Caspase-mediated Cleavage of the Ubiquitin-protein Ligase Nedd4 during Apoptosis*

Kieran F. HarveyDagger §, Natasha L. HarveyDagger , Julie M. Michael, Gayathri ParasivamDagger , Nigel Waterhouse, Emad S. Alnemriparallel , Dianne Watters, and Sharad KumarDagger **

From the Dagger  Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia,  Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Herston, Queensland 4029, Australia, and parallel  Center for Apoptosis Research, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, gamma -radiation, tumor necrosis factor-alpha , C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237down-arrow sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Apoptosis is characterized by organized dismantling of the cell structure and involves the action of various classes of proteases (1). The main players in the proteolytic cascade activated during apoptosis are caspases, a group of cysteine proteases related to the cell death protein CED-3 in Caenorhabditis elegans (reviewed in Refs. 2-5). Numerous studies have shown that activation of caspases is central to the execution of apoptosis and inhibition of caspases can suppress apoptosis in a variety of situations (2-5). Caspases are synthesized as zymogens which undergo proteolytic cleavage and processing prior to activation (2-5).

The cleavage by caspases requires an aspartate residue at the P-1 position in the substrate. Active caspases cleave a range of cellular substrates during apoptosis. The DNA repair enzyme poly(ADP-ribose) polymerase (PARP)1 was one of the first identified cellular substrates cleaved in apoptosis (6). Caspase-3 was subsequently shown to cleave PARP with high efficiency (7, 8). Several other proteins of diverse cellular functions are cleaved during apoptosis by caspases (reviewed in Ref. 4) and include the catalytic subunit of the DNA-dependent protein kinase (9, 10), U1 70-kDa ribonucleoprotein (11), heteronuclear ribonucleoproteins C1 and C2 (12), alpha -fodrin (13), nuclear lamins (14, 15), Gas2 (16), D4-GDI (17), PITSLRE kinases (18), Rb (19, 20), PKC-delta (21), MDM2 (22), PAK2 (23), the large subunit of replication factor C (24), huntingtin (25), transcription factors SREBP-1 and SREBP-2 (26), focal adhesion kinase (27, 28), DNA fragmentation factor DFF (29), Bcl-2 (30), MEKK-1 (31), and the inhibitor of caspase-activated DNase (32). Cleavage of these and other possible caspase targets is likely to result in changes that occur in a cell undergoing apoptosis by mediating events that are required further downstream, such as abrogation of the repair mechanisms, detachment of the apoptotic cell from surrounding cells/tissue, disruption of the cytoskeleton, initiation of DNA fragmentation, and the formation and engulfment of apoptotic bodies (4).

Apoptosis is not accompanied by random cleavage of a large number of proteins and electrophoresis patterns of proteins from early apoptotic cells do not significantly differ from nonapoptotic cells (12). Additionally, the specificity of caspases involved clearly argues against a large number of proteins being degraded during apoptosis. Thus the proteins that are targeted specifically for degradation by caspases are likely to play some vital role in the apoptotic process. Therefore, it is necessary to identify all caspase substrates that are cleaved during apoptosis so that a clearer picture can emerge about the significance of caspase-mediated proteolysis in apoptosis.

In the present study we describe a novel target, Nedd4, for caspase-mediated cleavage during apoptosis. Nedd4 was initially identified as a developmentally regulated gene in the mouse (33). Nedd4 is widely expressed in many tissues and cell types and encodes an evolutionarily conserved ubiquitin-protein ligase (34-37). In addition to a ubiquitin-protein ligase domain, Nedd4 protein contains multiple WW domains and a calcium/lipid-binding (CaLB) domain (34, 35). Through its WW domains, Nedd4 has been shown to interact with the epithelial sodium channel (ENaC) subunits (35), hematopoietic transcription factor p45/NF-E2, and RNA polymerase II (38), and probably mediates their turnover via ubiquitin-mediated pathways. Here we show that in cells undergoing apoptosis in response to various agents, Nedd4 is specifically cleaved in a caspase-dependent manner. The kinetics of Nedd4 cleavage are similar to that of caspase activation and the cleavage of another caspase substrate, PARP.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines and Culture Conditions-- The Burkitt's lymphoma cell lines BL29, BL30A, BM13674, and BL30K were maintained in RPMI 1640 medium containing either 20% (BL30A) or 10% (BL30K, BL29, and BM13674) fetal calf serum. Jurkat and Daudi cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, while HeLa, NIH-3T3, and COS cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cells were induced to undergo apoptosis by treatment with 200 ng/ml anti-APO-1/Fas antibody (Upstate Biotechnology) or by exposure to either tumor necrosis factor (TNF) (10 ng/ml) and cycloheximide (10 µg/ml), etoposide (40 µM), C-8 ceramide (20 µM), or 20 gray of gamma -radiation from a 137Cs source. The extent of apoptosis was monitored by microscopic examination of cellular nuclei stained with 4,6-diamino-2-phenylindole.

