JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M201322200 on March 1, 2002

J. Biol. Chem., Vol. 277, Issue 19, 16775-16781, May 10, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/19/16775    most recent
M201322200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, F.
Right arrow Articles by Cryns, V. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, F.
Right arrow Articles by Cryns, V. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Caspase Proteolysis of the Cohesin Component RAD21 Promotes Apoptosis*

Feng ChenDagger , Merideth KamradtDagger , Mary MulcahyDagger , Young ByunDagger , Huiling Xu§, Michael J. McKay§, and Vincent L. CrynsDagger

From the Dagger  Robert H. Lurie Comprehensive Cancer Center and the Department of Medicine, Northwestern University Medical School, Chicago, IL 60611 and the § Peter MacCallum Cancer Institute, Melbourne 8006, Australia

Received for publication, February 2, 2002, and in revised form, February 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp279 by caspases-3 and -7 in vitro to generate two major proteolytic products of ~65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

During development and in response to homeostatic challenges, cells are selectively eliminated by a genetically regulated suicide mechanism known as apoptosis or programmed cell death. Activation of this intrinsic cell death apparatus leads to a series of dramatic nuclear events, including chromatin condensation, degradation of chromosomal DNA into internucleosomal fragments, and disassembly of the nuclear membrane (1). Caspases are a conserved family of cysteine proteases that play a critical role in the execution of apoptosis by cleaving a subset of cellular proteins at aspartic acid residues and altering their function (2). Indeed, caspases have been directly linked to many of the nuclear (and other) manifestations of apoptosis. For instance, caspase-3 proteolyzes and activates acinus, a nuclear protein whose caspase cleavage product directly induces chromatin condensation (3). Caspases also activate caspase-activated DNase (CAD),1 an endonuclease normally present in the nucleus as an inactive heterodimer bound to its inhibitor ICAD/DFF-45. Caspase-3 cleaves and displaces ICAD/DFF-45 from CAD, thereby enabling CAD to degrade chromosomal DNA (4-6). In addition, proteolytic cleavage of the nuclear lamins by caspase-6 promotes the dismantling of the nuclear envelope (7-9). Importantly, caspase-9 proteolysis of yet to be identified proteins increases the permeability of nuclear pores, thereby allowing caspases to enter the nucleus and cleave these critical nuclear targets (10). Clearly, the nuclear manifestations of apoptosis reflect the concerted action of caspases on multiple proteolytic targets, only a subset of which have been identified and characterized.

Using an expression cloning strategy we have described recently to systematically identify cDNAs encoding caspase substrates (11-13), we report here the isolation of human RAD21/SCC1/MCD1 (14-16) (hereafter referred to as RAD21) as a novel nuclear caspase target. RAD21 was first identified as a nuclear phosphoprotein that repairs double-strand DNA breaks and is essential for viability in fission yeast (14, 17). More recently, RAD21 has been demonstrated to be a component of the conserved mitotic cohesin complex that holds sister chromatids together and ensures the faithful segregation of duplicated chromosomes to daughter cells; defects in this process lead to aneuploidy (18, 19). During mitosis, the separation of sister chromatids requires the removal of the cohesin "glue," which holds sister chromatids together from S phase until the beginning of anaphase. In yeast, RAD21 is specifically cleaved during the onset of anaphase by Esp1/separase, a cysteine protease distantly related to caspases, thereby allowing sister chromatid separation and the segregation of chromosomes to opposite poles (20-22). Interestingly, separase proteolysis of budding yeast RAD21 generates a short-lived carboxyl-terminal product that is rapidly degraded by the ubiquitin-proteasome pathway; stabilization of this cleavage product by a variety of methods leads to chromosome loss and is lethal (23). In metazoans, cohesin is removed from chromatin by two distinct processes. During prophase, the vast majority of cohesin is removed from chromosome arms by a cleavage-independent mechanism that likely contributes to the condensation of DNA into discrete chromatid arms, leaving only a small amount of cohesin bound to chromatids at the centromere (24-27). At the onset of anaphase, centromeric cohesin is cleaved by separase to trigger sister chromatid separation (25, 28). In both yeast and metazoans, the introduction of a mutant RAD21 that is resistant to separase cleavage inhibits sister chromatid separation (20, 28). Hence, the mitotic proteolysis of RAD21 by a caspase-like protease is essential for proper chromosome segregation in organisms as diverse as yeast and humans.

In contrast to its critical role in mitosis, RAD21 has not been implicated previously in apoptosis. In the present study, we report that RAD21 is specifically cleaved by caspases at Asp279 in vitro and in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Overall, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- HeLa and MCF-7 cells were maintained in DMEM (Mediatech) supplemented with 10% fetal calf serum (Invitrogen).

Small Pool Expression Cloning-- 35S-labeled protein pools were prepared from small pools (48 cDNAs/pool) of a human heart cDNA library (Invitrogen) using the TNT T7 Quick Coupled Transcription/Translation System (Promega) as described previously (11, 12). These 35S-labeled protein pools were then incubated with control buffer, 25 ng of caspase-3, or 25 ng of caspase-8, and individual cDNAs encoding caspase substrates in vitro were isolated as we have detailed elsewhere (11-13).

Proteolytic Cleavage Reactions in Vitro-- 35S-labeled full-length human RAD21 was prepared using the TNT T7 Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions. 35S-labeled human RAD21 was then incubated with buffer, 2.5 or 25 ng of caspases-1, -2, -3, -6, -7, or -8 for 1 h at 37 °C, and the reaction products were resolved by SDS-PAGE and visualized by autoradiography as described elsewhere (12, 29). To identify the major caspase cleavage site of RAD21 in vitro, we selectively altered the Asp residue at a potential cleavage site to a Glu residue (D279E) using the QuikChange site-directed mutagenesis kit (Stratagene) with the following oligonucleotide primers: 5'-GGCCTGATAGTCCTGAGTCAGTGGATCCCGTTG-3' and 5'-CAACGGGATCCACTGACTCAGGACTATCAGGCC-3'. The resulting RAD21 D279E construct was verified by DNA sequencing. 35S-labeled wild-type and mutant D279E RAD21 were then incubated with buffer or 10 or 25 ng of caspases-3 or -7 as above to determine whether the D279E RAD21 mutant was resistant to caspase proteolysis. In parallel experiments, 35S-labeled human REC8, a meiotic cohesin structurally related to RAD21, was incubated with buffer or 2.5 or 25 ng of caspases-3 or -7, and the reaction products were analyzed as above.

