Article |
Address correspondence to Marcus Peter, The Ben May Institute for Cancer Research, University of Chicago, 924 E. 57th Street, Chicago, IL 60637. Tel.: (773) 702-4728. Fax: (773) 702-3701. E-mail: MPeter{at}ben-may.bsd.uchicago.edu
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Abstract |
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Key Words: apoptosis; caspases; DEDD; intermediate filaments; monoubiquitination
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Introduction |
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We recently identified a protein that we called death effector domain containing DNA binding protein (DEDD). Among apoptosis signaling molecules, DEDD is unique, both in its abundance and its conservation between species, indicating an essential involvement in an evolutionarily universal process (Stegh et al., 1998). DEDD contains three nuclear localization signals and binds DNA and chromatin with high affinity but predominantly resides in the cytoplasm, suggesting that DEDD functions as a communicator between the cytoplasm and nucleus (Stegh et al., 1998; Schickling et al., 2001). Similar to several other DED proteins, overexpression experiments suggested that DEDD was a proapoptotic molecule. In contrast to all other DED proteins, DEDD requires nuclear localization to induce apoptosis, suggesting that it engages a novel nuclear apoptosis pathway (Schickling et al., 2001). In the nucleoli, it activates and colocalizes with active caspase-6 and interacts with a novel DED binding protein DEDAF (Zheng et al., 2001).
We now show that in many cells cytosolic DEDD is expressed mostly in a diubiquitinated form which associates with both keratin 18 and procaspase-3. Upon induction of apoptosis, cytosolic DEDD first aggregates and then stains in filamentous structures associated with the cytokeratin 8/18 network in a strictly caspase-3dependent fashion. We also report the cloning of a close homologue of DEDD, which was recently reported as DEDD2 (Roth et al., 2002) or FLAME3 (Zhan et al., 2002). Using an antibody against the DED of DEDD2, we demonstrate that DEDD2 can also be stained on IFs in apoptosing cells. As apoptosis progresses, K18 is cleaved by caspase-3 and reorganizes into intracellular inclusions that colocalize with DEDD, DEDD2, and active caspase-3. Expression of a cytosolic dominant negative version of DEDD inhibits activation of caspase-3 and the subsequent cleavage of K18. Therefore, we conclude that DEDD is important for the degradation of the IF system by caspase-3 and execution of apoptosis in epithelial cells.
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Results |
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DEDD is a mono- and diubiquitinated protein
Two different anti-DEDD antibodies, PRO29 and DED3, detected three protein forms of DEDD at 54, 44, and the expected 37 kD (Fig. 3, A and B; unpublished data). These three bands were spaced by 8 kD, suggesting a covalent modification of 8 kD, the size of ubiquitin. Therefore, we tested whether the higher DEDD bands corresponded to mono and diubiquitinated DEDD. This analysis was complicated by the fact that the higher forms of DEDD cannot be directly immunoprecipitated from cellular lysates due to their insolubility (unpublished data). To test whether higher DEDD bands could be detected upon overexpression of DEDD, we transiently transfected 3'FLAG-tagged DEDD (DEDD-FLAG) into 293T cells. Transfection of DEDD-FLAG resulted in a weakly reactive 55-kD band (Fig. 3 D, lane 4, bottom). Upon longer exposure of the same immunoblot, a faster migrating band at 45 kD was also detected (Fig. 3 D, lane 4, top). These two FLAG reactive bands likely represented the FLAG-tagged versions of the endogenous 54- and 44-kD bands. Cotransfection of the same amounts of DEDD-FLAG in the presence of HA-tagged ubiquitin (Fig. 3 D, lanes 68) resulted in a much more efficient formation of FLAG-reactive bands migrating above DEDD-FLAG (Fig. 3 D, lane 8). The detected band migrated
4 kD slower than the highest band in cells transfected with DEDD-FLAG, consistent with DEDD-FLAG modified by two HA-ubiquitin (10 kD) rather than two endogenous ubiquitin (8 kD) peptides. These data strongly suggested that DEDD was a diubiquitinated protein. We also detected higher molecular weight forms of DEDD likely corresponding to higher order ubiquitinated DEDD (Fig. 3 D, lane 8, top, arrowheads). To confirm the formation of monoubiquitinated DEDD in 293T cells, we cotransfected cells with DEDD-FLAG and HA-ubiquitin and immunoprecipitated DEDD with anti-FLAG (Fig. 3 E). A positive signal with anti-HA in Western blotting confirmed the identity of the 46-kD band as monoubiquitinated DEDD (Fig. 3 E, right).
