase-2 NLS Is Recognized by Importin
CARD Is Necessary for Caspase-2 Localization to Dot-like and
Filamentous Structures--
As mentioned above, nuclear localization
does not appear to modulate the ability of full-length caspase-2,
caspase-2 prodomain, or the caspase-2 CARD to aggregate in dot- or
filament-like structures. In previous work defining the first NLS of
caspase-2, we observed that deletion of the first 44 amino acids of the
caspase-2 prodomain (a deletion that removes most of helix 1 of the
caspase-2 CARD and also deletes the putative bipartite NLS) not only
impaired nuclear localization of the prodomain but also resulted in the loss of dot- and filament-like structures. We hypothesized that the
first helix of the CARD is critical in mediating the ability of
caspase-2 to localize in dot- or filament-like structures. To
investigate this region further, we constructed deletion mutants of
full-length caspase-2, where either the first 25 amino acids or the
first 44 amino acids were deleted from caspase-2 and fused to GFP (Fig
5A). As shown before,
caspase-2-GFP forms dot-like structures in the nucleus (Fig.
5A). Removal of the first 25 amino acids had little effect
on both nuclear localization of caspase-2-GFP and the presence of dots
or filaments in the nucleus (Fig. 5B). However, deletion of
the first 44 amino acids resulted in loss of dot-like or filamentous
structures (Fig. 5C), defining residues 25-44 as important
for promoting the assembly of caspase-2-GFP into dots and filaments.
Because residues 35-44 comprise most of the first helix of CARD
including several basic residues likely to participate in electrostatic
interactions, we propose that the first helix of the caspase-2 CARD is
required for the ability of caspase-2 to form dot- or filament-like
structures.
Formation of Aggregates Is Not a Consequence of Caspase-2
Homodimerization--
Because the PDM
To confirm this result, we performed yeast two-hybrid analysis on cells
harboring pAS2.1 and pACT2 PDM An Intact CARD but Not Nuclear Localization Is Necessary for
Apoptosis-induced by Caspase-2 Overexpression--
Our data suggest
that aggregate formation that occurs upon caspase-2 overexpression
requires helix 1 of the CARD, although dimerization is not dependent on
this region. To determine whether the ability of caspase-2 to aggregate
is important for cell killing, NIH3T3 cells were transfected with
wild-type caspase-2 or the prodomain mutants, PDM
It could also be argued that the PDM By immunofluorescence, endogenous caspase-2 is primarily localized
to the nucleus and also the Golgi complex, showing a diffuse localization pattern (26, 28). In contrast, ectopically expressed caspase-2 is mostly nuclear, where it aggregates into structures resembling dots and filaments (7, 9, 27). By using various deletion
mutants of the caspase-2 prodomain, we have been able to assign
specific regions of the caspase-2 prodomain to the functions of
apoptosis, nuclear localization, and the formation of nuclear and
cytoplasmic aggregates (summarized in Table
I).
/
heterodimer. The
mutation of a conserved Lys residue in the NLS abolishes nuclear
localization of caspase-2 and binding to the importin
/
heterodimer. Although caspase-2 is imported into the nucleus, mutants
lacking the NLS were still capable of inducing apoptosis upon
overexpression in transfected cells. We define a region in the
prodomain that regulates the ability of caspase-2 to form dot- and
filament-like structures when ectopically expressed, which in turn
promotes cell killing. Our data provides a mechanism for caspase-2
nuclear import and demonstrate that association of procaspase-2 into
higher order structures, rather than its nuclear localization, is
required for caspase-2 activation and its ability to induce apoptosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical
motif shared by other initiator caspases including CED-3 in the worm,
DRONC in Drosophila, and caspase-9 in mammals (3-6). A
structurally and functionally related motif, the death effector domain
(DED), is found in the prodomains of some other initiator caspases
including caspase-8 and -10 (3, 4). The proximal activation of
initiator caspases in vivo is regulated by adaptor proteins
that, like the caspases, contain CARDs or DEDs (3, 4). In response to
an apoptotic stimulus, adaptor proteins bind to initiator caspases via
their CARDs or DEDs and recruit them to specific death complexes. High
local concentrations of procaspase molecules are generated, allowing
their activation and autoprocessing. When expressed ectopically,
however, activation of initiator caspases usually occurs spontaneously
because of high intracellular concentrations of caspases (3, 4). Upon overexpression in transfected cells, via its CARD-containing prodomain, caspase-2 associates into distinctive dot-like and filamentous nuclear
structures that may represent sites of caspase-2 homodimerization and
activation (7-9). In a similar fashion, the DEDs of caspase-8 and -10 mediate their formation into structures in the cytoplasm called death
effector filaments (10). Because of their propensity to homodimerize at
high concentrations, overexpression of initiator caspases, including
caspase-2, is sufficient to trigger apoptosis in transiently
transfected cells (11-13). In fact, enforced dimerization of effector
caspases is sufficient to trigger their activation (14-16).