Induction of Apoptosis and Immunoblotting-- HeLa cells were treated with 50 µM etoposide for 0-48 h. Whole cell lysates were then prepared and analyzed by immunoblotting as described previously (29) using a polyclonal antibody that recognizes the carboxyl terminus of human RAD21 (1:400 dilution), PARP monoclonal antibody (BD PharMingen) (1:1000 dilution), or protein kinase Cdelta polyclonal antibody (Santa Cruz Biotechnology) (1:1000 dilution). The percentage of cells in each treatment condition that had apoptotic (condensed or fragmented) nuclei was determined in parallel experiments by fixing cells grown on glass coverslips with 100% methanol at -20 °C for 2 min and staining nuclei with 10 µg/ml Hoescht No. 33258 (Sigma) for 30 min. Nuclei were visualized by fluorescence microscopy using a Nikon Eclipse E400 microscope, and the percentage of apoptotic nuclei was determined in three independent experiments (at least 200 nuclei/experiment were scored) as described previously (13, 30). In the caspase inhibitor experiments, HeLa cells were untreated or were preincubated with 100 µM zVAD-fmk (Enzymes Systems Products) for 1 h prior to treatment with 10 ng/ml tumor necrosis factor (TNF)-alpha and 1 µg/ml cycloheximide for 18 h. RAD21 was then analyzed by immunoblotting as above. Chromatin-enriched pellets and supernatant fractions were obtained by centrifugation of whole cell lysates at 15,000 × g as described elsewhere (25).

Construction of GFP-tagged Rad21 cDNAs-- GFP-tagged Rad21 cDNAs encoding full-length RAD21, the amino-terminal (N-RAD21), and carboxyl-terminal (C-RAD21) caspase cleavage products were constructed by PCR amplifying human wild-type Rad21 with the following primers: 5'-GGCCCTCGAGCCAGCCAGAACAATGTTC-3' and 5'-GGCCCCGCGGTATAATATGGAACCTTGG-3' (full-length Rad21), 5'-GGCCCTCGAGCCAGCCAGAACAATGTTC-3' and 5'-GGCCCCGCGGTTAATCAGGACTATCAGGCCC-3' (N-RAD21 encoding amino acids 1-279), 5'-GGCCCTCGAGATGTCAGTGGATCCCGTTGAA-3' and 5'-GGCCCCGCGGTATAATATGGAACCTTGG-3' (C-RAD21 encoding amino acids 280-631). The PCR products were then digested with XhoI and SacII and cloned into the corresponding sites in pEGFP-N1 (CLONTECH). The sequence of each construct fused at its carboxyl terminus with GFP was confirmed by automated DNA sequencing.

Transient Transfections and Quantitation of Apoptosis-- HeLa cells were plated at ~50% confluence on glass coverslips and transiently transfected with 1.2 µg of pEGFP-N1 plasmid containing empty vector, full-length RAD21, the amino-terminal caspase cleavage product (N-RAD21), or the carboxyl-terminal caspase cleavage product (C-RAD21). Transfections were performed using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions. Twenty-four h later, cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Nuclei were then stained with 10 µg/ml Hoescht No. 33258 (Sigma) for 30 min. GFP-positive cells were scored for apoptotic (condensed/fragmented) nuclei by fluorescence microscopy as described previously (13). The percentage of GFP-positive cells with apoptotic nuclei was determined in three independent experiments (at least 200 nuclei/experiment were scored). In the co-transfection experiments, HeLa cells were transiently transfected with 0.4 µg of pEGFP-N1 plasmid containing C-RAD21 and 0.8 µg of pcDNA3 plasmid containing empty vector, p35, survivin, Bcl-xL, or wild-type RAD21. Twenty-four h later, GFP-positive cells were scored for apoptosis as above. In all experiments, the statistical significance of inter-group differences was assessed by a two-tailed paired Student's t test.

Cell Cycle Analyses-- HeLa cells were transiently transfected as above and subjected to a double thymidine block as follows. HeLa cells were plated at ~50% confluence and treated overnight with 2 mM thymidine. On the next day, cells were transiently transfected with GFP-tagged constructs, returned to normal growth media for the duration of the day, and then subjected to a second overnight incubation with 2 mM thymidine. On the following day, cells were washed in phosphate-buffered saline (PBS) and returned to normal growth media for 16 h. Cells were then trypsinized and incubated with 0.5% paraformaldehyde for 15 min at 37 °C. Next, cells were washed in PBS supplemented with 0.05% Tween 20 and resuspended in 70% ethanol for 15 min at 4 °C. After washing in PBS, cells were incubated with propidium iodide (50 µg/ml) and RNase (100 µg/ml) for 1 h at 37 °C. Washed cells were then analyzed by fluorescence-activated cell sorting; the cell cycle distribution of GFP-fluorescent cells was determined by DNA content.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of RAD21 as a Caspase-3 Substrate in Vitro by Small Pool Expression Cloning-- We have recently described an expression cloning strategy to systematically identify cDNAs encoding caspase substrates (11-13). In the present study, we prepared 35S-labeled protein pools from small pools (48 cDNAs/pool) of a human heart cDNA library by coupled transcription and translation in vitro. These 35S-labeled protein pools were then incubated with control buffer, caspase-3, or caspase-8, and the cleavage products were analyzed by SDS-PAGE. As demonstrated in Fig. 1A, 35S-labeled protein pool 156 contained an ~125-kDa protein (indicated by the asterisk) that was specifically cleaved by caspase-3 (C3) but not by caspase-8 (C8) into proteolytic products of ~65 and 32 kDa (indicated by arrows). As shown in Fig. 1B, the proteolytic activity of caspase-3 and -8 was confirmed by demonstrating their ability to cleave 35S-labeled vimentin (a known caspase-3 substrate) (13, 31) or 35S-labeled BID (a well characterized caspase-8 substrate) (32, 33). cDNA pool 156 was then subdivided into smaller pools, and the 35S-labeled protein pools were retested for their sensitivity to caspase-3 cleavage until a single cDNA encoding the putative caspase substrate was isolated (Fig. 1C). Sequencing of clone 156 revealed that it was a partial cDNA encoding the human mitotic cohesin component RAD21 (14-16). The protein encoded by clone 156 lacked the amino-terminal 34 amino acids of the full-length human RAD21 protein.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1.   Isolation of RAD21 as a caspase-3 substrate in vitro by small pool expression cloning. A, 35S-labeled protein pool 156 contains a 125-kDa protein (indicated by the asterisk) that is specifically proteolyzed into two products of ~65 and 32 kDa (indicated by arrows) by caspase-3 (C3) but not by control (C) buffer or caspase-8 (C8). B, selective proteolysis of 35S-labeled vimentin by caspase-3 (C3) and 35S-labeled BID by caspase-8 (C8) into their signature cleavage fragments (indicated by arrows). C, identification of a single cDNA from pool 156 that encodes a 125-kDa protein (indicated by the asterisk) that is cleaved by caspase-3 (C3) into the appropriately sized products (indicated by arrows). cDNA pool 156 was subdivided, and the corresponding 35S-labeled protein pools were re-examined as above until a single cDNA encoding the caspase-3 substrate was isolated. Sequencing of this clone revealed that it is a partial human rad21 cDNA. Small pool expression cloning, cleavage reactions, and analysis of proteolytic products were performed as detailed under "Experimental Procedures" and elsewhere (11-13). The molecular mass of markers in kDa is indicated at the left of each panel.