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Unlike polyubiquitination, the role of monoubiquitination does not appear to be to target proteins to the proteasome-mediated degradative pathway. In fact, treatment of cells with proteasome inhibitors has been reported to result in deubiquitination of monoubiquitinated proteins such as histone H2A and H2B (Mimnaugh et al., 1997) and the HIV-1 p6Gag protein (Schubert et al., 2000), likely due to the depletion of cells of free ubiquitin which gets bound to accumulating polyubiquitinated proteins. To test whether diubiquitinated DEDD would also be deubiquitinated in cells treated with proteasome inhibitors we transiently transfected 293T cells with HA-ubiquitin (resulting in detection of only diubiquitinated endogenous DEDD) and treated with 20 µM of MG132 for 12 h (Fig. 3 F, lane 6). A complete conversion of 55 kD DEDD into 37 kD DEDD was detected, consistent with DEDD being deubiquitinated. To confirm the existence of both the mono- and the diubiquitinated DEDD species in primary cells, we tested expression of DEDD in tissues of mice (Fig. 3 G). Kidney expresses mainly unmodified DEDD, and tongue and lung contain mainly mono- and diubiquitinated DEDD, respectively, and colon and lung expressed mixtures of different DEDD species. These data indicated tissue specificity in DEDD expression and ubiquitination.
We noticed that ubiquitinated DEDD is less soluble than unmodified DEDD (unpublished data) consistent with our observation that DEDD-FLAG was more efficiently immunoprecipitated than HA-Ubi-DEDD-FLAG (Fig. 3 E). Induction of apoptosis in Jurkat T cells by STS coincided with a disappearance of the 37-kD DEDD band in the detergent soluble fraction and an appearance of diubiquitinated DEDD in the detergent insoluble fraction (Fig. 3 H). This change in solubility was recapitulated in fractionation of HeLa cells (unpublished data). Further characterization of these fractions identified cytoskeletal proteins, such as keratin 8 (K8), specifically fractionated as detergent insoluble corresponding to the low solubility of keratins in NP-40. Thus, the newly formed DEDD2Ub cofractionated with K8 in HeLa cells indicating that DEDD2Ub possessed solubility properties of cytoskeletal proteins.
DEDD colocalizes and associates with cytokeratin 8/18
Apoptotic DEDD-positive filamentous structures strongly resembled filamentous structures formed by cytoskeletal proteins, suggesting that DEDD may interact with the cytoskeleton. To test this, we stained cells for DEDD, actin, microtubules, and cytokeratins. We did not detect a colocalization of DEDD with either actin or microtubules (unpublished data). However, DEDD appeared to colocalize with cytokeratins in STS treated MCF7-caspase-3 cells (Fig. 4 A). We chose to stain for cytokeratin 8 (K8), as it is known that K8, compared with K18, is much more resistant to caspase cleavage (Caulin et al., 1997; Ku et al., 1997). Interestingly, confocal microscopy indicated that not all IFs stained positive for DEDD, but virtually all DEDD-positive structures were stained with the K8 antibody. A similar result was obtained when cells were costained for DEDD and K18 (unpublished data). A costaining of DEDD with K18 in IF structures was also observed in HeLa cells treated with TRAIL for 2 h using electron microscopy and immunogold labeling (Fig. 4 B). Because DEDD is only detectable in apoptosing cells by microscopy, these experiments did not address whether DEDD was constantly associated with the IF system with the DED3 epitope becoming accessible during apoptosis or whether it associated with cytokeratins in a stimulation dependent manner. To determine whether DEDD was associated with K18 in nonapoptosing cells, we performed an immunoprecipitation experiment using an anti-K18 antibody with the detergent Empigen BB, reported to solubilize 40% of cellular cytokeratins in simple epithelial cell lines (Lowthert et al., 1995). In agreement with our cell fractionation results, DEDD coimmunoprecipitated with full length K18 (Fig. 4 C). Specifically, the coimmunoprecipitated band was DEDD2Ub and could again be detected with both anti-DEDD antibodies. When apoptosis was induced by treating these cells with etoposide for 48 h, K18 was completely degraded in these cells and consequently DEDD could no longer be immunoprecipitated with the anti-K18 antibody (Fig. 4 C, lane 8), suggesting that in these cells, DEDD2Ub stays bound to K18 throughout the apoptosis induction. Interestingly, we detected the appearance of higher forms of DEDD that could point to an apoptosis induced ubiquitination of DEDD during this treatment (Fig. 4 C, bottom, arrowhead).