/
recognition of "classical" NLSs, such as those described
for the SV40 large T-antigen and retinoblastoma tumor suppressor
protein (29). By binding to the NLS, importin-
captures the protein
to be imported, whereas importin-
targets this complex to the
nuclear pore (29).
/
heterodimer, whereas a single point mutation abolishes binding, suggesting that caspase-2 is likely to be imported into the nucleus via the well characterized classical pathway in
vivo. We also define a region in the prodomain at the N terminus of the CARD that regulates the formation of dots/filaments that correlate with the cell killing ability of caspase-2.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
25, PDM
44, and PDM
CARD, coding regions were
amplified using appropriate primers. An initiation codon was engineered in the forward primers immediately 5' of the codons encoding
Ile26, Val45, and Leu122,
respectively. To construct PDM
Link, which contains only the CARD
region (Met1-Thr121) and lacks the prodomain
linker region (Leu122-Asp169), CARD was
amplified and cloned into the XhoI and BamHI
sites of pEGFP-N1. Next, the region encoding the caspase-2 p18 and p14 subunits (the caspase domain) was amplified and subcloned into the
BamHI site of the CARD-GFP fusion construct resulting in an in-frame fusion of CARD with the caspase domain. Caspase-2 mutants containing a K152A substitution were created by PCR-based mutagenesis using WT or catalytically inactive caspase-2 GFP as templates. A
chimeric construct consisting of the caspase-2 prodomain linker fused
in-frame to the coding sequence of caspase-3 was constructed by PCR on
the previously described C2P-caspase-3
9-GFP plasmid template
(15).
25, and PDM
44
constructs were generated by amplifying DNA using the same forward
primers as used for the GFP fusions and a reverse primer incorporating an HA tag. All HA-tagged constructs were amplified using the caspase-2 C320G catalytically inactive mutant as template. Amplified products were cloned into the XhoI and EcoRV restriction
sites of the expression vector pcDNA3 (Invitrogen).
70 °C until use. Control
molecules used were His6-tagged GFP alone and SV40 large
T-antigen residues 111-135, including the importin
/
-recognized
NLS (PKKKRKV132), fused to GFP, expressed from a construct
generated in identical fashion to the caspase-2 constructs using the
GatewayTM technology, except that plasmid pPR28 (32) was
used as the PCR template. Mouse
1 and
1 importin proteins used in
binding experiments were expressed as GST fusion proteins and purified by affinity chromatography (33).
,
or predimerized
/
at a final concentration of 5-12.5 µM) in 25 µl were loaded onto 12% native polyacrylamide gels and
electrophoresed at 80 V for 4-7 h at 4 °C. Subsequently, the
positions of the GFP fusion proteins in the gel were determined by
fluorescent gel imaging using a Wallac Arthur 1422 Multi-wavelength Fluorimager, using side illumination, and exposure times of 0.25-2 s.
44) was amplified and
subcloned into the SmaI/BamHI restriction sites of the Gal4 DNA-binding domain vector, pAS2.1, and the Gal4 activation domain vector, pACT2. The pAS2.1-PDM
44 and pACT2-PDM
44 constructs were co-transformed into Saccharomyces cerevisiae strain
Y190 as was pAS2.1-PDM
44 and empty pACT2 vector and pACT2-PDM
44
with empty pAS2.1 vector. Colonies containing both vectors were
selected on SD medium lacking Leu and Trp as described previously (13). Interacting fusion proteins were screened for
-galactosidase activity in a colony-lift filter assay using
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
according to the instructions provided by Clontech.
Positive and negative controls for two-hybrid assays were as described earlier (13).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
The mouse caspase-2 prodomain.
