From the 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
Center for Apoptosis Research, Kimmel Cancer Institute, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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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, -radiation, tumor necrosis factor-
, 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
DQPD237
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.
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INTRODUCTION |
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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), -fodrin (13), nuclear lamins (14,
15), Gas2 (16), D4-GDI (17), PITSLRE kinases (18), Rb (19, 20), PKC-
(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.
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EXPERIMENTAL PROCEDURES |
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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 -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--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--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 -galactosidase
expression plasmid (pEF-
-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).
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RESULTS |
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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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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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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
-radiation, BM13674 cells treated with
-radiation, and HeLa cells
treated with TNF-
, 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-
, 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
-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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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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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 Asp237 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 DVTD41
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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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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DISCUSSION |
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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 (,
, and
) 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 DQPD 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 DQPD
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.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. N. Nomura for providing the human Nedd4 cDNA.
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FOOTNOTES |
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* 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 -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.
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REFERENCES |
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