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DEDD partially colocalizes with caspase-3cleaved K18
During apoptosis induced by various methods, many type I keratins are cleaved by caspases (Oshima, 2002) resulting in disintegration of the IF system. Two cleavage sites have been identified in K18 (Fig. 5 A). Asp238, in the center of the molecule is cleaved by both caspase-3/7 and caspase-6 and Asp397, at the COOH terminus, is specific for caspase-3/7 cleavage. Cleavage at D397 occurs early during apoptosis and generates a neoepitope reactive with the commercially available antibody M30, that can be used to monitor caspase-3 activation (Leers et al., 1999). Caspase-mediated cleavage of K18 (48.1 kD) generates 45-, 26.3-, and 19-kD fragments (Caulin et al., 1997; Ku et al., 1997). In both MCF7-vec and MCF7-C3 cells, STS treatment generated the 45 kD large fragment (D397 cleavage), which migrates close to the full-length K18, demonstrating that caspase-3 is not absolutely required for cleavage at D397 (Fig. 5 B, lanes 2, 4, 6, and 8). However, complete cleavage of K18 at both D238 and D397 was markedly different between MCF7-vec and MCF7-C3. STS treatment (8 h) of MCF7-C3 cells resulted in a significant amount of K18 cleavage based on the generation of the 26.3-kD fragment, identifying the epitope of the anti-K18 mAb in the NH2-terminal half of the protein (Fig. 5 B, lane 4). Under the same conditions, the M30 Ab detected two fragments (45 and 19 kD), both of which must contain the neoepitope formed after cleavage of K18 at position D397 (Fig. 5 B, lane 8). The absence of both the 26.3- (Fig. 5 B, lane 2) and the 19-kD (Fig. 5 B, lane 6) fragments in MCF7-vec cells indicates that the K18 cleavage at D238 requires caspase-3.
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DEDD aggregation precedes activation of caspase-3 and cleavage of K18
To determine the sequence of events involving DEDD changes and K18 cleavage, and to determine whether the ability of DEDD to stain IF like structures required the presence of cytokeratins, we compared the keratin deficient cell line SW13 with SW13 cells stably expressing K8 and K18 (Fig. 6). In the SW13 cells that do not express keratins, induction of apoptosis by STS resulted in detection of DEDD in intracellular aggregates (Fig. 6 A, arrowhead). This result indicated that the aggregation of DEDD is independent of its association with cytokeratins. However, in the K8/K18-expressing SW13 cells, DEDD stained in filaments that again colocalized largely with K18 (Fig. 6 B; unpublished data). To test whether the change of DEDD was generally occurring in cells lacking IF proteins, we treated a number of lymphoid cells with STS and stained for intracellular DEDD in a FACS-based analysis (Fig. 6 C). In all cases, DEDD staining increased during the course of apoptosis. The increase in intracellular DEDD staining could be prevented by pretreating cells with the poly-caspase inhibitor zVAD-fmk as shown for HeLa cells (Fig. 6 D), again confirming that active caspases were required for the increased staining of DEDD. Taken together, all data indicated the change of DEDD is independent of cytokeratins, suggesting that it precedes changes in the IF system during apoptosis, and that it may regulate these changes. To test whether DEDD played a regulatory role in the caspase-mediated cleavage of K18, we compared the kinetics of the DEDD apoptotic staining with activation of caspase-3 and cleavage of K18 by caspase-3 in HeLa cells (Fig. 6 E). After 1 h of STS treatment we detected few M30-positive cells by intracellular staining, whereas the DED3 staining had already reached 70% of its maximal response. In this particular experiment, after 4 h of treatment, almost all cells positive for DEDD also contained caspase-3cleaved K18. Comparison of these events with the appearance of processed caspase-3 revealed that the kinetics of caspase-3 activation in these cells were indistinguishable from the kinetics of K18 cleavage, confirming that M30 staining was effective in quantifying the activation of caspase-3. These data were also confirmed by quantifying immunofluorescence data (unpublished data). The observation that the increase in staining and aggregation of DEDD are both independent of keratins and precedes activation of caspase-3 and K18 cleavage by caspase-3 suggests that DEDD may function in facilitating caspase-3 cleavage of K18.
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To determine the association of processed caspase-3 and DEDD during different stages of apoptosis, we performed an EM analysis of HeLa cells treated with TRAIL. In early stages of apoptosis, DEDD colocalized with active caspase-3 on IFs (Fig. 7 C, left). At intermediate stages when IF filaments started to disintegrate, DEDD colocalized with active caspase-3 on the remaining sheaves of IF structures (Fig. 7 C, middle, arrowhead) and in small inclusions. At later stages, these small inclusions fused to larger structures still containing DEDD and active caspase-3 (Fig. 7 C, right). This analysis was supported by a kinetic analysis of STS-treated MCF7-C3 cells using immunofluorescence microscopy (unpublished data) and demonstrated that the colocalization of caspase-3 subunits with DEDD is not restricted to a cell line that stably expresses exogenous procaspase-3 but can also be found between endogenous DEDD and endogenous active caspase-3. These data strongly suggested a direct interaction between DEDD and caspase-3 that results in activation of caspase-3.