A, the N-terminal region is shown in
italics, and the putative bipartite NLS is indicated.
The locations of the six -helices of the CARD are
underlined. The linker region, which lies between the CARD
and the caspase domain, is shown in gray. B,
schematic representation of caspase-2 GFP fusion proteins used in
localization and immunoprecipitation studies. The GFP tag is located at
the C terminus of caspase-2. Link, linker region (shown as a
black box); p18/p14, the caspase-2 catalytic
region.
Link), the GFP
fusion protein forms aggregates in the cytoplasm (Fig. 2C),
indicating that the CARD in conjunction with the caspase domains cannot
mediate nuclear transport. On the other hand, a GFP fusion lacking the
CARD but containing the linker region connected to the caspase domains
(PDM
CARD) localizes diffusely in the nucleus, in the absence of the
previously characterized NLS (Fig. 2D).
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Fig. 2.
The linker region of the prodomain mediates
nuclear localization of caspase-2. COS cells were transfected with
the indicated GFP constructs and 24 h later were fixed and
analyzed by confocal microscopy. A, prodomain-GFP;
B, CARD-GFP; C, PDM Link-GFP;
D, PDM
CARD-GFP; E,
caspase-3-GFP; and F, caspase-2Link + caspase-3-GFP.
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Fig. 3.
Identification of a classical NLS at
the C terminus of the caspase-2 prodomain. Alignment of amino acid
residues (149-156) of mouse caspase-2 with human and rat caspase-2 and
human c-Myc protein. A, conserved residues are
indicated in boldface type. COS cells transfected with the
indicated wild-type or mutant (K152A) GFP constructs were fixed and
analyzed using a confocal microscope. B, caspase-2-GFP;
C, caspase-2(K152A)-GFP; D,
prodomain-GFP; E, prodomain (K152A)-GFP;
F, PDM CARD-GFP; G,
PDM
CARD(K152A)-GFP.
CARD-GFP construct that
normally localizes diffusely in the nucleus (Fig. 3F). Once
again, mutation of Lys152 resulted in a localization
pattern exclusive of the nucleus (Fig. 3G). Collectively,
these mutagenesis studies clearly define another nuclear localization
signal in the mouse caspase-2 prodomain in which Lys152 is
essential for the nuclear localization of full-length caspase-2, the
caspase-2 prodomain, and a mutant of caspase-2 lacking the CARD.
/
-Dependent on Lys152--
NLSs normally
mediate nuclear entry by conferring interaction with the cellular
nuclear import machinery and, in particular, recognition by members of
the NLS-recognizing importin superfamily (29). The best understood
nuclear import pathways represent those mediated by either the importin
/
heterodimer, where importin
interacts with the NLS directly
or importin
alone (29). To assess the ability of caspase-2 to be
recognized by importins, GFP fusion protein expressing constructs were
derived for the caspase-2 prodomain (residues 19-167) and the
caspase-2 linker (residues 122-167) with or without the K152A
mutation. Proteins were purified by affinity chromatography and then
tested for their ability to bind to importin
, importin
, or the
importin
/
dimer using native gel electrophoresis and
fluorimaging. Both wild-type caspase-2 constructs were recognized by
both importin
alone and the importin
/
heterodimer, as
indicated by reduced mobility of the complexes of GFP fusion proteins
with importins in the gel (Fig. 4).
Binding of the importin
/
heterodimer resulted in a greater shift
in mobility than that of importin
alone due to the larger molecular
weight of the complex. Importin
, in contrast, did not show any
binding, consistent with the idea that caspase-2 contained a
conventional NLS recognized by importin
, the importin
/
heterodimer, and thus is completely comparable with the "classical"
nuclear import substrate SV40 large T antigen. Results for the NLS of
the latter fused to GFP in this respect are indicated in Fig. 4
(rightmost lanes and not shown); GFP alone did not show any
shift in mobility due to the addition of importins (not shown).
Experiments were also performed for caspase-2-GFP fusion protein
constructs containing the single point mutation K152A (Fig. 4), results
indicating a complete lack of binding of either importin
or the
importin
/
heterodimer to the mutant proteins. Clearly,
Lys152 is required for importin recognition of caspase-2,
consistent with the idea that Lys152 is a critical residue
within the second caspase-2 NLS. These results suggest that in
vivo caspase-2 nuclear localization is likely to be mediated by
the classical importin
/
-mediated nuclear import pathway.
