Correspondence to: Gergely L. Lukacs, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. Tel:(416) 813-5125 Fax:(416) 813-5771 E-mail:glukacs{at}sickkids.on.ca.
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Abstract |
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Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CADICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3dependent regulation of CAD activity takes place.
Key Words: apoptosis, chromosomal DNA degradation, caspase-activated DNase, nuclear targeting, caspase-3
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Introduction |
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Apoptosis is an essential process that controls cell numbers during development and participates in the elimination of cells that have undergone irreparable genomic damage (
The DNA fragmentation factor consists of two subunits, the 40-kD caspase-3activated DNase (CAD1 or DFF40) and the 45-kD inhibitor of CAD (ICAD or DFF45;
Heterodimerization of ICAD/CAD is obligatory not only to prevent chromosomal DNA degradation in growing cells, but also to ensure co- or posttranslational folding of CAD in the cytoplasm (
While compelling evidence has characterized the endonuclease activity of mouse CAD (mCAD) and its human orthologue (hCAD;
To better understand the subcellular compartmentalization of the apoptotic DNase, our objectives were to establish the cellular distribution and to elucidate the targeting determinants of the CADICAD complex. Immunolocalization of the endogenous ICAD with two polyclonal antibodies provides the direct evidence for the constitutive nuclear targeting of ICAD. Using deletional mutagenesis and fusion proteins, we have identified an NLS at the COOH terminus of ICAD and confirmed the function of the NLS of CAD. The two NLSs appear to contribute in an additive manner to the nuclear targeting of the ICADCAD heterodimer, implying that the caspase-3dependent activation of CAD takes place in the nucleus.
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Materials and Methods |
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Cell Lines and Transfection
HeLa, COS-1, MCF7, HTE, PANC, MDCK, CHO, and BHK-21 cells were grown in -modified Eagle's or DME medium supplemented with 10% FCS at 37°C under an atmosphere of 5% CO2. Transient transfections were performed with the calcium phosphate precipitation method, Effecten (QIAGEN), or Fugene (Roche) on 6070% confluent cells. Cells were harvested after 48 h of transfection.
To generate HeLa cells stably expressing hICAD-C-myc, cells were transfected with the pcDNA3-hICAD-C-myc expression vector as calcium phosphate precipitates, and selected in the presence of 0.5 mg/ml G418 (Geneticin; GIBCO BRL). Clones were screened by indirect immunostaining using the mouse monoclonal anti-myc (9E10, Covance Research Products Inc.) antibody.
Plasmid Constructions
The cDNA of human ICAD was isolated by PCR cloning, using a cDNA library prepared from Caco-2 cells (American Type Culture Collection, accession number HTB37) as a template. The cDNAs encoding the mCAD and hCAD were provided by Dr. S. Nagata (Osaka University Medical School, Osaka, Japan) and Dr. R. Halenbeck (Chiron Corp., Emeryville, CA), respectively. The membrane-targeted EGFP construct, encoding EGFP and the ras farnesylation site (EGFPF) and the cDNA of procaspase-3 were the gift of Dr. W. Jiang (265-330 (ICAD-S), ICAD
306-331 (ICAD
NLS), mCAD
329-344 (CAD
NLS), and hCAD
329-338 (hCAD
NLS) were generated by PCR mutagenesis. cDNA of fusion proteins, comprising EGFP (
Bacterial Expression of hICAD and mCAD
To generate polyclonal anti-ICAD antibody, the full-length coding region of hICAD was fused in-frame with GST in the pGEX-4T1 vector and transformed in HB101 bacteria. Production of the fusion protein was induced with 0.1 mM isopropyl ß-D-thiogalactopyranoside. The bacteria suspension was lysed by sonication in 0.5 M NaCl, 20 mM Hepes, 10% glycerol, 0.1 mM EDTA, and 1 mM DTT, pH 7.5. GST-hICAD was purified from the soluble fraction using glutathione Sepharose 4B (Sigma Chemical Co.), eluted with sonication buffer supplemented with 10 mM reduced glutathione, and further purified with SDS-PAGE. Gel slices, containing GST-hICAD were crushed for immunization of rabbits. Recombinant hICAD, hICAD-His6, and mCAD-His6 were expressed in BL21(DE3) cells using the pET15b (Novagen) expression plasmid and purified according to the supplier's recommendations using metal affinity chromatography.