Caspase-3 is recruited to K8/K18 during apoptosis
Though procaspase-3 is evenly distributed in the cytosol in most cells, active caspase-3 localizes with certain keratins to intracellular inclusions in late stage apoptosing epithelial cells (Caulin et al., 1997; MacFarlane et al., 2000). If keratin-bound DEDD is involved in the caspase-3 cleavage of keratins caspase-3 should be detectable at IFs. Due to the lack of anticaspase-3 antibodies for adequate procaspase-3 detection in immunofluorescence microscopy, we transiently transfected MCF7 cells with a procaspase-3GFP fusion protein and induced apoptosis by treating cells with STS. After fixation with paraformaldehyde cells were stained with anti-K8 (Fig. 8 A). In untreated cells caspase-3GFP was detected throughout the cytosol with no significant colocalization with K8 and expression of the fusion protein did not affect the integrity of the IF system (Fig. 8 A, top row). Late after induction of apoptosis at 4 h of STS treatment both caspase-3GFP and K8 were found in the same intracellular inclusions (Fig. 8 A, bottom row) in agreement with previous reports. However, at an earlier stage of apoptosis (2 h after STS treatment), caspase-3GFP was found in small structures lining IF filaments mostly in the center of the cells (Fig. 8 A, middle row). The association of caspase-3 with K8 in early apoptosing cells was most evident in a three-dimensional movie (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200112124/DC1). To directly test whether DEDD associates with procaspase-3 prior to induction of apoptosis we immunoprecipitated procaspase-3 from MCF7-C3 cell lysates and immunoblotted with biotinylated PRO29 (Fig. 8 B). The only DEDD-reactive band that specifically associated with caspase-3 was the 54-kD band of DEDD. Because our analysis of this band had identified it as diubiquitinated DEDD, we now reprobed that blot with an anti-ubiquitin mAb and confirmed that this protein is both reactive with anti-DEDD and anti-ubiquitin antibodies. Our data suggested that procaspase-3 in MCF7-C3 cells is associated with diubiquitinated DEDD, and that DEDD mediates recruitment of procaspase-3 to the IFs during apoptosis, leading to cleavage of IF proteins such as K18 or vimentin.
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The dominant negative effect of DEDDNLS suggests that it could either bind to IF proteins, directly blocking access of caspase-3 to K18, or it could prevent activation of caspase-3 in the cytosol. To test whether cleavage of K18 by caspase-3 was inhibited by DEDD
NLS we transiently transfected HeLa cells with the CD8:C8 construct with DEDD
NLS (Fig. 9 D). Expression of the CD8:C8 construct was monitored by staining for surface CD8 by flow cytometry. Again, coexpression of DEDD
NLS did not affect expression of CD8:C8 or the amount of caspase-8 generated in these cells (Fig. 9 D). As in 293T cells, DEDD
NLS inhibited cleavage of K18 by caspase-3 and prevented the appearance of caspase-3 like activity in the cytosol. These data strongly argue that DEDD
NLS does not simply block K18 cleavage by binding to K18, thereby blocking access of caspase-3 to K18 but rather that DEDD
NLS regulates activation or activity of caspase-3 in the cytosol. This argument is further supported by transient expression of DEDD
NLSGFP fusion proteins in MCF7-C3 cells which showed a diffuse cytosolic localization with no costaining with the IF system (unpublished data). Our experiments in Fig. 3 have demonstrated that DEDD
NLS cannot be ubiquitinated. Therefore, the dominant negative effect of DEDD
NLS may be due to its inability to move to the nucleus and/or its inability to be ubiquitinated, suggesting that ubiquitination may serve as an important functional regulator of DEDD. Taken together the data strongly point to DEDD as a regulator of the cleavage of K18 by caspase-3 and suggest that DEDD is involved in activation of caspase-3 which leads to DNA fragmentation in the tested cells.
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Discussion |
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DEDD2 has a similar structure to DEDD (Roth et al., 2002; Zhan et al., 2002; and this work). Like DEDD, DEDD2 localizes to nucleoli when overexpressed and acts proapoptotically from within the nucleus. Our experiments detecting endogenous proteins demonstrated that an antibody against the DED in DEDD2 showed the same remarkable staining as the DED3 antibody against the DED in DEDD. In both cases, the increase in staining and detection of brightly stained filamentous structures depended on activation of caspase-3. This strongly suggests that both proteins act together and may even respond to the same stimuli.