Preparation of Apoptotic Cell Extracts-- Etoposide or Fas antibody-treated Jurkat cells were washed with phosphate-buffered saline and then resuspended in extraction buffer (50 mM PIPES, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, and CompleteTM protease inhibitors (Boehringer Mannheim). Cells were allowed to swell on ice for 20 min then lysed by freeze/thawing. Lysates were centrifuged at 200 × g for 5 min at 4 °C, and supernatants were then centrifuged at 9000 × g for 15 min at 4 °C. Supernatants were stored in aliquots at -70 °C until used.

Nedd4 Antibodies-- A polyclonal antibody (N4ab1) raised against the region of Nedd4 encompassing amino acids 210-720 of mouse Nedd4 has been described previously (34). An additional polyclonal rabbit serum (N4ab2) was raised against a glutathione S-transferase fusion protein containing the three WW domains (amino acid residues 235-511) of mouse Nedd4.2 Escherichia coli cultures were induced for 3-5 h with 1 mM isopropyl-1-thio-beta -D-galactopyranoside and glutathione S-transferase-WW fusion protein purified by affinity chromatography according to the instructions provided by Pharmacia Biotech Inc. To raise rabbit antisera, 300 µg of affinity purified glutathione S-transferase-WW protein mixed with Freund's complete adjuvant were inoculated subcutaneously at multiple sites. Boosters, at monthly intervals, consisted of 200 µg of the fusion protein in Freund's incomplete adjuvant introduced subcutaneously. Rabbits were bled 6-10 days post-booster, and antibody titers were determined by immunoblotting. All injections and animal handling were carried out according to approved protocols. N4ab2 was affinity-purified on an antigen-coupled Sepharose column.

SDS-PAGE and Western Blotting-- Cell extracts (10-20 µg of protein) were boiled in SDS-PAGE loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 20% glycerol, 4% SDS, 0.2% bromphenol blue) for 5 min then centrifuged at 9000 × g for 5 min. Proteins were resolved on SDS-PAGE gels and transferred to polyvinylidine difluoride (Dupont) or nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked in 5% skim milk in phosphate-buffered saline containing 0.05% Tween 20 at 4 °C overnight. Blots were probed with either N4ab1 (total serum) at 1/5000 dilution for 1 h, affinity-purified N4ab2 at 1/1000 for 1 h, an anti-ICH-1L rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) at a 1/500 dilution for 4 h, an anti-PARP polyclonal antiserum (Boehringer Mannheim) at a dilution of 1/2000 for 1 h or an anti-CPP32 monoclonal antibody (Transduction Laboratories) at a dilution of 1/1000 for 4 h, at room temperature. Following incubation with appropriate horseradish peroxidase-coupled secondary antibodies, signals were detected using the ECL system (Amersham Corp.).

Recombinant Caspases-- Recombinant caspases were expressed in E. coli as described previously (9). Briefly, exponentially growing bacteria carrying the protease expression plasmids were induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3-6 h and lysed by sonication in a lysis buffer containing 25 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM dithiothreitol, 10% sucrose, and 0.1% CHAPS. The lysates were centrifuged at 10,000 × g for 10 min, and clear bacterial extracts were collected and stored at -20 °C. To confirm the activity of proteases, aliquots of the extracts were incubated with 100 µM DEVD-AFC [Z-Asp-Glu-Val-Asp-(7-amino-4-trifluoromethyl coumarin)] for caspase-2, -3, -6, and -7 or 100 µM YVAD-AFC [Z-Tyr-Val-Ala-Asp-(7-amino-4-trifluoromethyl coumarin)] for caspase-1, for 30 min at 37 °C and release of AFC was measured by spectrofluorometry at excitation and emission wavelengths of 400 and 505 nm, respectively (39). Both substrates and caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) were purchased from Enzyme Systems Products (California). N-acetyl-Tyr-Val-Ala-Asp-chrolomethyl ketone (YVAD-cmk) and N-acetyl-Asp-Glu-Ala-Asp aldehyde (DEVD-CHO) were from Bachem (Switzerland).