Polyclonal Antibody Production
Purified GST-hICAD fusion protein was sent to Harlam Bioproducts for Science for inoculation into rabbits. Immunization was achieved with four boost of injections (0.5 mg protein/rabbit). The specificity of the rabbit antibodies was determined by comparing the activity of the immune and preimmune serum. For immunoblotting and immunofluorescence, the antibody was used at 1:1,0001:3,000 dilution, respectively.
Immunofluorescence Microscopy
Fluorescence staining of transfected and nontransfected cells was carried out on glass coverslips after fixing (4% paraformaldehyde for 20 min) and permeabilizing (0.2% Triton X-100 in PBS for 5 min) the cells as previously described (
Immunoblotting and Coimmunoprecipitation
For immunoblottings, cells were washed with ice-cold PBS and lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0) containing 10 µg/ml of leupeptin and pepstatin, 10 mM iodoacetamide, and 1 mM PMSF for 20 min at 4°C. Nuclei and unbroken cells were removed by centrifugation (15,000 g for 15 min at 4°C). Soluble proteins were denatured in Laemmli sample buffer, separated with SDS-PAGE, and transferred to a nitrocellulose membrane. Immunoblotting, using anti-HA, anti-myc, or anti-hICAD primary antibodies and enhanced chemiluminescence Western blot kit (Amersham), were performed as previously described (
Detection of Apoptosis
TUNEL assay was performed as previously described (
Online Supplemental Materials
Supplemental Figure 1.
Exogenous hICAD is confined to the nucleus at a low level of expression. HeLa cells were transiently transfected with hICAD-C-myc and immunostained with anti-myc and polyclonal anti-ICAD antibodies. (Available at http://www.jcb.org/cgi/content/full/150/2/321/DC1)
Supplemental Figure 2.
Expression level of ICAD-S and ICAD-L. (a and b) The expression level of ICAD-S relative to ICAD-L was determined with quantitative immunoblotting. (c) The weak cytosolic immunostaining of endogenous ICAD is eliminated in HeLa cells expressing exogenous mCAD-N-HA, presumably because of the nuclear translocation of the hICAD-S/mCAD complex. (Available at http://www.jcb.org/cgi/content/full/150/2/321/DC1)
Supplemental Figure 3.
The NLSs of both hCAD and hICAD contribute to the nuclear accumulation of the ICADCAD complex. Coimmunolocalization of full-length or truncated hCAD-N-HA and hICAD-C-myc was carried out as depicted on Fig 7 a. (Available at http://www.jcb.org/cgi/content/full/150/2/321/DC1)
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Supplemental Figure 4.
Distribution of endogenous and exogenous hICAD in HeLa cells after biochemical fractionation. The ICAD content of nuclear (nu), microsomal (mi), and cytosolic (cy) fractions of HeLa cells, obtained by differential centrifugation, were determined with immunoblot analysis. (Available at http://www.jcb.org/cgi/content/full/150/2/321/DC1)
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Results |
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Localization of ICAD in Nonapoptotic Cells
The subcellular localization of endogenous ICAD was examined using a polyclonal anti-hICAD antibody, developed by immunizing rabbits with purified GST-hICAD fusion protein. Western blot analysis showed that the rabbit anti-hICAD immune serum, but not the preimmune serum, recognizes recombinant hICAD with an apparent molecular mass of ~45 kD (Fig 1 a), corresponding to the predicted molecular mass of hICAD (
Two major polypeptides were recognized by the anti-hICAD immune serum, but not by the preimmune serum, in whole cell lysates of HeLa, COS-1, MCF7, HTE, and PANC cells, all of human or primate origin (Fig 1 b). The slower migrating band, with an apparent molecular mass of ~45 kD, corresponds to the predicted mass of the full-length hICAD (ICAD-L). The less abundant polypeptide, with an apparent molecular mass of ~36 kD, is consistent in size with the alternatively spliced variant, hICAD-S (
The subcellular distribution of endogenous hICAD was established with indirect immunofluorescence microscopy in five cell lines, including HeLa, HTE, PANC, COS-1, and MCF7, using two independent anti-ICAD antibodies. The representative fluorescence micrographs of HeLa and HTE cells show that hICAD resides predominantly within the nucleus and is excluded from the nucleolus, with a consistently weak fluorescence signal in cytoplasm (Fig 2, a and b). No nuclear staining was detectable with the rabbit preimmune serum or after the adsorption of the specific antibody to recombinant ICAD-His6 (Fig 2 a). Importantly, the strong nuclear staining of ICAD could be observed with the affinity-purified goat polyclonal anti-ICAD antibody, as illustrated in the HeLa cells (Fig 2 c). These data indicate that hICAD constitutively accumulates in the nucleus in a variety of cells with the maintenance of a reduced level in the cytoplasm.