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Fig. 4.
Caspase-2 NLS is recognized by importin
/
. Recognition of caspase 2 by
the importin
/
heterodimer as indicated by native gel
electrophoresis and fluorescent gel imaging. The indicated GFP fusion
proteins were incubated in the absence or presence of importins for 15 min at room temperature, prior to native electrophoresis on a 12%
polyacrylamide gel. After 5 h electrophoresis at 80 V, the gel was
imaged using a Fluorimager. The position of the different importin
(Imp)-GFP fusion protein complexes are indicated.
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Fig. 5.
A short region of CARD is necessary for
higher order structures but not for homodimerization of caspase-2.
COS cells transfected with the indicated GFP constructs were fixed and
analyzed using a confocal microscope. A, WT
caspase-2-GFP; B, PDM 25-GFP; C,
PDM
44-GFP. D, HEK293T cells were co-transfected with
GFP-tagged and HA-tagged caspase-2 expression constructs and lysed
24 h following transfection. Expression of HA-tagged proteins in
each lysate was checked by immunoblot analysis using an anti-HA
monoclonal antibody (top panel). GFP fusion proteins were
immunoprecipitated (IP) from each sample using an anti-GFP
monoclonal antibody. Co-precipitated HA-tagged proteins were detected
by immunoblot analysis using an anti-HA monoclonal antibody
(middle panel). Immunoprecipitation of GFP constructs was
checked by immunoblot analysis with an anti-GFP monoclonal antibody
(bottom panel). WB, Western blot.
E, PDM
44 Gal4 DNA binding domain and Gal4 activation
domain fusion constructs were co-transfected together, or with the
appropriate empty vector, into the S. cerevisiae Y190
strain. Colonies were assayed for
-galactosidase activity by colony
filter assay using X-gal as a substrate.
44 mutant lacks most of helix
1 of the CARD, we hypothesized that the loss of aggregate formation may
reflect an inability of the PDM
44 mutant to homodimerize via its
CARD. We have demonstrated in yeast that caspase-2 can homodimerize in
a prodomain-dependent manner (13). Co-immunoprecipitation experiments were carried out to evaluate whether the PDM
44 mutant could homodimerize in mammalian cells. GFP-tagged caspase-2 constructs were co-transfected with equal amounts of HA-tagged constructs and GFP
fusions precipitated with an anti-GFP monoclonal antibody. As expected,
both wild-type caspase-2 and the PDM
25 mutants co-precipitated their
HA-tagged counterparts (Fig. 5D), demonstrating that
caspase-2 can homodimerize in mammalian cells. Surprisingly, however,
the PDM
44 mutant was also able to homodimerize, despite lacking part of the first helix of CARD. The ability of PDM
44 to homodimerize in
mammalian cells suggests that homodimerization between two caspase-2
molecules does not require helix 1 of the CARD and that caspase-2
homodimerization does not correlate with aggregate formation.
44 fusion constructs. Fig.
5E shows that co-expression of pAS2.1 and pACT2 fusion
constructs results in a positive
-galactosidase assay (bottom
panel), whereas cells harboring either fusion construct with the
corresponding empty vector did not contain
-galactosidase activity
(top and middle panels). Thus whereas PDM
44 is
unable to form aggregates it is still capable of homodimerization. This
indicates that homodimerization between two caspase-2 molecules is not
necessarily sufficient in itself to mediate aggregate formation.
25 or PDM
44, and
assessed for the presence of apoptotic morphology 24 h later. As
shown previously, overexpression of caspase-2 kills NIH3T3 cells very
efficiently (>95% death within 24 h) (Fig.
6). The PDM
25 mutant is slightly less
effective than wild-type caspase-2, inducing cell death of ~70% of
transfected cells. Interestingly, the PDM
44 mutant kills transfected
cells very inefficiently with less than 20% of transfected cells
displaying apoptotic morphology at this time point. Thus an intact CARD
is essential for the formation of higher order caspase-2 structures as
well as its ability to kill efficiently transfected cells, suggesting
that formation of dots or filaments correlates with cell killing
potential, at least in the context of the precursor molecule.
View larger version (13K):
[in a new window]
Fig. 6.