Due to its dual localization, DEDD may have different functions in the nucleus and the cytoplasm. We have shown that overexpression of DEDD in the nucleus affects DNA Pol I-dependent transcription of rDNA (Stegh et al., 1998; Schickling et al., 2001). Consistent with a nuclear function of DEDD, it was recently reported that DEDD (and DEDD2) interact with the TFIIIC102 subunit of the human transcription factor IIIC (Zhan et al., 2002). This finding together with our recent identification of endogenous DEDD as being in part a nucleolar protein (Schickling et al., 2001) suggests that DEDD plays a role in the general transcription machinery in the nucleus. This might explain why downregulation of total cellular DEDD in HeLa and MCF7 cells using DEDD-specific siRNA was more toxic to the cells than treatment with control siRNAs. However, the lack of induced DNA fragmentation after STS treatment suggests that DEDD is required for STS induced apoptosis. We conclude that this reflects DEDD's cytoplasmic function since overexpression of purely cytosolic DEDDNLS inhibited caspase-3 activation and cleavage of K18 in cells cotransfected with the caspase-8 activating killer construct CD8:caspase-8 (Martin et al., 1998). This activity of DEDD
NLS could be due to two functional changes when compared to wild-type DEDD: the inability of the mutant protein to enter the nucleus or the lack of monoubiquitination. The most well-known function of ubiquitination is to target proteins for degradation by the proteasome. However, monoubiquitination has been shown to be involved in endocytosis of a number of cellular and viral proteins (for review see Hicke, 2001). The best-known monoubiquitinated proteins are histones (Busch and Goldknopf, 1981). Monoubiquitination of H2A and H2B has been shown to be involved in gene regulation (Spencer and Davie, 1999). However, only a fraction of histones are monoubiquitinated. In contrast, DEDD represents the most quantitatively monoubiquitinated protein known. Cleavage of diubiquitinated DEDD with cyanogen bromide revealed that two different fragments are modified (unpublished data), indicating that DEDD is monoubiquitinated at two different sites. Interestingly, it is diubiquitinated DEDD that coimmunoprecipitated with K18 or procaspase-3. We are currently testing whether monoubiquitination of DEDD is required for these interactions to occur. Interestingly, no ubiquitinated DEDD could be detected in nucleoli-enriched fractions (Schickling et al., 2001; unpublished data), whereas in the cytosol of the same cells DEDD was mainly ubiquitinated. Therefore, mono- and diubiquitination could also regulate DEDD's intracellular localization.
Disintegration of the IF network is a general feature of apoptosing cells and cleavage of IF proteins is important for progression of apoptosis. Recent data indicate that IF proteins play a dynamic role during apoptosis by actively regulating the apoptotic process both positively and negatively. Vimentin is cleaved by caspases during apoptosis (Prasad et al., 1998; Morishima, 1999), generating a proapoptotic NH2-terminal fragment (Byun et al., 2001). Cytokeratins 8 (K8) and 18 (K18), IF proteins present in primary and transformed simple epithelia, seem to negatively regulate the apoptosis sensitivity of certain cells such that K8 or K18 deficient epithelial cells are 100 times more sensitive to TNF than wild-type cells (Caulin et al., 2000). The moderation of TNF-induced apoptosis by K18 may be due to competition between K18 and TNF-RI for TRADD, a key TNF-RI adaptor protein (Inada et al., 2001). Another study reported that the lack of cytokeratins strongly sensitized hepatocytes to CD95 mediated apoptosis by allowing CD95 to be transported more efficiently to the cell surface (Gilbert et al., 2001). Many of the IF proteins contain conserved cleavage sites for caspase-3 or -7 (at the COOH terminus and in the center of the proteins) and for caspase-6 (in the center) (Caulin et al., 1997; Ku et al., 1997). Once IF filaments have collapsed after cleavage by caspases, many of the IF proteins can be found in specific intracellular inclusions (Caulin et al., 1997; MacFarlane et al., 2000). These spheroidal inclusions contain K8, cleaved K18, cleaved K19 but not K13/15/16, vimentin, and ß-actin or tubulin (Caulin et al., 1997; MacFarlane et al., 2000). Interestingly, not only cleaved cytokeratins but also most, if not all, of the cytosolic pool of active caspase-3 were found sequestered in these structures during TRAIL induced apoptosis in MCF7 cells (MacFarlane et al., 2000). We now show that these inclusions which occur during a number of other apoptotic stimuli that activate caspase-3 contain both DEDD and DEDD2.
Detection of filamentous structures of active caspase-3 on IF branches precedes the formation of these inclusions, which also precisely colocalize with activated DEDD and DEDD2. Because DEDD is constitutively associated with K18 in nonapoptosing cells, DEDD likely changes its conformation while bound to K18. This conclusion is further supported by the finding that in keratin-free SW13 cells DEDD can be found in intracellular aggregates and not in K8/18-like filamentous structures. Reconstitution of SW13 cells with both K8 and K18 resulted in detection of DEDD filaments. We could detect no DEDD structures bound to vimentin in a vimentin expressing variant of SW13 cells (unpublished data), suggesting that binding of DEDD to the IF system is predominantly mediated through cytokeratins.
DEDD was found to be associated with K18 and significantly colocalized with K18 during immunofluorescence microscopy. DEDD almost completely colocalized with active caspase-3. Unexpectedly, colocalization of DEDD with caspase-3cleaved K18 was only partial. However, consistent with DEDD's localization, active caspase-3 also only partially colocalized with caspase-3cleaved K18. This finding could be explained by the properties of the IF system which has been shown to be highly dynamic (Prahlad et al., 1998; Windoffer and Leube, 1999). We propose that keratins after the initial caspase cleavage move away from caspase-3/DEDD complexes, explaining the apparent dissociation of active caspase-3/DEDD with cleaved K18. At the end stage of apoptosis when most inclusions have fused to larger structures DEDD, cleaved K18 and active caspase-3 again colocalize in most of these inclusions.