Human RAD21 Is Specifically Cleaved by Caspases-3 and -7 at Multiple Sites, Including Asp279, to Yield Several Proteolytic Products in Vitro-- Because clone 156 was a partial Rad21 cDNA, we next examined the sensitivity of full-length human RAD21 to proteolysis in vitro by a panel of recombinant caspases. As shown in Fig. 2A, 35S-labeled full-length human RAD21 was preferentially cleaved by caspase-3 (C3) and to a lesser extent by caspase-7 (C7) into two abundant proteolytic products of ~65 and 48 kDa and two fainter products of 32 and 22 kDa (indicated by arrows). Prolonged exposure also revealed a few additional faint products between 25 and 60 kDa (data not shown). In addition, caspases-6 (C6) and -8 (C8) weakly cleaved RAD21 to generate faint 65- and 48-kDa products. Importantly, the catalytic activity of each caspase was verified by incubation with a known substrate (data not shown). To identify the major caspase cleavage site in RAD21 responsible for producing the observed 65- and 48-kDa fragments, we examined its sequence for consensus caspase-3 cleavage (DXXD) motifs (34, 35) and found two adjacent, potential cleavage sites (DSPDdown-arrow S280 and DSVDdown-arrow P283) that would generate cleavage products of the observed size. Each of the critical Asp residues (Asp279 and Asp282) at these potential cleavage sites was individually altered to a Glu residue by site-directed mutagenesis. As shown in Fig. 2B, 35S-labeled wild-type (WT) RAD21, but not mutant D279E RAD21, was cleaved into its signature 65- and 48-kDa products by caspases-3 and -7 in vitro (indicated by arrows). Instead, the D279E mutant was cleaved into a much larger size product (indicated by the asterisk) that was not observed when wild-type RAD21 was incubated with caspases-3 or -7. In contrast, 35S-labeled mutant D282E RAD21 was readily proteolyzed by caspases-3 and -7 into the expected size products (data not shown). Interestingly, as demonstrated in Fig. 2C, 35S-labeled full-length human REC8, a meiotic cohesin structurally related to RAD21 that is also cleaved by separase (36, 37, 42), was not proteolyzed by caspases-3 (C3) or -7 (C7) in vitro. Although the identity of the minor cleavage site(s) of RAD21 in vitro has not been delineated, these findings indicate unambiguously that RAD21 is preferentially cleaved at Asp279 by caspases-3 and -7 in vitro.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 2.   Human RAD21 is specifically cleaved by caspases-3 and -7 at multiple sites, including Asp279, to yield several proteolytic products in vitro. A, 35S-labeled full-length human RAD21 was incubated with control (C) buffer or 2.5 or 25 ng of caspase-1, -2, -3, -6, -7, or -8 (C1-C8) for 1 h at 37 °C, and the reaction products were resolved by SDS-PAGE. The cleavage fragments are indicated by arrows. B, RAD21 mutant D279E is resistant to proteolysis at Asp279 by caspases-3 and -7 in vitro. 35S-labeled WT or mutant D279E RAD21 were incubated with control (C) buffer or 10 or 25 ng of caspases-3 (C3) or -7 (C7) for 1 h at 37 °C. WT RAD21, but not the D279E mutant, was proteolyzed into major fragments of 65 and 48 kDa (indicated by arrows). The D279E mutant was cleaved into a single larger product (indicated by the asterisk) that was not observed when WT RAD21 was incubated with caspases-3 or -7 in vitro. C, Human REC8, a homologue of RAD21, is not cleaved by caspases-3 or -7 in vitro. 35S-labeled human REC8 was incubated with control (C) buffer or 2.5 or 25 ng of caspase-3 (C3) or caspase-7 (C7) for 1 h at 37 °C. The molecular mass of markers in kDa is indicated at the left of each panel.