The Nuclear Localization Signal of ICAD
The nuclear import ICAD could be achieved passively in association with CAD or another carrier molecule. Alternatively, an unrecognized NLS residing in ICAD could mediate nuclear uptake. Primary sequence analysis of the mouse and human ICAD revealed two series of basic residues separated by a 10amino acid spacer at the extreme COOH terminus, which is reminiscent of the consensus sequence of the bipartite NLS of nucleoplasmin (Fig 3 a;
Overexpressed hICAD consistently appeared largely nuclear, with a low level of cytosolic expression in different expression systems (HeLa, COS-1, and BHK), regardless of the epitope (c-myc, flag, or HA [influenza hemagglutinin]) introduced at the COOH or NH2 termini (Fig 3 c, top and data not shown). Several lines of evidence indicate that NLS-dependent active uptake accounts for the nuclear localization of overexpressed hICAD. First, the nuclear accumulation of hICAD was clearly compromised without NLS. Strong cytosolic immunostaining was detected for hICADNLS-C-myc, a recombinant version of hICAD prepared by deleting the last 26amino acid residues, comprising the NLS consensus (Fig 3 c, bottom). Further, prominent cytosolic retention was observed for the epitope-tagged ICAD-S, representing the alternatively spliced isoform, which lacks the last 66 amino acid residues (Fig 3 c, bottom right), suggesting that the nuclear import of exogenous hICAD cannot be attributed to association with CAD. Secondly, both stably and transiently transfected hICAD-C-myc was predominantly nuclear at expression levels, which were comparable to that of endogenous ICAD in HeLa cells (Fig 4 a, left, and Figure S1 [available at http://www.jcb.org/cgi/content/full/150/2/321/ DC1]). Finally, depletion of the cellular ATP content by metabolic inhibitors dissipated the nucleocytosolic gradient of hICAD-C-myc (Fig 4 a, middle), supporting the hypothesis that nuclear accumulation of hICAD involves active transport. Irreversible deterioration of the nuclear envelope could not account for this phenomenon, since hICAD-C-myc reconcentrated in the nuclei upon the recovery of the cellular ATP content (Fig 4 a, right). These observations are consistent with the activity of a previously unrecognized NLS at the COOH terminus of hICAD. Furthermore, they also suggest that the weak cytosolic immunostaining, obtained with the polyclonal anti-ICAD antibodies (Fig 2), is, most likely, because of the presence of low levels of monomeric ICAD-S, confined to the cytoplasm (Figure S2, a and b [available at http://www.jcb.org/cgi/content/full/150/2/321/DC1]). Supporting this notion, the cytosolic immunostaining of ICAD-S was virtually abolished upon transient expression of CAD-N-HA, with a concomitant increase in the nuclear immunostaining of ICAD (Figure S2, c).
The ICAD/CAD Heterodimer Is Nuclear in Nonapoptotic Cells
It has been established previously that co- or posttranslational heterodimerization of CAD and ICAD is necessary for the expression of CAD. The possible impact of heterodimerization on the subcellular targeting of ICAD was assessed by transient coexpression of epitope-tagged human ICAD and mouse CAD.
Three lines of evidence indicated that epitope-tagged human ICAD, a highly conserved orthologue of mouse ICAD (
The subcellular distribution of hICAD in the ICADCAD complex was investigated by immunofluorescence staining of HeLa cells, transiently cotransfected with epitope-tagged hICAD and mCAD. The nearly exclusive nuclear expression of heterologous hICAD and mCAD was independent of the epitope (Fig 6 a), which is consistent with the localization of endogenous hICAD (Fig 2). Similar subcellular distribution was observed in COS-1 and BHK cells (data not shown). Association or binding to nuclear constituents is unlikely to play a major role in the nuclear retention of ICAD/CAD. In contrast to the nuclear proteins, which are resistant to detergent extraction because of their association with the nucleoskeleton or chromosomal DNA (e.g., lamina-associated polypeptide 2, (
Nuclear accumulation of ICAD/CAD could not be attributed to activation of apoptosis, since neither increased chromosomal DNA fragmentation nor phosphatidylserine translocation into the outer leaflet of the plasma membrane was detectable by terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL) assay or annexin V staining, respectively. Finally, identical localization was demonstrated for the epitope-tagged hICAD/hCAD heterodimer (Fig 6 b), supporting our hypothesis that nuclear targeting of ICAD/CAD is an inherent characteristic of the complex, rather than the consequence of heterologous expression, and dimerization ICAD does not impair its nuclear accumulation.