Several models may explain the function of the DEDD containing inclusions. It has been suggested that these inclusions could be the final graveyard for active caspases such as caspase-3, -7, and -9 (unpublished data). In this model, they would represent structures used to inactivate and dispose of active caspases. However, why only simple epithelial cells and not numerous other cell types would use this mechanism to dispose of caspases remains to be elucidated. It is more likely that these structures serve a specific function. Late during etoposide- and UV-induced apoptosis, most of these structures were found in cytoplasmic blebs which seemed to develop into apoptotic bodies (Fig. 5 D; unpublished data). This effect was not seen in STS treated cells, as STS has been shown to inhibit blebbing (Mills et al., 1998).
Based on the new findings in this work, we have developed a model of DEDD's function (Fig. 10). DEDD resides in the cytoplasm in an inactive form bound to a population of K18. Upon induction of apoptosis, a small amount of caspase-3 is activated (step I), causing a conformational change in DEDD (step II). This may attract and activate more caspase-3 (step III), which then leads to cleavage of DEDD-associated cytokeratins (step IV). This step is clearly evident by the complete colocalization of active caspase-3, DEDD, and K18. After cleavage of K18, the IF network collapses and DEDD, active caspases, and cleaved cytokeratins are found in intracellular inclusions which may subsequently migrate into cytoplasmic blebs (step V). Our data indicate that activation of caspase-3 is both upstream and downstream of DEDD. Caspase-3 activity is required both for the conformational change of DEDD as well as the cleavage of DEDD-associated IF proteins. Therefore, DEDD would provide a unique way for one effector caspase to be activated in two steps in the same compartment. According to our model, DEDD could act as an intracellular timer to ensure degradation of substrates by caspase-3 in an ordered fashion. The regulation of these two steps requires further study.
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Materials and methods |
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Cloning of DEDD2 and generation of polyclonal anti-DEDD2 antiserum
TBLASTN searches of the human expressed sequence tag data base using the DEDD open reading frame identified a clone BE797255 from a small cell lung carcinoma library that contained a sequence that was highly homologous to 80% of the DEDD open reading frame. Sequencing of this clone which was obtained from the ATCC revealed an open reading frame coding for a protein of 326 amino acids that was 48% identical to DEDD (EMBL/GenBank/DDBJ accession no. for DEDD2 cloned in this work: AY125488). We then generated an antibody to the corresponding region within the DED (APGAAGGLARARSGLE) of DEDD2 which was used to generate the anti-DEDD antibody DED3. Although the DED between DEDD and DEDD2 are 47.5% identical the region of this 16amino acid peptide only shared 2 identical amino acids. Subsequent tests revealed that the antibodies DED3 and anti-DEDD2 were specific for DEDD and DEDD2, respectively.
Induction of apoptosis
Apoptosis induction in cells used in immunofluorescence and intracellular FACS stainings was performed with the following conditions: 1 µM staurosporine (Sigma-Aldrich) for the indicated times, 1 µg/ml TRAIL (MacFarlane et al., 2000) for the indicated times, 200 µM etoposide (Sigma-Aldrich) for 24 h, 1 µg/ml cycloheximide plus 20 ng/ml TNF for 12 h, or UV irradiated (100 kJ) and cultivated for 24 h after irradiation before harvesting. To block apoptosis, cells were pretreated for 30 min with 50 µM zVAD-fmk.
Western blot
Western blotting was performed as previously described (Schickling et al., 2001) with the following antibodies: PRO29 (previously called anti-DEDD1; Schickling et al., 2001) (1 µg/ml), biotinylated PRO29 (1 µg/ml), DED3 (previously called anti-DEDD2; Schickling et al., 2001) (1 µg/ml), anti-K8 (1 µg/ml; Sigma-Aldrich), anti-FADD (1 µg/ml; Transduction Laboratories), anti-FLAG (1 µg/ml; Sigma-Aldrich), anti-HA (1 µg/ml; Covance), anti-K18 (0.4 µg/ml; Santa Cruz Biotechnology, Inc.), anti-lamin A/C (0.4 µg/ml; Santa Cruz Biotechnology, Inc.), and anti-active caspase-3 (1:1,000; Cell Signaling Technologies). Goat antirabbit-Ig-HRP (1:15,000; Santa Cruz Biotechnology, Inc.) or isotype specific goat antimouse IgG-HRP (1:15,000; Southern Biotechnology) were used as secondary antibodies.