Human RAD21 Is Rapidly Proteolyzed by Caspases during the Induction of Apoptosis, Leading to Its Partial Dissociation from Chromatin-- Having demonstrated that RAD21 is cleaved by caspases in vitro, we wanted to determine whether it is also cleaved by caspases in cells undergoing apoptosis in vivo. To this end, HeLa cells were treated with 50 µM etoposide for varying time periods, and RAD21 was detected by immunoblotting with a carboxyl-terminal polyclonal antibody. As shown in Fig. 3A, RAD21 was rapidly proteolyzed into a 65-kDa fragment (indicated by arrows) within 12 h of treatment with etoposide. By 36 h, all of the full-length RAD21 had been cleaved into the 65-kDa product. Of note, the observed RAD21 apoptotic cleavage product was the same size as that generated by RAD21 proteolysis by caspase-3 in vitro, suggesting that the apoptotic proteolysis of RAD21 is also mediated by caspases. Interestingly, RAD21 was cleaved more rapidly during etoposide-induced apoptosis than the well characterized nuclear caspase-3 substrate PARP (38); PARP proteolysis was detected only after 36 h of etoposide treatment. Instead, the time course of RAD21 proteolysis during apoptosis more closely resembled that of the cytosolic caspase-3 substrate protein kinase Cdelta (39). Indeed, RAD21 proteolysis could be detected after 12 h of etoposide treatment when only 1% of HeLa cells had apoptotic nuclear morphology, and RAD21 was completely cleaved into its 65-kDa product after 36 h of treatment when only 25% of cells had apoptotic nuclei. Hence, the specific proteolysis of RAD21 into its signature 65-kDa product precedes apoptotic chromatin condensation/fragmentation.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Human RAD21 is rapidly proteolyzed by caspases during the induction of apoptosis, leading to its partial dissociation from chromatin. A, time course for proteolysis of RAD21, PARP, and protein kinase Cdelta during etoposide-induced apoptosis. HeLa cells were treated with 50 µM etoposide for the indicated amounts of time. The percentage of cells with apoptotic nuclear morphology was determined for each treatment condition in parallel experiments as detailed under "Experimental Procedures" and is indicated at the bottom. B, the caspase inhibitor zVAD-fmk blocks the apoptotic proteolysis of RAD21. HeLa cells were untreated or preincubated with vehicle or 100 µM zVAD-fmk for 1 h, and then they were treated with 10 ng/ml TNF-alpha and 1 µg/ml cycloheximide for 18 h. C, RAD21 is specifically cleaved in caspase-3-deficient MCF-7 cells undergoing TNF-alpha -induced apoptosis. MCF-7 breast carcinoma cells were untreated or treated with 10 ng/ml TNF-alpha and 1 µg/ml cycloheximide for 24 h. D, caspase cleavage of RAD21 partially releases it from chromatin into the soluble fraction. Chromatin-enriched pellet (P) and supernatant (S) fractions were prepared from untreated HeLa cells and HeLa cells incubated with 10 ng/ml TNF-alpha and 1 µg/ml cycloheximide as described under "Experimental Procedures" and elsewhere (25). In panels A-D, immunoblotting was performed as described under "Experimental Procedures." The molecular mass of markers in kDa is indicated at the left of each panel.

To determine whether the specific proteolysis of RAD21 during apoptosis is, indeed, mediated by caspases, HeLa cells were untreated or preincubated with the broad spectrum caspase inhibitor zVAD-fmk (100 µM) for 1 h, and the cells were then treated with 10 ng/ml TNF-alpha and 1 µg/ml cycloheximide for 18 h. As shown in Fig. 3B, the apoptotic cleavage of RAD21 induced by TNF-alpha (indicated by the arrow) was completely inhibited by zVAD-fmk, thereby indicating that caspases are responsible for the proteolysis of RAD21 during apoptosis in vivo. zVAD-fmk also inhibited RAD21 proteolysis induced by etoposide (data not shown). To determine whether caspase-3 was necessary for the apoptotic proteolysis of RAD21, we treated MCF-7 breast carcinoma cells, which lack caspase-3 (40), with 10 ng/ml TNF-alpha and 1 µg/ml cycloheximide for 24 h. As shown in Fig. 3C, RAD21 was proteolyzed into its characteristic 65-kDA cleavage product in MCF-7 undergoing TNF-alpha -induced apoptosis, indicating that caspase-3 is not essential for the apoptotic proteolysis of RAD21 and that other caspases (such as caspase-7) can cleave RAD21 in vivo. Taken together, our results demonstrate unequivocally that RAD21 is rapidly and specifically cleaved by caspases during the induction of apoptosis triggered by a broad spectrum of stimuli.

We next examined whether caspase cleavage of RAD21 altered its association with chromatin. To this end, we prepared low speed chromatin-enriched pellet and supernatant fractions from untreated HeLa cells or HeLa cells incubated with TNF-alpha and cycloheximide for 48 h. As demonstrated in Fig. 3D, full-length RAD21 bound to chromatin and was found exclusively in the chromatin-enriched pellet (P) in both untreated and TNF-alpha -treated HeLa cells. In contrast, the 65-kDa RAD21 caspase cleavage product (indicated by the arrow) partly dissociated from chromatin and was found in both the supernatant (S) and pellet (P) fractions in TNF-alpha -treated cells. These findings were consistent with our observations that GFP-tagged full-length RAD21 was present in the nucleus of interphase cells, whereas its carboxyl-terminal cleavage product was present in both the cytosol and nucleus (Fig. 4A). Taken together, these results indicate that caspase cleavage of RAD21 leads to its partial removal from chromatin and relocalization to the cytoplasm.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.   Caspase cleavage of RAD21 promotes apoptosis. In panels A and B, HeLa cells were transiently transfected with GFP-tagged cDNAs encoding full-length Rad21 (RAD21), the amino-terminal (N-RAD21), or the carboxyl-terminal (C-RAD21) caspase cleavage products, and GFP-positive cells were scored for apoptotic nuclei as described under "Experimental Procedures." A, representative photomicrographs showing GFP fluorescence (upper panels) and nuclear morphology (lower panels). Apoptotic nuclei are indicated by arrows. B, the data from the three independent experiments (mean ± S.D.) (*, p < 0.0005). C, C-RAD21-induced apoptosis is inhibited by several antiapoptotic proteins but not by wild-type RAD21. HeLa cells were transiently co-transfected with GFP-tagged C-RAD21 and a pcDNA3 plasmid containing empty vector, p35, survivin, Bcl-xL, or WT Rad21 as described under "Experimental Procedures," and GFP-positive cells were scored for apoptotic nuclei. The data are presented as the mean ± S.D. of three independent experiments (*, p < 0.05 and **, p < 0.005).