The Role of ICAD in the Nuclear Targeting of the ICADCAD Complex
Polypeptides >45 kD require an NLS and interactions with nuclear transport receptors to be targeted specifically into the nucleus (NLS-C-myc heterodimer (Fig 7 a). Similar subcellular distribution of mCAD was observed in the presence of ICAD-S-C-myc (Fig 7 a), suggesting that the NLS of hICAD is necessary for the efficient nuclear accumulation of the CADICAD complex. Furthermore, these results also imply that nuclear uptake of CAD may be influenced by the expression level of ICAD-S. This latter possibility was verified by determining the subcellular distribution of mCAD in complex with hICAD-S. Nuclear accumulation of mCAD was observed only in 52% of the expressors and showed substantial cytosolic staining in the rest of the transfectants upon coexpression with hICAD-S (Fig 7 c, empty bars).
The possibility that hICADNLS or hICAD-S is unable to chaperone the folding of mCAD, leading to a nonnative, and perhaps mistargeted mCAD seems unlikely since the expression level of mCAD was augmented by either the full-length or truncated variants of hICAD more than six-fold, as measured by quantitative immunoblot analysis (Fig 8 a, lanes 14). Similar distribution patterns were observed when human CAD-N-HA was coexpressed with the full-length or truncated hICADs (Figure S3 [available at http://www.jcb.org/cgi/content/full/150/2/321/DC1]), precluding the possibility that dimerization of hICAD with mCAD induced the exposure of a buried NLS. Hence, we conclude that the NLS of hICAD has an important role in the constitutive nuclear localization of the ICAD/CAD heterodimer.
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The Role of CAD in the Nuclear Targeting of the ICADCAD Complex
To examine the possible role of CAD in the nuclear targeting of the ICADCAD complex, the function of its putative NLS was also evaluated. The COOH-terminal tail of mCAD, containing the positively charged amino acid cluster (326RRKQPARKKRPARKR344), was fused in-frame to EGFP. The transiently expressed EGFP-CAD326-344 chimeric polypeptide was nuclear in HeLa cells, indicating that the COOH terminus of mCAD is sufficient to confer nuclear import capacity to EGFP (Fig 8 b).
The significance of the NLS in the context of mCAD was demonstrated by immunolocalization of the COOH terminally truncated mCAD lacking its NLS (CADNLS). Deletion of the NLS disrupted the nuclear import of the CAD
NLS-N-HA as well as the coexpressed ICAD-C-myc (Fig 7 a). In contrast to the exclusive nuclear localization of mCAD and hICAD (Fig 6 and Fig 7, a and b, filled bar), the complex comprising the mCAD
NLS and hICAD remained cytosolic for >50% of the transfectants (Fig 7 c, filled bar). The rest of the transfectants displayed incomplete nuclear accumulation with significant amount of cytosolic mCAD
NLS-NHA (Fig 7 c). Importantly, deletion of the NLS in mCAD does not seem to interfere with its biosynthesis and folding, since the expression level of mCAD
NLS was comparable to that of mCAD, in the presence of either the full-length or the truncated hICADs (Fig 8 a, lanes 68). Furthermore, deletion of the putative NLS of the human CAD caused a similar subcellular distribution of the ICADCAD
NLS complex (Figure S3). Thus, perturbation of the nuclear localization of the ICAD/CAD heterodimer by CAD
NLS provides direct evidence for the role of CAD NLS. Therefore, we conclude that NLSs of both ICAD and CAD contribute to the constitutive nuclear import of the complex. Our results also imply that translocation of activated CAD from the cytoplasm into the nucleus is not occurring specifically to achieve fragmentation of chromosomal DNA during apoptosis.