Site-directed Mutagenesis-- Site-directed mutagenesis of mouse Nedd4 cDNA in a pBluescript vector (Stratagene) was carried out according to a published protocol (40). Asp residues at position 237 (DQPD237), 288 (DLTD288), 293 (DNDD293), and 316 (DGPD316) of mouse Nedd4 (34) were replaced with Gly.

In Vitro Cleavage Assays-- Various wild-type and mutant Nedd4 cDNA constructs in pBluescript (SK+) were used as templates for coupled transcription/translation using a T7 kit (Promega) and [35S]methionine (ICN). In addition to full-length mouse Nedd4, modified versions of wild-type and mutant cDNAs encoding carboxyl-terminally truncated proteins were generated by removing a 1.0-kilobase pair HindIII fragment from the 3'-end of the coding frame. 35S-Labeled Nedd4 protein was incubated with the appropriate recombinant caspase or extract from apoptotic Jurkat cells for 3 h at 37 °C. Approximately equivalent amounts of caspases (as determined by active site titration using Z-VAD-fmk) or up to 50 µg of cell extracts were used. The reactions were terminated by the addition of SDS-PAGE loading buffer and heating at 100 °C for 5 min. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes and exposed to x-ray film.

Transient Transfection and Cell Killing Experiments-- For cell death assays, NIH-3T3 cells were plated at 2.5 × 105/well in 6-well dishes the day before transfection. For each well, we used 2 µg of the Nedd4 or Nedd2 cDNA cloned into pCXN2 expression plasmid (41) mixed with 0.5 µg of the beta -galactosidase expression plasmid (pEF-beta -galactosidase). All transfections were carried out using Superfect reagent (Qiagen) according to the manufacturer's protocol. Cells were fixed, stained with X-gal 18-24 h post-transfection, and observed by light microscopy as described previously (42). The pCXN2-Nedd2 construct was used as a positive control and has been described previously (42).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cleavage of Nedd4 Protein during Fas-mediated Apoptosis in Jurkat Cells-- To check whether Nedd4 cleavage occurs in vivo during apoptosis mediated by a physiological stimulus, we exposed Jurkat T cells to an apoptosis-inducing Fas antibody and monitored the cleavage of Nedd4 by immunoblotting. For immunoblotting we employed two different polyclonal Nedd4 antibodies (N4ab1 and N4ab2), both of which detect the mouse and the human Nedd4 protein of 120~,130 kDa. Although the predicted size of mouse Nedd4 is 103 kDa, while human Nedd4 is approximately 5 kDa larger than the mouse protein due to the presence of an additional WW domain, in SDS-polyacrylamide gels these proteins migrate more slowly, giving a larger apparent molecular weight (34). In some human cell lines, a smaller band of approximately 110 kDa is also detected by both antibodies, which we believe is derived from alternative splicing or represents a proteolytic fragment of Nedd4 (Fig. 1). This smaller band has also been noted in rat tissues using two independently raised antibodies against rat Nedd4 protein (37). Both antibodies detected identical Nedd4 cleavage products during Fas-mediated apoptosis in Jurkat cells, therefore data with N4ab2 only are shown (Fig. 1). Within 2 h of Fas antibody treatment, disappearance of both Nedd4 bands was clearly visible concomitant with the appearance of a 95-kDa band. By 4 h no intact Nedd4 protein was visible.


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Fig. 1.   Nedd4 protein undergoes cleavage during Fas-mediated apoptosis in Jurkat T cells. Jukat T-cells were treated with an anti-Fas monoclonal antibody for the indicated duration of time. Cell extracts were subjected to immunoblot analyses using antibodies against Nedd4 (N4ab2), PARP, the small subunit of caspase-2, or the large subunit of caspase-3. Percent apoptotic cells as assessed by 4,6-diamino-2-phenylindole staining of the sample used for immunoblotting, are indicated. Although not shown in these blots, Jurkat cells preincubated with Z-VAD-fmk prior to the addition of Fas antibody, did not show any activation of caspase-2 and -3.