A short region of CARD is necessary for cell
killing, but nuclear localization is not required for cell
killing. NIH3T3 cells transfected with the indicated GFP
constructs were fixed and scored for apoptotic morphology using a
fluorescence microscope 24 h following transfection. Data (± S.E.) are means of two separate experiments performed in
duplicate.
44 mutant cannot kill due to its
impaired nuclear localization. However, the PDM
Link caspase-2 mutant
that lacks the newly identified NLS and does not localize to the
nucleus was still able to induce significant levels of cell death (Fig.
6). In addition, the K152A mutants with impaired nuclear localization
can also kill transfected cells at comparable levels to the wild-type
versions (data not shown). Thus it is unlikely that impaired nuclear
localization per se of the PDM
44 mutant can explain its
inability to kill transfected cells. This is consistent with the
observation that caspase-2 aggregate formation still occurs in the
absence of nuclear localization.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A summary of the results obtained using various caspase-2
expression constructs
Our previous studies had identified a putative bipartite NLS that
resides at the N terminus of caspase-2 and overlaps the first helix of
the CARD (7). Deletion of this region results in the loss of nuclear
localization as well as a loss of higher order structures, but it is
not sufficient to direct nuclear transport of GFP (7). In this study we
identify and characterize a second NLS present in the linker region of
the caspase-2 prodomain. Our data show that in contrast to the
previously proposed NLS, this signal is sufficient to drive nuclear
import of another protein that normally resides in the cytoplasm. We
identify a critical Lys residue within this NLS that is absolutely
required for nuclear transport of caspase-2, suggesting that this
sequence represents a genuine, functional NLS. Mutation of this Lys
residue abolishes interaction with importin and an importin
/
heterodimer in vitro, suggesting a mechanism for
NLS-mediated caspase-2 nuclear import. Given that this second NLS is
sufficient to mediate nuclear transport, the role of previously
identified N-terminal bipartite NLS is currently unclear.
Our findings of the second NLS are consistent with a recent study (27)
that reported a homologous sequence in the human caspase-2 prodomain.
Two Lys residues were mutated in this sequence to demonstrate a
functional role in nuclear transport of caspase-2. Comparison of the
human and mouse caspase-2 NLS sequences reveals the presence of only a
single Lys in the mouse sequence that is also conserved in the rat.
This Lys corresponds to the same position of the critical Lys in the
c-Myc NLS. Thus by first establishing the conserved nature of the
caspase-2 NLS, we have been able to distinguish exactly what Lys
residue is the most critical component of this signal. More
importantly, we have been able to propose a mechanism for caspase-2
nuclear import in vivo by demonstrating interaction of the
caspase-2 NLS with the importin- moiety of the importin
/
heterodimer. Together our results suggest that caspase-2 is imported
into the nucleus via this NLS by the same mechanism used by well
characterized nuclear import substrates such as the SV40 T antigen,
c-Myc oncoprotein, and the retinoblastoma tumor suppressor
protein (29).
What is the role of caspase-2 in the nucleus? Surprisingly, the caspase-2 mutants defective in nuclear localization are still able to form aggregates and induce cell death upon overexpression, suggesting that nuclear localization may be dispensable for caspase-2 function. As these data were obtained using ectopically expressed protein, it remains formally possible that endogenous nuclear caspase-2 serves a specific function in apoptosis. One possibility is that caspase-2 may be sequestered in the nucleus to prevent its unfettered activation, given its propensity to oligomerize and autoactivate. As we demonstrate in this study, however, caspase-2 forms dots and filaments in the nucleus that correlate with cell killing ability, suggesting that this is the site for procaspase-2 activation. Supporting this, a recent study shows that caspase-2 is retained in the nucleus until the late stages of cell death and that apoptosis can be triggered by caspase-2 overexpression in the presence of leptomycin B, an inhibitor of nuclear export (27).
With these studies in mind, caspase-2 may serve as a sensor in the nucleus for receiving signals that are transmitted via this subcellular compartment. If true, we would predict that caspase-2 must cleave at least one nuclear substrate to trigger cell death. It is therefore puzzling that caspase-2 overexpression still kills cells when nuclear localization of caspase-2 is abrogated, suggesting that there must be a similar caspase-2 substrate in the cytoplasm. In one study, Bid translocation to mitochondria and cytochrome c release occurred while caspase-2 remained in the nucleus suggesting that nuclear caspase-2 causes mitochondrial permeabilization indirectly (27). In contrast, other studies (18, 19) have shown that either nuclear or recombinant caspase-2 is sufficient to cause cytochrome c release from isolated mitochondria directly. Thus, caspase-2 seems to be able to induce mitochondrial permeabilization both indirectly from the nucleus as well as directly when supplied exogenously.