The NLSs of ICAD and CAD Contribute to the Nuclear Uptake of ICADCAD Complex in an Additive Manner
To assess whether the NLS of each constituent participates in the nuclear targeting of the ICADCAD complex, the subcellular distribution of the heterodimer composed of mCADNLS and ICAD
NLS was examined. In striking contrast to the nuclear colocalization of mCAD-N-HA and hICAD-C-myc (Fig 9 a), the nuclear targeting of the complex lacking both NLSs was virtually abolished, as illustrated by the laser confocal fluorescent micrographs (Fig 9 b). Comparable results were obtained with the mCAD
NLS-N-HA/hICAD-S-C-myc complex (Fig 7 a). Nuclear accumulation of the mCAD
NLS-N-HA/hICAD-S-C-myc complex could not be recognized in 81% of the transfected cells, and appeared to be excluded from the nucleus in 19% of the expressors (Fig 7 b, empty bar). Since the cytosolic expression of the ICAD/CAD was substantially more pronounced when both NLSs were absent, in contrast to single NLS deletion, we conclude that the NLSs of both ICAD and CAD are required for efficient nuclear import of the heterodimer.
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Staurosporine-induced Apoptosis Is Associated with the Caspase-dependent Release of ICAD from the Nucleus
Immunolocalization of endogenous hICAD suggests that proteolytic activation of ICAD/CAD takes place in the nucleus, rather than in the cytoplasm as proposed previously (
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In sharp contrast to HeLa cells, the nuclear expression of the endogenous hICAD was preserved upon staurosporine treatment of MCF7 cells, a cell line which lacks functional caspase-3 (
The lack of a staurosporine effect on the degradation of ICAD in MCF7 cells could not be explained by the absence of the upstream apoptotic signaling cascade, since hICAD could be eliminated from the nuclei after the complementation of the cells with procaspase-3. MCF7 cells were cotransfected with plasmids encoding procaspase-3 and membrane-targeted EGFP (EGFPF) at a molar ratio of 4:1. The inclusion of EGFPF facilitated the identification of procaspase-3 expressors. Activation of procaspase-3 with staurosporine caused the disappearance of endogenous ICAD from the nuclei of MCF7 cells expressing both EGFPF and caspase-3, but not EGFPF alone, as detected with immunostaining (Fig 11). These in situ immunolocalization studies suggest that expression of caspase-3 is indispensable for proteolytic activation of ICAD/CAD, which takes place in the nucleus.
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Discussion |
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Based on in vitro reconstitution of chromosomal DNA degradation, it was initially postulated that proteolytic cleavage of ICAD by caspase(s) would allow the dissociation of ICAD from CAD in the cytosol and unmasking the NLS of CAD, permitting the nuclear import of the active nuclease (
A number of observations indicate that the ICAD/CAD heterodimer resides, predominantly, in the nucleus of nonapoptotic cells. A panel of epitope-tagged ICAD and CAD variants was expressed in combinations or individually to demonstrate that they localize and colocalize in the nucleus. Nuclear localization was independent of the expression system or the epitope employed, and did not coincide with either DNA fragmentation or phosphatidylserine translocation (Fig 4, Fig 6, and Fig 9). Moreover, inhibition of caspase activity with DEVD-CHO had no effect on the nuclear localization of exogenous ICAD/CAD or endogenous ICAD (data not shown). Finally and most importantly, we have demonstrated that endogenous hICAD is predominantly confined to the nucleus in diverse cell types with two independent polyclonal anti-hICAD antibodies (Fig 2). Although we were unable to determine the subcellular localization of endogenous CAD, given the obligatory heterodimerization of the inactive CAD with ICAD, these experiments strongly suggest that the ICADCAD complex is targeted constitutively into the nucleus.
One of the most important findings of our study is the elucidation of the nuclear targeting signals of the ICADCAD complex. These experiments relied on the observation that coexpression of hICAD ensured a dramatic increase in the level of heterologous CAD, enabling investigations of the role of the NLSs without significant interference from the endogenous ICAD/CAD. In addition to identifying the NLS of ICAD, we have functionally verified the NLS in both mCAD and hCAD using two criteria. First, the NLSs of mCAD and hICAD were transferable. Both EGFP-ICAD306-331 and EGFP-CAD326-344 chimeras were targeted to the nucleus, in contrast to the homogenous cellular distribution of the EGFP (Fig 3 b and 8 b). Second, the deletion of the bipartite NLS from the COOH terminus of hICAD manifested in the cytosolic accumulation of monomeric hICAD-S-C-myc and hICADNLS-C-myc as opposed to the full-length ICAD, which was predominantly nuclear (Fig 3 c).