Nedd4 Cleavage Is Likely to Be Mediated by Caspases-- The time course of cleavage of Nedd4 was similar to that of PARP cleavage in these cells (Fig. 1). Both the cleavage of Nedd4 and PARP was completely inhibited when Jurkat cells were treated with anti-Fas antibody in the presence of the general cell-permeable caspase inhibitor Z-VAD-fmk (Fig. 1). We further investigated whether cleavage of Nedd4 correlated with the activation of caspases. As revealed by the appearance of caspase subunits and disappearance of precursors, both caspase-2 and caspase-3 were rapidly activated following antibody ligation. The time course of activation of both these caspases was similar to the time course of Nedd4 and PARP cleavage, suggesting that cleavage of Nedd4 is dependent on the onset of apoptosis and is likely to be mediated by caspases.

Cleavage of Nedd4 Occurs in Apoptosis Induced by Etoposide-- To check whether Nedd4 cleavage occurs in response to treatment by other apoptosis inducing agents, we subjected Jurkat T cells to 40 µM etoposide treatment. Cleavage of Nedd4 protein, similar to that seen when Jurkat cells were treated with anti-Fas antibody was evident, although the kinetics of cleavage were slower reflecting slower apoptotic induction (Fig. 2). For example at 8 h after treatment with etoposide, 43% of cells showed apoptotic morphology, as compared with 72% observed for Fas antibody treated cells at the same time point. Again, the time course of Nedd4 cleavage was identical to PARP cleavage and the appearance of caspase-2 and caspase-3 subunits, as detected by immunoblotting (Fig. 2).


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Fig. 2.   Cleavage of Nedd4 in Jurkat T cells following treatment with 40 µM etoposide. At the indicated times following treatment with etoposide, cell extracts were subjected to immunoblot analysis using various antibodies as in Fig. 1.

Cleavage of Nedd4 in Various Cell Lines Undergoing Apoptosis-- We further analyzed whether other cell types undergoing apoptosis also show cleavage of Nedd4 protein. In the Burkitt's lymphoma cell line BL30A treated with either C-8 ceramide or gamma -radiation, BM13674 cells treated with gamma -radiation, and HeLa cells treated with TNF-alpha , Nedd4 cleavage similar to that seen in Jurkat cells was clearly evident (Fig. 3, A and B). Although the major cleavage product in all cases was a 95-kDa band, in BM13674 cells, and to some extent BL30A cells, intermediate size bands were also visible (Fig. 3A). In HeLa cells treated with TNF-alpha , significant Nedd4 cleavage was evident by 4 h when only a fraction of cells (<15%) appeared apoptotic as assessed by nuclear staining (Fig. 3B). In three Burkitt's lymphoma cell lines that are resistant to apoptosis induced by gamma -radiation (43) no cleavage of Nedd4 was evident (Fig. 3C). These results suggest that Nedd4 cleavage is a general apoptosis-related phenomenon and not restricted to a particular cell type or to a specific apoptosis-inducing agent.


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Fig. 3.   Nedd4 cleavage occurs in different cell types and in response to various apoptosis inducing signals. Various cultured cells, as indicated, were exposed either to 20 gray of gamma -radiation and harvested 8 h post-treatment (A and C), 20 µM C-8 ceramide for 24 h (A), or 10 ng/ml TNF-alpha and 10 µg/ml cycloheximide for the indicated duration of time (B). Three Burkitt's lymphoma cell lines that are resistant to apoptosis induced by gamma -radiation did not show any appreciable cleavage of Nedd4 protein (C). At 8 h following irradiation, 1-5% of BL29, BL30K, and Daudi cells exhibited apoptotic morphology as compared with 80-85% for BL30A and BM13674 (not shown). Note that BL29 cells express considerably lower amounts of Nedd4 protein compared with other cell lines. Cell extracts were subjected to immunoblotting using N4ab2.