Caspase Cleavage of RAD21 Promotes Apoptosis-- To determine whether caspase cleavage of RAD21 promotes apoptosis, we transiently transfected HeLa cells with GFP-tagged full-length RAD21 or with cDNAs encoding RAD21's amino-terminal (amino acids 1-279 labeled N-RAD21) or carboxyl-terminal (amino acids 280-631 labeled C-RAD21) caspase cleavage products resulting from cleavage at Asp279. As demonstrated in Fig. 4A, neither full-length RAD21 (present in the nucleus of interphase cells) nor N-RAD21 (present in both the nucleus and cytoplasm) induced apoptotic nuclear alterations. In contrast, transient expression of C-RAD21 induced cellular rounding, a reduction in cytosolic volume (upper panel) and fragmentation of the nucleus (lower panel, apoptotic nuclei indicated by arrows) that typify apoptotic cell death. These data are presented quantitatively in Fig. 4B. Similar results were obtained in MCF-7 breast carcinoma cells (data not presented). To begin to delineate the mechanisms by which C-RAD21 induces apoptosis, we examined whether co-transfecting HeLa cells with a variety of antiapoptotic cDNAs could inhibit C-RAD21-induced apoptosis. As shown in Fig. 4C, both Bcl-xL and survivin potently inhibited C-RAD21-induced apoptosis, whereas p35, a broad spectrum caspase inhibitor, partially inhibited C-RAD21-induced cell death. In contrast, wild-type RAD21 did not antagonize C-RAD21-induced apoptosis, thereby suggesting that C-RAD21 does not induce apoptosis by inhibiting the function of the full-length protein. Taken together, our findings indicate that caspase cleavage of RAD21 plays an active role in the execution of apoptosis by specifically generating a proapoptotic carboxyl-terminal cleavage product.

Caspase Cleavage of RAD21 Does Not Inhibit Cell Cycle Progression-- Because inhibition of RAD21 has recently been shown to cause mitotic arrest as a consequence of misaligned chromosomes at metaphase (41), we examined the effect of RAD21 and each of its caspase cleavage products on cell cycle progression. To this end, HeLa cells were transiently transfected with GFP vector, GFP-tagged RAD21, N-RAD21, or C-RAD21 and released from a double thymidine (G1/S) block for 16 h. As shown in Fig. 5, none of these RAD21 constructs inhibited cell cycle progression. Similar results were obtained at 28 h after release from a double thymidine block except that the sub-G1 population of C-RAD21-transfected cells increased, consistent with the induction of apoptosis (data not presented). These findings indicate that caspase cleavage of RAD21 does not induce mitotic arrest under these conditions.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 5.   Caspase cleavage of RAD21 does not inhibit cell cycle progression. HeLa cells were transiently transfected with GFP vector or GFP-tagged cDNAs encoding WT Rad21, the amino-terminal (N-RAD21), or the carboxyl-terminal (C-RAD21) caspase cleavage products, and cells were released from a double thymidine block for 16 h as described under "Experimental Procedures." GFP-positive cells were sorted by flow cytometry, and their cell cycle phase was determined as detailed under "Experimental Procedures." The data are presented as the mean ± S.D. of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have identified the mitotic cohesin component RAD21 as a novel nuclear caspase substrate using a small pool expression cloning strategy we have described previously (11-13). RAD21 is rapidly cleaved by caspases-3 and -7 at DSPDdown-arrow S280 during the induction of apoptosis by diverse stimuli. Interestingly, the cleavage site Asp279 has been remarkably conserved in RAD21 from Caenorhabditis elegans to humans (15), suggesting that the apoptotic proteolysis of RAD21 at Asp279 may occur in diverse species. In contrast, the structurally related meiotic cohesin REC8 that is also cleaved by separase (42) is not cleaved by caspases, underscoring the specificity of the proteolysis of RAD21 during apoptosis. Moreover, caspase cleavage of RAD21 precedes apoptotic chromatin condensation; we observed RAD21 proteolysis into its characteristic 65-kDa product when 99% of nuclei were intact. We have also demonstrated that caspase cleavage of RAD21 has important functional consequences; caspase-cleaved RAD21 partially dissociates from chromatin and redistributes to the cytosol. Because RAD21 proteolysis precedes apoptotic chromatin condensation, our findings suggest that caspase cleavage of RAD21 and its subsequent removal from chromatin could contribute to this process.

Indeed, we have demonstrated that caspase proteolysis of RAD21 at Asp279 actively participates in the apoptotic destruction of the nucleus by specifically generating a proapoptotic carboxyl-terminal cleavage product (C-RAD21). Strikingly, neither full-length RAD21 nor its amino-terminal cleavage product induces apoptosis, thereby underscoring the specificity of our findings and providing additional support for the notion that caspase cleavage of RAD21 activates a latent proapoptotic function. Our first hypothesis as to the proapoptotic mechanism of C-RAD21 was that it might function as an inhibitor of wild-type RAD21; however, our results do not support this mechanism. First, co-transfection of excess wild-type RAD21 does not suppress C-RAD21-induced apoptosis. Second, RAD21-deficient chicken cells undergo mitotic arrest; cohesion defects lead to chromosome misalignment at metaphase, thereby blocking entry into anaphase and subsequent mitotic exit (41). However, C-RAD21 does not induce mitotic arrest under the conditions we examined, suggesting that it does not disrupt the function of wild-type RAD21. Third, disruption of separase cleavage of RAD21 by a variety of approaches leads to aneuploidy as a result of chromosome segregation defects coupled with DNA re-replication (23, 28, 43). In contrast, C-RAD21 transfected cells show little evidence of aneuploidy even 72 h after transfection when the vast majority of transfected cells are apoptotic (data not shown). Taken together, these findings indicate that C-RAD21-induced apoptosis is not mediated by mitotic arrest or by chromosome segregation defects.