The role of the NLS of CAD was assessed in cells cotransfected with hICAD and truncated CAD. Incorporation of mCADNLS, or hCAD
NLS into the heterodimer partially disrupted its nuclear accumulation, similar to that observed in the presence of hICAD
NLS, implying that the NLS of CAD is also recognized by the nuclear import machinery in the complex. Importantly, when the NLSs of both ICAD and CAD were deleted, the nuclear exclusion of mCAD
NLS/hICAD-S and mCAD
NLS/hICAD
NLS became obvious (Fig 9). The more pronounced cytosolic accumulation of the double mutants relative to that of single NLS deletion suggests that the two NLSs have an additive effect on the nuclear targeting efficiency of the ICADCAD complex. A cumulative effect of multiple nuclear localization signals on the targeting of soluble polypeptides is not without precedent. A number of proteins harbor two or more NLSs (e.g., c-myc, Mat
2, and p53), which are required to achieve complete nuclear localization. Multiple copies of NLS apparently ensure more efficient targeting than do single copies (
In the light of the nuclear localization of the ICADCAD complex, it is reasonable to assume that the DNA fragmentation activity, which was isolated from the cytosol of apoptotic cells, was released from the nucleus during the preparation procedure, when ATP-dependent nuclear import was mitigated (
Activation of the ICAD/CAD Heterodimer Occurs in the Nucleus
The constitutive nuclear accumulation of ICAD/CAD dimer implies that regulation of CAD activity, most likely, takes place in the nucleus. How nuclear ICAD/CAD is activated upon apoptosis is an important question in understanding the mechanism of chromosomal DNA degradation. Convincing in vivo and in vitro data demonstrate that activation of CAD requires the proteolytic cleavage of ICAD by the caspase cysteine proteases (
In the absence of evidence for the constitutive recycling of ICAD/CAD between the cytoplasm and the nucleus, the apoptotic signaling cascade must either trigger nuclear uptake of the heterotetrameric, activated caspase-3 or activate procaspase-3 within the nucleus to direct the proteolytic cleavage of the heterodimeric ICAD. While we are unable to distinguish between these possibilities at the present time, circumstantial and direct evidence suggest that nuclear translocation of both procaspases and active caspases can occur. Immunolocalization experiments have convincingly shown the nuclear import of caspase-9 in apoptotic neurons and suggested a similar process for caspase-3 in neuroblastoma cells (
What are the biological advantages of the constitutive accumulation of ICAD/CAD in the nucleus? First, programmed cell death is often associated with a loss of mitochondrial membrane potential and impaired ATP production by the respiratory chain (
In conclusion, the experimental data presented in this study indicate that two independent NLSs, identified in ICAD and CAD, are necessary and sufficient to render highly efficient, constitutive nuclear targeting of the ICAD/ CAD heterodimer. These observations, together with the nuclear accumulation of endogenous hICAD, and its redistribution on staurosporine-induced apoptosis in both HeLa and caspase-3 transfected MCF7 cells, imply that regulation of the apoptotic nuclease by the proteolytic cleavage of ICAD takes place in the nucleus.
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: CAD, caspase-3activated DNase; DFF, DNA fragmentation factor; EGFP, enhanced green fluorescent protein; GST, glutathione-S-transferase; hICAD-L, full-length human ICAD; hICAD-S, alternatively spliced variant of hICAD; ICAD, inhibitor of caspase-activated DNase; NLS, nuclear localization signal.
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Acknowledgements |
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We are grateful to Drs. S. Nagata, R. Halenbeck, V. Dixit, and W. Jiang for providing the cDNAs of mouse CAD, human CAD, procaspase-3, and EGFPF, respectively, and to Drs. S. Grinstein and M. Manolson for careful reading of the manuscript. We are indebted to Dr. R. Halenbeck for sharing unpublished results. We also thank Dr. H. O'Brodovich for his continuous support and Drs. D.W. Andrews and W. Trimble for helpful suggestions. The valuable assistance of K.-J. Sohn, J. So, and J. Sandu is highly appreciated.
This study was supported by the Medical Research Council (MRC) of Canada and the Canadian Cystic Fibrosis Foundation (CCFF; Sparx II Program). D. Lechardeur was supported, in part, by a CCFF Postdoctoral Fellowship. The instrumentation was partially covered by an Ontario Thoracic Society Block Term grant. J.M. Rommens and G.L. Lukacs are Scholars of MRC of Canada.
Submitted: 8 November 1999
Revised: 7 June 2000
Accepted: 7 June 2000
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References |
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