In Vitro Cleavage of Nedd4 by Various Caspases-- An examination of Nedd4 protein sequence showed that it contains several DXXD (consensus sequence for cleavage by downstream caspases such as caspase-3 and -7, Ref. 44) sequences which are conserved in mouse, rat and human proteins (34). We therefore investigated whether Nedd4 protein can be cleaved by caspases in vitro. As shown in Fig. 4, indeed both human and mouse Nedd4 proteins were cleaved by caspase-1, -3, -6, and -7, and extracts from apoptotic cells, but not by caspase-2. The major cleavage products in all cases were roughly similar in size suggesting that the recombinant caspases, and caspases activated in apoptotic Jurkat cells cleave Nedd4 either at the same sites or in the same vicinity. Mouse Nedd4 generated fragments of 90 kDa and a doublet of around 25 kDa when treated with caspase-1, -7, and apoptotic cell extracts, while caspase-3 and -6 produced a single band around 25 kDa in addition to the 90-kDa fragment (Fig. 4A). Cleavage of mouse Nedd4 by caspases and cell extracts also generated a cleavage intermediate of around 115 kDa. With human Nedd4, the main cleavage products were approximately 95 and 20 kDa in size (Fig. 4B). The intermediate corresponding to the 115-kDa mouse Nedd4 product was not evident and a doublet of around 20 kDa was seen with caspase-1 and caspase-6. These results showed that there is at least one caspase cleavage site common to both mouse and human proteins. Incubation of apoptotic cell extracts with 100 nM DEVD-CHO, but not 100 nM YVAD-cmk abolished their Nedd4 cleaving activity, suggesting that a caspase-3-like protease in cell extracts mediates the cleavage of Nedd4.


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Fig. 4.   In vitro cleavage of mouse and human Nedd4 protein by various caspases. 35S-Labeled mouse (A) and human (B) Nedd4 proteins were generated by in vitro translation and incubated with recombinant caspases or extracts prepared from apoptotic Jurkat cells. Where indicated, cell extracts (CE) were incubated with 100 nM of either YVAD-cmk or DEVD-CHO for 30 min prior to the addition of Nedd4 protein. Major cleavage products are shown on the left-hand side of the panels. Minor cleavage bands and intermediates are indicated by open arrowheads on the right-hand side.

Mapping of the Caspase Cleavage Sites in Nedd4-- The two antibodies used in this study were raised against residues 210-720 (N4ab1) and residues 235-511 (N4ab2) of the Nedd4 protein (Fig. 5). Both detected the same 95-kDa human Nedd4 cleavage product which was identical in size to that seen in in vitro cleavage experiments. The smaller fragments of Nedd4 (20-25 kDa) seen in in vitro cleavage assays were not detected by either antibody. Therefore we predicted that the major cleavage site may be located 20-25 kDa from either the amino or carboxyl terminus of the Nedd4 protein. Although there are three DXXD sequences conserved in mouse, human and rat Nedd4 sequences (Fig. 5), only one (DQPD237 in mouse or DQPD206 in known human sequence) would generate the expected size fragments. We therefore generated a truncated form of mouse Nedd4 protein lacking the two carboxyl-terminal DXXD sites (DVND774 and DGVD887). When subjected to digestion by recombinant caspases, the truncated Nedd4 protein was cleaved generating fragments of about 25 kDa, possibly representing the amino terminus of the protein, and 55 kDa from the region downstream of the 25-kDa fragment (Fig. 6). In the mouse Nedd4 sequence, besides the DQPD237, three other DXXD sequences DLTD288, DNDD293, and DGPD316 are located in the vicinity of the putative cleavage site (Fig. 5). We altered all four putative cleavage sites by replacing the P-1 Asp with a Gly. While D288G, D293G, and D316G had no effect on cleavage of mouse Nedd4 by various caspases (data not shown), the D237G mutation in mouse sequence abolished the generation of both 55- and 25-kDa fragments by caspase-1, -3, -7, and apoptotic cell extracts, without affecting cleavage by caspase-6 (Fig. 6B). These results suggest that in vitro cleavage by caspases-1, -3, and -7 occurs at Asp237down-arrow in mouse protein (Asp238 in rat and Asp206 in the incomplete human sequence lacking the amino terminus, Ref. 34). In all mutants, the generation of the 75-kDa intermediate (equivalent to the 115-kDa intermediate of full-length Nedd4) was not affected (Fig. 6B). We believe that this fragment arises due to cleavage of mouse Nedd4 at the DVTD41down-arrow site. No corresponding site is present in the known human sequence, and accordingly, the intermediate product is not seen in human Nedd4 incubated with various caspases or apoptotic cell extracts (Fig. 4B).