How, then, might RAD21 cleavage promote apoptosis? One possibility is that caspase proteolysis of RAD21, as well as its partial removal from chromatin, might render chromosomal DNA more vulnerable to attack by acinus, CAD, or other factors capable of inducing apoptotic DNA fragmentation, such as endonuclease G or apoptosis-inducing factor (AIF) (44-46). Although we observed that the RAD21 D279E mutant did not inhibit etoposide-induced chromatin condensation/fragmentation (data not shown), the interpretation of this finding is complicated by the presence of the endogenous cleavage-sensitive wild-type RAD21 in all the cancer cell lines we examined. Alternatively, C-RAD21 (which remains partly associated with chromatin) might directly alter chromatin structure through dysregulated interactions with its known binding proteins SMC1 and SMC3, which play a key role in maintaining chromatin structure (18, 19). Caspase cleavage of RAD21 might also contribute to the execution of apoptotic cell death by disrupting the DNA repair function of RAD21. RAD21 repairs double-strand DNA breaks induced by ionizing radiation and/or other DNA damaging agents in fission yeast and chicken cells, and it is essential for viability in fission yeast (14, 17, 41). These findings suggest that the close proximity of sister chromatids resulting from their cohesion facilitates homologous recombination repair of double-strand DNA breaks (41). In addition, mutations in RAD21 sensitize yeast to cell death induced by DNA damage or microtubule-disrupting agents (47). Caspase cleavage of RAD21 at Asp279 separates the conserved amino-terminal domain necessary for DNA repair from other functional domains, including the putative nuclear localization motifs (317KRKRK and 401RKRRK) (15). Consequently, caspase proteolysis of RAD21 likely impairs its DNA repair capabilities by directly inactivating its repair activity and/or by triggering its partial dissociation from chromatin. RAD21, thus, can be added to a growing list of DNA repair enzymes that are cleaved by caspases, including PARP, the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS), the ataxia telangiectasia gene product, and RAD51 (38, 48-53). Hence, the coordinated destruction of the cellular DNA repair machinery by caspases is a central theme in apoptosis that likely expedites apoptotic cell death by allowing CAD-mediated DNA fragmentation to proceed unimpeded by repair processes.

Furthermore, caspase proteolysis of RAD21 likely amplifies the apoptotic signal. Indeed, apoptosis induced by C-RAD21 is partially suppressed by the baculoviral p35 gene product, a broad spectrum caspase inhibitor (54). Hence, caspase proteolysis of RAD21 amplifies the cell death signal by generating a carboxyl-terminal cleavage product that activates more caspases, creating a positive feedback loop. This mechanism for amplifying the apoptotic cell death signal has been described for other caspase substrates such as MEKK-1 and vimentin (13, 55). In addition, we observed that the antiapoptotic protein survivin, an inhibitor of apoptosis (IAP) family member frequently overexpressed in cancer (56), inhibits C-RAD21-induced apoptosis. Although the antiapoptotic mechanism of survivin is unclear, it may directly inhibit caspases (57, 58). Intriguingly, survivin is a chromosomal passenger protein that forms a multimeric complex with other such proteins including inner centromere protein (INCENP) and Aurora-B; these proteins play important roles in chromosome segregation and cytokinesis (59, 60). Recent evidence suggests that RAD21 also behaves like a chromosomal passenger protein; it moves from the centromere to the spindle midzone during anaphase and to the midbody during telophase, and it is necessary for INCENP localization to centromeres (27, 41). Taken together, these studies suggest an intimate relationship between RAD21 and survivin that might account for the ability of survivin to antagonize C-RAD21-induced apoptosis. Clearly, the elucidation of the precise mechanism(s) by which caspase cleavage of RAD21 promotes apoptosis will require further study.

Another intriguing aspect of our findings is the striking parallel between the actions of RAD21 during apoptosis and mitosis. In mammalian cells, the vast majority of RAD21/cohesin is removed from sister chromatid arms prior to their condensation by a poorly understood cleavage-independent mechanism during prophase (25, 27, 61). Indeed, the observation that chromatin arm condensation begins immediately after cohesin removal has led to the speculation that chromatin-associated cohesin may inhibit condensation. During the onset of anaphase, the remaining centromeric cohesin is subsequently removed by the proteolysis of RAD21 by a caspase-like separase that triggers sister separation to opposite poles (25, 28). Moreover, the cleavage of RAD21/SCC1 is necessary for sister chromatid separation; the introduction of a cleavage-resistant RAD21/SCC1 into budding yeast or human HeLa cells blocks sister separation (20, 21, 28). As in mitosis, the apoptotic removal of RAD21 precedes chromatin condensation, although in apoptosis (but not mitosis), the dissociation of RAD21 from chromatin is initiated by proteolysis. Furthermore, in both apoptosis and mitosis, RAD21 is cleaved by distantly related proteases. Taken together, these striking similarities suggest that components of the apoptotic and mitotic machinery have been shared during the course of evolution.

    ACKNOWLEDGEMENTS

We are indebted to Dr. R. Talanian for providing the recombinant caspases used in this study and to Drs. H. Li and H. Perlman for the critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by Grant DAMD 17-00-1-0096 from the Department of Defense Prostate Cancer Research Program, by National Institutes of Health grants NS31957 (to V. L. C.), T32-CA79447 (to M. M.), and 5T32-CA70085 (to M. K.), by a grant from the National Health and Medical Research Council Australia (to M. J. M.), by institutional research grants to Northwestern University from the Howard Hughes Medical Institute and the American Cancer Society (to V. L. C.), and by the Elizabeth Boughton Trust (to V. L. C.).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: Tarry 15-755, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-0644; Fax: 312-908-9032; E-mail: v-cryns@northwestern.edu.