Immunofluorescence microscopy
Cells were grown on poly-prep slides (Sigma-Aldrich) and fixed with 2% paraformaldehyde for 20 min at room temperature. After the fixation the slides were washed three times with 50 mM NH4Cl (in PBS) for 3 min, permeabilized in ice-cold PBS with 0.3% Triton X-100 (Sigma-Aldrich) for 1 min, washed with PBS-Mg (PBS + 1 mM MgCl2), and incubated for 10 min in blocking solution (PBS, 0.01% saponin, 0.25% BSA and 0.02% NaN3). The slides were incubated for 2 h at room temperature or overnight at 4°C with the indicated antibodies: DED3 (8 µg/ml), anti-K8 (5 µg/ml; Sigma-Aldrich), M30 (1:50; Roche), anti-active caspase-3FITC (9 µg/ml; Pharmingen), and anti-DEDD2 (10 µg/ml). Peptide competition for the DED3 antibody was performed as previously described (Schickling et al., 2001). After washing with PBS-Mg, slides were incubated with secondary antibodies (5 µg/ml; Southern Biotechnology) and DAPI (2 µg/ml) for 1 h at room temperature, washed, and dehydrated in 100% ethanol. Coverslips were mounted onto the slides with Vectashield mounting medium (Vector Laboratories) and analyzed with an Axiovert S100 immunofluorescence microscope equipped with an Axiocam digital camera and software (Zeiss). Confocal pictures were taken and analyzed with a confocal microscope LSM 510 (Zeiss).
Intracellular FACS analysis
Cells were fixed for 30 min in methanol (-20°C), pelleted for 5 min at 500 g, and blocked for 30 min at room temperature in incubation buffer (PBS, 0.1% Tween 20, and 1% BSA). Primary antibodies were diluted in incubation buffer (DED3 10 µg/ml, M30 1:50, anti-CD8 10 µg/ml [Pharmingen], anti-active caspase-3FITC 1:10) and incubated with the cells for 1 h at room temperature. Subsequently, the cells were washed with washing buffer (PBS, 0.1% Tween 20) and incubated with FITC-conjugated secondary antibodies (10 µg/ml; Southern Biotechnology) for 30 min at room temperature. The cells were washed twice and analyzed by fluorescence-activated cell sorter (FACScan; Becton Dickinson).
Electron microscopy and immunogold cytochemistry
Cells were fixed with a mixture of 4% formaldehyde and 0.1% glutaraldehyde in Dulbecco's PBS, pH 7.4, for 1 h at room temperature, spun down in a swing-out rotor at 3,000 g, rinsed in PBS, dehydrated in ethanol, and infiltrated with Unicryl resin from British Biocell International Ltd. The resin was polymerized with UV radiation (360 nm), at 4°C, according to the manufacturer's instructions. Ultrathin sections were blocked with normal goat serum, diluted 1:50 in PBS containing 1% BSA and 1% Tween 20, for 4 h at room temperature. Serial sections were incubated, for 18 h at 4°C, with antibodies to DEDD (DED3), ß-actin, tubulin, vimentin, cytokeratins 8 and 18, and to the caspase-3 cleavage fragment of cytokeratin 18 (M30). These primary antibodies were all diluted (1 + 5) in PBS containing 1% normal goat serum, 1% BSA and 1% Tween 20 (PBSGAT). In control incubations, the primary antibodies were replaced with mouse IgG1 or normal rabbit serum (Dako Ltd.). Thorough washing in PBSGAT was followed by incubation, for 18 h at 4°C, in the appropriate goat-derived antimouse or rabbit IgG (British Biocell International Ltd.). Secondary antibodies, adsorbed against human serum proteins and conjugated with 10 nm colloidal gold, were diluted 1:50 in PBSGAT. Sections incubated with mixtures of mouse- and rabbit-derived antibodies were subsequently labeled with a mixture of antimouse antibody conjugated with 10 nm gold and antirabbit antibody conjugated with 5 nm gold, respectively. Ultrathin sections were examined unstained or after staining with lead citrate and/or uranyl acetate.
Ubiquitination of DEDD
293T cells were transiently cotransfected by the calcium-precipitation method as previously described (Stegh et al., 1998) with DEDD-FLAG or DEDDNLS-FLAG in the presence of vector or pMT123 at the indicated amounts. Cells were harvested 24 h after transfection, lysed in sample buffer, and Western blots were probed with anti-FLAG. For IP, cells were lysed with 2% empigen (as described in Lowthert et al., 1995). Lysates were spun down and incubated with 10 µg anti-FADD 1C4 or anti-FLAG M2 (Sigma-Aldrich) and 50 µl of antimouse IgG1 beads (Sigma-Aldrich) at 4°C for 1 h rotating end to end. Beads were washed, resuspended in sample buffer, and Western blots were probed with anti-FLAG and HA.11 (Covance). 293T cells transiently transfected with vector or pMT123 were cultured for 4 h and treated with DMSO or 20 µM MG132 (Calbiochem) for 12 h. Cells were harvested, lysed in sample buffer, and Western blots were probed with PRO29. E36 and ts20 cells were maintained at the permissive temperature (30°C) or shifted to the restrictive temperature (40°C) for 12 h. Cells were harvested, lysed in sample buffer, and Western blots were probed with PRO29.