Deletion of the first 44 amino acids of caspase-2 resulted in caspase-2-GFP localizing diffusely in the cytoplasm without forming dots and filaments. Although disrupted in its nuclear localization, we have shown that nuclear localization is not required for caspase-2 to form higher order structures. Loss of higher order structure formation correlated with a loss of cell killing ability, suggesting that formation of dots/filaments is a visual indicator of caspase-2 activity and consequent cell death. Surprisingly, despite its inability to aggregate into higher order structures and its inability to kill cells, this mutant was still capable of homodimerization. These observations suggest that although caspase-2 aggregation into a large complex is mediated by the CARD, caspase-2 dimerization can occur in the absence of the prodomain, i.e. caspase-2 dimerization and aggregation are distinct events.
One possibility is that in overexpressing cells, formation of
aggregates represents oligomers of dimerized procaspase-2 molecules. Supporting this idea, gel filtration analysis shows that purified recombinant caspase-2 elutes as an oligomeric complex, the formation of
which is dependent on the
prodomain.2 This suggests
that caspase-2 may not require an adaptor molecule for its
oligomerization and activation as high localized concentrations in vivo would be sufficient to form a large complex, and
formation of a large complex may be sufficient for caspase-2
autoactivation (24). Thus CARD-dependent aggregation is a
likely mechanism for procaspase-2 activation and apoptosis induced by
caspase-2 overexpression. As caspase-2 activation occurs rapidly in
response to a variety of cell death signals (34), oligomerization may be an efficient mechanism to mediate activation in vivo.
However, the mechanisms that facilitate and control caspase-2
oligomerization in vivo remain to be discovered.
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ACKNOWLEDGEMENTS |
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We thank members of our laboratory for sharing reagents and helpful discussions and Ghafar Sarvestani for help with confocal microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council of Australia and the Cancer Council of South Australia.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.
To whom correspondence should be addressed: Hanson Institute,
P. O. Box 14, Rundle Mall, Adelaide, South Australia, 5000, Australia.
Tel.: 61-8-8222-3738; Fax: 61-8-8222-3139; E-mail: sharad.kumar@imvs.sa.gov.au.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M211512200
2 S. H. Read, B. C. Baliga, and S. Kumar, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
CARD, caspase recruitment domain;
NLS, nuclear localization signal;
GFP, green fluorescent protein;
DED, death effector domain;
WT, wild type;
PDM, caspase-2 prodomain;
HA, hemagglutinin;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Nicholson, D. W. (1999) Cell Death Differ. 6, 1028-1042[CrossRef][Medline] [Order article via Infotrieve] |
2. | Wang, X. (2000) Gene Dev. 15, 1060-1066 |
3. | Kumar, S., and Colussi, P. A. (1999) Trends Biochem. Sci. 24, 1-4[CrossRef][Medline] [Order article via Infotrieve] |
4. | Shi, Y. (2002) Mol. Cell 9, 459-470[Medline] [Order article via Infotrieve] |
5. |
Dorstyn, L.,
Colussi, P. A.,
Quinn, L. M.,
Richardson, H.,
and Kumar, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4307-4312 |
6. | Lamkanfi, M., Declercq, W., Kalai, M., Saelens, X., and Vandenabeele, P. (2002) Cell Death Differ. 9, 358-361[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Colussi, P. A.,
Harvey, N. L.,
and Kumar, S.
(1998)
J. Biol. Chem.
273,
24535-24542 |
8. | Kumar, S. (1999) Cell Death Differ. 6, 1060-1066[CrossRef][Medline] [Order article via Infotrieve] |
9. | Shikama, Y., Miyashita, T., and Yamada, M. (2001) Exp. Cell Res. 264, 315-325[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Siegel, R. M.,
Martin, D. A.,
Zheng, L., Ng, S. Y.,
Bertin, J.,
Cohen, J.,
and Lenardo, M. J.