Published, JBC Papers in Press, March 1, 2002, 2002, DOI 10.1074/jbc.M201322200

    ABBREVIATIONS

The abbreviations used are: CAD, caspase-activated DNase; ICAD, inhibitor of CAD; TNF, tumor necrosis factor; GFP, green fluorescent protein; WT, wild-type; PARP, poly(ADP-ribose) polymerase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Steller, H. (1995) Science 267, 1445-1449[Abstract/Free Full Text]
2. Cryns, V. L., and Yuan, J. (1998) Genes Dev. 12, 1551-1570[Free Full Text]
3. Sahara, S., Aoto, M., Eguchi, Y., Imamota, N., Yoneda, Y., and Tsujimoto, Y. (1999) Nature 401, 168-173[CrossRef][Medline] [Order article via Infotrieve]
4. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43-50[CrossRef][Medline] [Order article via Infotrieve]
5. Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) Cell 89, 175-184[CrossRef][Medline] [Order article via Infotrieve]
6. Sakahira, H., Enari, M., and Nagata, S. (1998) Nature 391, 96-99[CrossRef][Medline] [Order article via Infotrieve]
7. Lazebnik, Y. A., Takahashi, A., Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9042-9046[Abstract/Free Full Text]
8. Rao, L., Perez, D., and White, E. (1996) J. Cell Biol. 135, 1441-1455[Abstract/Free Full Text]
9. Takahashi, A., Alnemri, E. S., Lazebnik, Y. A., Fernandes-Alnemri, T., Litwack, G., Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8395-8400[Abstract/Free Full Text]
10. Faleiro, L., and Lazebnik, Y. (2000) J. Cell Biol. 151, 951-960[Abstract/Free Full Text]
11. Lustig, K. D., Stukenberg, T., McGarry, T., King, R. W., Cryns, V. L., Mead, P., Zon, L., Yuan, J., and Kirschner, M. W. (1997) Methods Enzymol. 283, 83-99[CrossRef][Medline] [Order article via Infotrieve]
12. Cryns, V. L., Byun, Y., Rana, A., Mellor, H., Lustig, K., Ghanem, L., Parker, P. J., Kirschner, M. W., and Yuan, J. (1997) J. Biol. Chem. 272, 29449-29453[Abstract/Free Full Text]
13. Byun, Y., Chen, F., Chang, R., Trivedi, M., Green, K., and Cryns, V. L. (2001) Cell Death Differ. 8, 443-450[CrossRef][Medline] [Order article via Infotrieve]
14. Birkenbihl, R. P., and Subramani, S. (1992) Nucleic Acids Res. 20, 6605-6611[Abstract/Free Full Text]
15. McKay, M. J., Troelstra, C., van der Spek, P., Kanaar, R., Smit, B., Hagemeijer, A., Bootsma, D., and Hoeijmakers, J. H. (1996) Genomics 36, 305-315[CrossRef][Medline] [Order article via Infotrieve]
16. Guacci, V., Koshland, D., and Strunnikov, A. (1997) Cell 91, 47-57[CrossRef][Medline] [Order article via Infotrieve]
17. Birkenbihl, R. P., and Subramani, S. (1995) J. Biol. Chem. 270, 7703-7711[Abstract/Free Full Text]
18. Nasmyth, K., Peters, J. M., and Uhlmann, F. (2000) Science 288, 1379-1385[Abstract/Free Full Text]
19. Hirano, T. (2000) Annu. Rev. Biochem. 69, 115-144[CrossRef][Medline] [Order article via Infotrieve]
20. Uhlmann, F., Lottspeich, F., and Nasmyth, K. (1999) Nature 400, 37-42[CrossRef][Medline] [Order article via Infotrieve]
21. Uhlmann, F., Wernic, D., Poupart, M.-A., Koonin, E., and Nasmyth, K. (2000) Cell 103, 375-386[CrossRef][Medline] [Order article via Infotrieve]
22. Tomonaga, T., Nagao, K., Kawasaki, Y., Furuya, K., Murakami, A., Morishita, J., Yuasa, T., Sutani, T., Kearsey, S. E., Uhlmann, F., Nasmyth, K., and Yanagida, M. (2000) Genes Dev. 14, 2757-2770[Abstract/Free Full Text]
23. Rao, H., Uhlmann, F., Nasmyth, K., and Varshavsky, A. (2001) Nature 410, 955-959[CrossRef][Medline] [Order article via Infotrieve]
24. Losada, A., Hirano, M., and Hirano, T. (1998) Genes Dev. 12, 1986-1997[Abstract/Free Full Text]
25. Waizenegger, I. C., Hauf, S., Meinke, A., and Peters, J. M. (2000) Cell 103, 399-410[CrossRef][Medline] [Order article via Infotrieve]
26. Warren, W. D., Steffensen, S., Lin, E., Coelho, P., Loupart, M., Cobbe, N., Lee, J. Y., McKay, M. J., Orr-Weaver, T., Heck, M. M., and Sunkel, C. E. (2000) Curr. Biol. 10, 1463-1466[CrossRef][Medline] [Order article via Infotrieve]
27. Hoque, M. T., and Ishikawa, F. (2001) J. Biol. Chem. 276, 5059-5067[Abstract/Free Full Text]
28. Hauf, S., Waizenegger, I. C., and Peters, J. M. (2001) Science 293, 1320-1323[Abstract/Free Full Text]
29. Cryns, V. L., Bergeron, L., Zhu, H., Li, H., and Yuan, J. (1996) J. Biol. Chem. 271, 31277-31282[Abstract/Free Full Text]
30. Kamradt, M., Chen, F., and Cryns, V. L. (2001) J. Biol. Chem. 276, 16059-16063[Abstract/Free Full Text]
31. Morishima, N. (1999) Genes Cells 4, 401-414[Abstract]
32. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[CrossRef][Medline] [Order article via Infotrieve]
33. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[CrossRef][Medline] [Order article via Infotrieve]
34. Talanian, R. V., Quinlan, C., Trautz, S., Hackett, M. C., Mankovich, J. A., Banach, D., Ghayur, T., Brady, K. D., and Wong, W. W. (1997) J. Biol. Chem. 272, 9677-9682[Abstract/Free Full Text]
35. Thornberry, N., Rano, T., Peterson, E., Rasper, D., Timkey, T., Garcia-Calvo, M., Houtzager, V., Nordstrom, P., Roy, S., Vaillancourt, J., Chapman, K., and Nicholson, D. (1997) J. Biol. Chem. 272, 17907-17911[Abstract/Free Full Text]
36. Watanabe, Y., and Nurse, P. (1999) Nature 400, 461-464[CrossRef][Medline] [Order article via Infotrieve]