Characterization of DEDD in murine tissue
A piece of tissue (size dependent on organ) from C3H or Nit ± C57BL/6Jx129 mice (one female and one male each) was diced finely with a scalpel and put in 200500 µl of lysis buffer (30 mM Tris, pH 7.5; 10% glycerol; 150 mM NaCl; 1% NP-40) plus inhibitors and incubated on ice for 30 min to 1 h before a brief sonication. Lysates were spun at 13,000 rpm for 7 min and the supernatant transferred to a new tube. 60 µg of protein were loaded per lane for Western blotting with PRO29.
Subcellular fractionation
Cells were harvested and lysed in ice-cold total lysis buffer composed of 250 mM NaCl; 50 mM HEPES, pH 7.0; 5 mM EDTA; 1% NP-40, Complete® protease inhibitors (Roche Molecular Biochemicals) for 60 min and sonicated (30 s, 20% intensity). Insoluble material was pelleted with 20,000 g for 15 min at 4°C. Supernatant (S) and pellet (P) were separated and resuspended in reducing sample buffer containing 5 M urea.
Immunoprecipitation of keratin 18 and caspase-3
HeLa cells were treated with 400 µM of etoposide, harvested, and lysed with 2% empigen lysis buffer as described previously (Lowthert et al., 1995). Lysates were spun (14,000 rpm, 15 min) and protein amount was quantified (Bio-Rad Laboratories). 3 mg of protein were incubated with 18 µg of anti-K18 (Santa Cruz Biotechnology, Inc.) or anti-FADD 1C4 at 4°C 1 h rotating end to end. Subsequently, 50 µl of resuspended antimouse IgG1-agarose beads (Sigma-Aldrich) were added to the lysate/antibody tubes and incubated overnight at 4°C rotating end to end. Following incubation, beads were washed four times with lysis buffer and resuspended in sample buffer. MCF7-C3 cells were lysed in 1% NP-40 lysis buffer (250 mM NaCl; 50 mM Hepes, pH 7.0; 5 mM EDTA; 1% Nonidet P-40, Complete®) for 1 h on ice. Lysates were (14,000 rpm, 15 min) and protein amount was quantified (Bio-Rad Laboratories). 3 mg of protein were incubated with 10 µg of anticaspase-3 (Cell Signaling Technologies) or normal rabbit Ig (Santa Cruz Biotechnology, Inc.) at 4°C 1 h rotating end to end. Subsequently, 35 µl of resuspended protein A-Sepharose beads (Sigma-Aldrich) were added to the lysate/antibody tubes and incubated overnight at 4°C rotating end to end. After incubation, beads were washed four times with lysis buffer and resuspended in sample buffer.
RNAi and cytotoxicity assay
RNAi experiments were performed as previously described (Elbashir et al., 2001). Briefly, HeLa cells were transfected with DEDD, lamin A/C, or Cy3-luciferase siRNAs (Dharmacon) at the indicated amounts with Transit-TKO (Mirus) in 24-well plates according to manufacturer's instructions and incubated for 48 h. Cells were harvested and lysed in sample buffer for Western blotting or quantified for DNA fragmentation as previously described (Stegh et al., 1998).
Influence of DEDDDNLS on keratin 18 cleavage
293T or HeLa cells were transfected with the indicated amount of plasmid DNA either using the calcium-phosphate (293T) or SuperfectTM (HeLa) following the manufacturer's protocol (QIAGEN). 24 h after transfection the cells were harvested and either intracellularly stained for cleaved keratin with M30 or lysed for quantification of caspase-3 and -8 activities with fluorogenic caspase substrates as previously described (Stegh et al., 2000).
Online supplemental material
Video 1 is available online at http://www.jcb.org/cgi/content/full/jcb.200112124/DC1. The three-dimensional image represented as a QuickTime video is taken from Fig. 8 (second row, right), and shows GFP-positive structures (green) aligning on intermediate filament strands (red) stained with anti-K8 after treating HeLa cells transfected with caspase-3GFP with staurosporine for 2 h.
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Footnotes |
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J.C. Lee and O. Schickling contributed equally to this work.
* Abbreviations used in this paper: DEDD, death effector domain containing DNA binding protein; IF, intermediate filament; STS, staurosporine.
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Acknowledgments |
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J.C. Lee was supported by the Medical Scientist Training Grant from the National Institutes of Health. A.H. Stegh was supported by a stipend from the Boehringer Ingelheim Fonds.
Submitted: 24 December 2001
Revised: 18 July 2002
Accepted: 31 July 2002
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