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Fig. 5.   Structure of Nedd4 proteins used for in vitro translation and the location of various putative caspase cleavage sites. CaLB, WW domains (numbered 1-4), and ubiquitin-protein ligase domain (Hect) are indicated. The three DXXD sequences conserved between mouse, human, and rat Nedd4 are shown (Asp206, Asp815, and Asp927 (D206, D815, and D927) in human and Asp237, Asp774, and Asp887 (D237, D774, and D887) in mouse). The position of DXXD sites mutated in mouse (Asp237, Asp288, Asp293, and Asp316 (D237, D288, D293, and D316)) are indicated as well as Asp41 (D41), which is believed to be cleaved in vitro and is only present in mouse Nedd4. The regions of mouse Nedd4 protein that were used to raise the two polyclonal rabbit antisera (N4ab1 and N4ab2) are indicated by bars. Note that the human Nedd4 clone used for in vitro translation is incomplete at the amino terminus.


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Fig. 6.   Mapping of the caspase cleavage sites in Nedd4 protein. Truncated versions of the wild type (A) or D237G mutant (B) Nedd4 proteins were translated from cDNA constructs lacking the coding region for the carboxyl-terminal 352 residues of the mouse Nedd4 protein. 35S-Labeled proteins were exposed to various caspases and extracts from apoptotic Jurkat cells (CE) as described in Fig. 4.

Nedd4 Cleavage Products Do Not Induce Apoptosis-- To check whether Nedd4 cleavage can alter the apoptotic response in cells, we attempted to generate mammalian cells stably expressing high levels of transfected wild-type and D237G mutant Nedd4. Despite several attempts, we were unable to generate such cells which led us to conclude that constitutively high level expression of Nedd4 may be cytotoxic and such cells are progressively deleted during G418 selection of transfectants. In further experiments to understand the significance of Nedd4 cleavage in apoptosis, we analyzed whether full length Nedd4 or its cleavage fragments can induce cell death. The rationale for this was based on the knowledge that caspase cleavage of proteins such as PAK-2 and Bcl-2 can generate products which are able to promote apoptotic changes (25, 30). We transiently transfected NIH-3T3 cells with various Nedd4 expression constructs. While a Nedd2 (caspase-2) expression construct efficiently killed transfected cells, neither the wild-type, mutant, or a truncated Nedd4 showed any significant cell killing activity (Fig. 7).


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Fig. 7.   Expression of wild type and mutant Nedd4 does not induce apoptosis. Various expression constructs in pCXN2 vector were co-transfected with a beta -galactosidase expression vector. At 18 h post-transfection, cells were washed, fixed, and stained with X-gal. beta -Galactosidase-positive blue cells were scored for apoptotic morphology. Data, derived from two separate experiments performed in duplicate, are presented as mean percent morphologically apoptotic cells in the total beta -galactosidase-positive cells. Nedd4c represents the construct containing the coding region for an amino-terminally truncated mouse Nedd4 protein (amino acids 238-887).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Several proteins have recently been shown to be cleaved during apoptosis by caspases. Here we have discovered Nedd4 as another target of caspases. Nedd4 was first identified as a developmentally down-regulated gene in the mouse central nervous system (33). It contains a region homologous to the E6AP carboxyl terminus (the hect domain), three WW domains (four in the human protein) and a CaLB domain (34, 35). Recently, Nedd4 was shown to have ubiquitin-protein ligase activity (36). Rat Nedd4 binds to all three subunits (alpha , beta , and gamma ) of the ENaC via its WW domains (35). Nedd4 binds to the ENaC by contacting the proline rich PY motifs, located in the C-terminal region of the subunits of ENaC shown to be mutated in Liddle's syndrome, an autosomal dominant form of hypertension (35). In mandibular gland duct cells, Nedd4 mediates the down-regulation of ENaC activity in response to increased intracellular sodium.2 A recent study shows that the WW domains of Nedd4 also interact with RNA polymerase II and the hematopoietic transcription factor p45/NF-E2 in vitro (38). Although Nedd4 is widely expressed in many tissues (34, 35, 37), its expression is temporally and spatially regulated during embryonic development and it is likely that it interacts with other as yet undiscovered proteins.3 Therefore, it is predicted that Nedd4 is involved in regulating a number of cellular proteins through ubiquitination.