37. Parisi, S., McKay, M. J., Molnar, M., Thompson, M. A., van der Spek, P. J., van Drunen-Schoenmaker, E., Kanaar, R., Lehmann, E., Hoeijmakers, J. H., and Kohli, J. (1999) Mol. Cell. Biol. 19, 3515-3528[Abstract/Free Full Text]
38. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347[CrossRef][Medline] [Order article via Infotrieve]
39. Emoto, Y., Manome, Y., Meinhardt, G., Kisaki, H., Kharbanda, S., Robertson, M., Ghayur, T., Wong, W. W., Kamen, R., Weichselbaum, R., and Kufe, D. (1995) EMBO J. 14, 6148-6156[Medline] [Order article via Infotrieve]
40. Janicke, R. U., Sprengart, M. L., Wati, M. R., and Porter, A. G. (1998) J. Biol. Chem. 273, 9357-9360[Abstract/Free Full Text]
41. Sonoda, E., Matsusaka, T., Morrison, C., Vagnarelli, P., Hoshi, O., Ushiki, T., Nojima, K., Fukagawa, T., Waizenegger, I. C., Peters, J. M., Earnshaw, W. C., and Takeda, S. (2001) Dev. Cell 1, 759-770[CrossRef][Medline] [Order article via Infotrieve]
42. Buonomo, S. B., Clyne, R. K., Fuchs, J., Loidl, J., Uhlmann, F., and Nasmyth, K. (2000) Cell 103, 387-398[CrossRef][Medline] [Order article via Infotrieve]
43. Jallepalli, P. V., Waizenegger, I. C., Bunz, F., Langer, S., Speicher, M. R., Peters, J. M., Kinzler, K. W., Vogelstein, B., and Lengauer, C. (2001) Cell 105, 445-457[CrossRef][Medline] [Order article via Infotrieve]
44. Li, L. Y., Luo, X., and Wang, X. (2001) Nature 412, 95-99[CrossRef][Medline] [Order article via Infotrieve]
45. Parrish, J., Li, L., Klotz, K., Ledwich, D., Wang, X., and Xue, D. (2001) Nature 412, 90-94[CrossRef][Medline] [Order article via Infotrieve]
46. Susin, S. A., Daugas, E., Ravagnan, L., Samejima, K., Zamzami, N., Loeffler, M., Costantini, P., Ferri, K. F., Irinopoulou, T., Prevost, M. C., Brothers, G., Mak, T. W., Penninger, J., Earnshaw, W. C., and Kroemer, G. (2000) J. Exp. Med. 192, 571-580[Abstract/Free Full Text]
47. Tatebayashi, K., Kato, J., and Ikeda, H. (1998) Genetics 148, 49-57[Abstract/Free Full Text]
48. Casciola-Rosen, L. A., Anhalt, G. J., and Rosen, A. (1995) J. Exp. Med. 182, 1625-1634[Abstract/Free Full Text]
49. Han, Z., Malik, N., Carter, T., Reeves, W. H., Wyche, J. H., and Hendrickson, E. A. (1996) J. Biol. Chem. 271, 25035-25040[Abstract/Free Full Text]
50. Song, Q., Lees-Miller, S. P., Kumar, S., Zhang, Z., Chan, D. W., Smith, G. C., Jackson, S. P., Alnemri, E. S., Litwack, G., Khanna, K. K., and Lavin, M. F. (1996) EMBO J. 15, 3238-3246[Medline] [Order article via Infotrieve]
51. Smith, G., di Fagagna, F., Lakin, N., and Jackson, S. (1999) Mol. Cell. Biol. 19, 6076-6084[Abstract/Free Full Text]
52. Huang, Y., Nakada, S., Ishiko, T., Utsugisawa, T., Datta, R., Kharbanda, S., Yoshida, K., Talanian, R., Weichselbaum, R., Kufe, D., and Yuan, Z.-M. (1999) Mol. Cell. Biol. 19, 2986-2997[Abstract/Free Full Text]
53. Hotti, A., Jarvinen, K., Siivola, P., and Holtta, E. (2000) Oncogene 19, 2354-2362[CrossRef][Medline] [Order article via Infotrieve]
54. Bump, N. J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen, P., Ferenz, C., Franklin, S., Ghayur, T., Li, P., Licari, P., Mankovich, J., Shi, L., Greenberg, A. H., Miller, L. K., and Wong, W. W. (1995) Science 269, 1885-1888[Abstract/Free Full Text]
55. Cardone, M. H., Salvesen, G. S., Widmann, C., Johnson, G., and Frisch, S. M. (1997) Cell 90, 315-323[CrossRef][Medline] [Order article via Infotrieve]
56. Ambrosini, G., Adida, C., and Altieri, D. C. (1997) Nat. Med. 3, 917-921[CrossRef][Medline] [Order article via Infotrieve]
57. Tamm, I., Wang, Y., Sausville, E., Scudiero, D. A., Vigna, N., Oltersdorf, T., and Reed, J. C. (1998) Cancer Res. 58, 5315-5320[Abstract/Free Full Text]
58. Shin, S., Sung, B. J., Cho, Y. S., Kim, H. J., Ha, N. C., Hwang, J. I., Chung, C. W., Jung, Y. K., and Oh, B. H. (2001) Biochemistry 40, 1117-1123[CrossRef][Medline] [Order article via Infotrieve]
59. Skoufias, D. A., Mollinari, C., Lacroix, F. B., and Margolis, R. L. (2000) J. Cell Biol. 151, 1575-1581[Abstract/Free Full Text]
60. Uren, A. G., Wong, L., Pakusch, M., Fowler, K. J., Burrows, F. J., Vaux, D. L., and Choo, K. H. A. (2000) Curr. Biol. 10, 1319-1328[CrossRef][Medline] [Order article via Infotrieve]
61. Sumara, I., Vorlaufer, E., Gieffers, C., Peters, B. H., and Peters, J. M. (2000) J. Cell Biol. 151, 749-762[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. M. Strohecker, F. Yehiely, F. Chen, and V. L. Cryns
Caspase Cleavage of HER-2 Releases a Bad-like Cell Death Effector
J. Biol. Chem., June 27, 2008; 283(26): 18269 - 18282.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
H. Yang, Q. Ren, and Z. Zhang
Cleavage of Mcd1 by Caspase-like Protease Esp1 Promotes Apoptosis in Budding Yeast
Mol. Biol. Cell, May 1, 2008; 19(5): 2127 - 2134.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
L. M. Miller Jenkins, S. J. Mazur, M. Rossi, O. Gaidarenko, Y. Xu, and E. Appella
Quantitative Proteomics Analysis of the Effects of Ionizing Radiation in Wild Type and p53K317R Knock-in Mouse Thymocytes
Mol. Cell. Proteomics, April 1, 2008; 7(4): 716 - 727.
[Abstract] [Full Text] [PDF]