(1998)
J. Cell Biol.
141,
1243-1253 |
11. | Kumar, S., Kinoshita, M., Noda, M., Copeland, N. G., and Jenkins, N. A. (1994) Genes Dev. 8, 1613-1626[Abstract] |
12. | Kumar, S., Kinoshita, M., Dorstyn, L., and Noda, M. (1997) Cell Death Differ. 4, 378-387[CrossRef] |
13. |
Butt, A. J.,
Harvey, N. L.,
Parasivam, G.,
and Kumar, S.
(1998)
J. Biol. Chem.
273,
6763-6768 |
14. |
MacCorkle, R. A.,
Freeman, K. W.,
and Spencer, D. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3655-3660 |
15. |
Colussi, P. A.,
Harvey, N. L.,
Shearwin-Whyatt, L. M.,
and Kumar, S.
(1998)
J. Biol. Chem.
273,
26566-26570 |
16. | Shearwin-Whyatt, L., Baliga, B., Doumanis, J., and Kumar, S. (2001) Biochem. Biophys. Res. Commun. 282, 1114-1119[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Lassus, P.,
Opitz-Araya, X.,
and Lazebnik, Y.
(2002)
Science
297,
1352-1354 |
18. |
Robertson, J. D.,
Enoksson, M.,
Suomela, M.,
Zhivotovsky, B.,
and Orrenius, S.
(2002)
J. Biol. Chem.
277,
29803-29809 |
19. |
Guo, Y.,
Srinivasula, S. M.,
Druilhe, A.,
Fernandes-Alnemri, T.,
and Alnemri, E. S.
(2002)
J. Biol. Chem.
277,
13430-13437 |
20. |
Kumar, S.,
and Vaux, D. L.
(2002)
Science
297,
1290-1291 |
21. | Duan, H., and Dixit, V. M. (1997) Nature 385, 86-89[CrossRef][Medline] [Order article via Infotrieve] |
22. | Shearwin-Whyatt, L. M., Harvey, N. L., and Kumar, S. (2000) Cell Death Differ. 7, 155-165[CrossRef][Medline] [Order article via Infotrieve] |
23. | Dowds, T. A., and Sabban, E. L. (2001) Cell Death Differ. 8, 640-648[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Read, S. H.,
Baliga, B. C.,
Ekert, P. G.,
Vaux, D. L.,
and Kumar, S.
(2002)
J. Cell Biol.
159,
739-745 |
25. | Zhivotovsky, B., Samali, A., Gahm, A., and Orrenius, S. (1999) Cell Death Differ. 6, 644-651[CrossRef][Medline] [Order article via Infotrieve] |
26. | O'Reilly, L. A., Ekert, P., Harvey, N., Marsden, V., Cullen, L., Vaux, D. L., Hacker, G., Magnusson, C., Pakusch, M., Cecconi, F., Kuida, K., Strasser, A., Huang, D. C., and Kumar, S. (2002) Cell Death Differ. 9, 832-841[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Paroni, G.,
Henderson, C.,
Schneider, C.,
and Brancolini, C.
(2002)
J. Biol. Chem.
277,
15147-15161 |
28. |
Mancini, M.,
Machamer, C. E.,
Roy, S.,
Nicholson, D. W.,
Thornberry, N. A.,
Casciola-Rosen, L. A.,
and Rosen, A.
(2000)
J. Cell Biol.
149,
603-612 |
29. | Jans, D. A., Lam, M. H. C., and Xiao, C.-Y. (2000) Bioessays 22, 532-544[CrossRef][Medline] [Order article via Infotrieve] |
30. | Dorstyn, L., and Kumar, S. (1997) Cell Death Differ. 4, 570-579[CrossRef] |
31. |
Forwood, J. K.,
Harley, V.,
and Jans, D. A.
(2001)
J. Biol. Chem.
276,
46575-46582 |
32. | Rihs, H.-P., Jans, D. A., Fan, H., and Peters, R. (1991) EMBO J. 10, 633-639[Abstract] |
33. |
Hu, W.,
and Jans, D. A.
(1999)
J. Biol. Chem.
274,
15820-15827 |
34. |
Harvey, N. L.,
Butt, A. J.,
and Kumar, S.
(1997)
J. Biol. Chem.
272,
13134-13139 |