From the mutagenesis studies, one of the cleavage sites in Nedd4 protein was mapped as a DQPDdown-arrow sequence, present in both mouse and human proteins. This sequence is also conserved in rat Nedd4 (35). As DXXD is the preferred cleavage sequence for caspase-2, -3, and -7 (44), it would appear that in vivo one or more of these caspases mediate the cleavage of Nedd4 during apoptosis. In vitro cleavage data suggest that both mouse and human Nedd4 proteins are cleaved by caspase-1, -3, -6, and -7, but not caspase-2. Caspase-2 is an upstream protease based on its ability to autoprocess following homodimerization (45) and thus it was not surprising that it did not cleave Nedd4. Inhibitor studies indicated that caspase-1 is unlikely to be involved, at least in Jurkat cells, and one or more caspase-3-like proteases are involved (Fig. 4). Caspase-6 does not appear to cleave at the DQPDdown-arrow sequence as the D237G mutation did not affect cleavage by this caspase (Fig. 5). Moreover, DXXD is not a preferred cleavage sequence for caspase-6 (44). Caspase-6 may thus cleave Nedd4 at another aspartate residue in the vicinity of Asp237. One likely candidate is Asp216 in mouse Nedd4 (or the corresponding Asp186 in human protein), cleavage at which would generate a fragment slightly smaller than 25 kDa, which is indeed the case (Fig. 4). Based on in vitro cleavage and inhibitor studies, most of the proteins known to be degraded in apoptotic cells are predicted to be cleaved by the downstream "effector" caspases which include caspase-3, -6, and -7 (3-5). It is thus reasonable to assume that in vivo cleavage of Nedd4 is also mediated by one or more of these caspases.

Interestingly, the cleavage of Nedd4 removes the amino-terminal CaLB domain from the rest of the protein without disrupting the WW domains and the ubiquitin-protein ligase domain of Nedd4. As evident from ENaC studies, the WW domains in Nedd4 are responsible for binding to the target protein, while a region between the WW domains and the hect domain binds the ubiquitin-conjugating enzyme (36). This would suggest that cleavage of Nedd4 by caspases during apoptosis is unlikely to disrupt substrate recognition and enzymatic activity of Nedd4. The CaLB domain of Nedd4 expressed in E. coli binds phospholipid vesicles in a calcium dependent manner (46). In vivo, the CaLB domain is responsible for the Ca2+-dependent redistribution of Nedd4 from cytoplasm to membrane in canine kidney cells, possibly bringing it near its membrane associated targets, such as ENaC (46). Therefore, the removal of CaLB domain during apoptosis would render Nedd4 protein unable to redistribute and bind to some of its physiological targets. We notice that in later stages of apoptosis, the 95-kDa Nedd4 cleavage product is also degraded. Thus it is also possible that removal of the amino-terminal region of Nedd4 makes it unstable and the cleaved product is degraded, perhaps by a proteasome-mediated pathway. Abrogation of Nedd4 function in apoptosis may be an energy-saving exercise, or alternatively, Nedd4 may normally be required to mediate the turnover of a protein(s) which is required for the apoptotic function. Although the significance of Nedd4 cleavage during apoptosis is not entirely clear at present, this report shows for the first time that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

    ACKNOWLEDGEMENT

We are grateful to Dr. N. Nomura for providing the human Nedd4 cDNA.

    FOOTNOTES

* This work was supported by funds from the Wellcome Trust and National Health and Medical Research Council of Australia (to S. K.) and in part by National Institute of Health Grant AG13487 (to E. S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Dawes Postgraduate Scholarship from the Royal Adelaide Hospital Research Fund.

** Wellcome Trust Senior Fellow in Medical Science. To whom correspondence should be addressed at the Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, PO Box 14, Rundle Mall, Adelaide, SA 5000, Australia. Tel.: 61-8-8222-3738; Fax: 61-8-8222-3139; E-mail: sharad.kumar{at}imvs.sa.gov.au.

1 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; CHAPS, 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate; PIPES, 1,4-piperazinediethanesulfonic acid; TNF, tumor necrosis factor; PAGE, polyacrylamide gel electrophoresis; hect, homologous to E6AP C-terminal; ENaC, epithelial sodium channel; CaLB, calcium/lipid-binding domain; AFC, 7-amino-4-trifluoromethyl coumarin; fmk, fluoromethyl ketone; cmk, chrolomethyl ketone; DEVD-CHO, N-acetyl-Asp-Glu-Ala-Asp aldehyde; X-gal, 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside.

2 A. Dinudom, K. F. Harvey, P. Komwatana, J. A. Young, S. Kumar, and D. I. Cook, submitted for publication.

3 S. Kumar and K. F. Harvey, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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