From the Department of Pharmacology and Cancer
Biology, Duke University, Durham, North Carolina 27710 and the
¶ Division of Biology, California Institute of Technology,
Pasadena, California 91125
Received for publication, September 23, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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In most cases, apoptotic cell death culminates in
the activation of the caspase family of cysteine proteases, leading to
the orderly dismantling and elimination of the cell. The IAPs
(inhibitors of apoptosis) comprise a family of
proteins that oppose caspases and thus act to raise the apoptotic
threshold. Disruption of IAP-mediated caspase inhibition has been shown
to be an important activity for pro-apoptotic proteins in
Drosophila (Reaper, HID, and Grim) and in mammalian cells
(Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these
proteins are able to stimulate the ubiquitination and degradation of
IAPs by a mechanism involving the ubiquitin ligase activity of the IAP
itself. In this report, we show that the Drosophila RHG
proteins (Reaper, HID, and Grim) are themselves substrates for
IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP
ubiquitin-ligase activity and a stable interaction between Reaper and
the IAP. Additionally, degradation of Reaper can be blocked by mutating
its potential ubiquitination sites. Most importantly, we also show that
regulation of Reaper by ubiquitination is a significant factor in
determining its biological activity. These data demonstrate a novel
function for IAPs and suggest that IAPs and Reaper-like proteins
mutually control each other's abundance.
Apoptosis is a regulated form of cell death that can be triggered
by a variety of intracellular and extracellular signals. Although
apoptosis research has revealed a plethora of signaling pathways that
can contribute to the decision of a cell to die, the ultimate
responsibility for completing the cell death program (in most cases)
resides with the cysteine proteases known as caspases (reviewed in
Refs. 1-3). Caspase-mediated cleavage of cellular substrates underlies
many of the ordered processes that occur as the dying cell shrinks,
degrades its DNA, and packages the remains for subsequent phagocytosis.
As such, regulation of caspase activation is a key control point for
the apoptotic machinery.
Acting in opposition to the caspases are the IAPs
(inhibitors of apoptosis). These proteins
function, at least in part, by directly binding to and inhibiting
active caspases (Refs. 4-6; reviewed in Refs. 7 and 8). Many of the
IAPs also contain a RING finger domain, and like other RING finger
proteins, these IAPs can function as ubiquitin ligases (9, 10).
Ubiquitin ligases work in conjunction with ubiquitin-activating and
ubiquitin-conjugating enzymes to covalently link ubiquitin to lysines
present in the target protein. Sequential linkage of multiple ubiquitin
moieties (polyubiquitination) then results in targeting of the
ubiquitinated protein for destruction by the proteasome (reviewed in
Ref. 11). Although it is well established that IAPs can inhibit
caspases through physical binding, the significance of IAP
ubiquitin-ligase activity for the anti-apoptotic function of IAPs is
not yet fully understood. For example, IAPs have been reported to
ubiquitinate and promote the degradation of caspases, which could
clearly favor cell survival (9, 12). Conversely, IAPs can
auto-ubiquitinate and thereby promote their own destruction, which
might be expected to favor cell death (9, 10, 12-17).
In Drosophila, the caspase-inhibiting function of IAPs is
antagonized by the pro-apoptotic proteins Reaper, HID, and Grim (the
RHG1 proteins). The genes
encoding these proteins are closely linked, and their combined deletion
results in a generalized failure of apoptosis during development of the
Drosophila embryo (18). Expression of these proteins is
tightly controlled at the level of transcription, and ectopic
expression of the RHG proteins in either insect or vertebrate cells can
initiate apoptosis (19-26). Although Reaper, HID, and Grim do not
share overall homology, they do share a short region at their extreme N
termini that is responsible for binding IAPs (the so-called RHG motif).
The interaction between the RHG motif and the IAP has been suggested to
preclude the IAP/caspase interaction, thereby alleviating caspase
inhibition (27-30). The N-terminal RHG motif is also present in
several vertebrate apoptotic regulators, including Smac/Diablo and
Omi/HtrA2 (18, 19, 22, 28, 29, 31-34). As a consequence, these
proteins can directly bind and inhibit IAPs in a manner similar to that of Reaper, HID, and Grim.
Recently, we and others have reported that Reaper, HID, and Grim can
also promote apoptosis by stimulating IAP ubiquitination and
degradation (12-17). Both IAP ubiquitin-ligase activity and the RHG
motif are required for this particular interaction, but it is not yet
clear whether other regions of the RHG protein or the IAP will also be
required. Moreover, although Smac and Omi bind IAPs, there have been no
reports that they also stimulate IAP degradation.
In this report, we provide evidence that the interaction between the
IAPs and the RHG proteins is a two-way street with regard to
ubiquitination and proteasome-mediated degradation; that is, not only
do the RHG proteins stimulate the ubiquitination and degradation of
IAPs, but the IAPs also stimulate the ubiquitination and degradation of
the RHG proteins. Our data demonstrate that the RHG motif is required
for IAP-mediated degradation of Reaper, which suggests that a stable
interaction between Reaper and the IAP is required for this form of
regulation. Moreover, Reaper degradation can be blocked by inhibiting
the proteasome, and when Reaper is stabilized by mutating potential
ubiquitination sites, it becomes a markedly more potent inducer of
apoptosis. Collectively, these data indicate that IAP regulators such
as Reaper are targeted for degradation by IAP ubiquitin-ligase
activity, and that this regulation is a significant factor in
determining their biological activity.
Immunofluoresence and in Situ Hybridization--
The following
genetic crosses were used to generate the imaginal discs in Fig. 1:
EnG4 × UAS-P35 (panels A and D), EnG4 × UAS-HID, UAS-P35 (panels B, E, and G),
and EnG4 × UAS-Rpr, UAS-P35 (panels C, F,
H, and I). DIAP1 and HID proteins were detected by indirect antibody fluorescence using the appropriate antibodies and
standard techniques. HID and Reaper RNAs were detected by hybridization
of digoxigenin-labeled probes, followed by dye staining.
Preparation of Recombinant Drosophila Proteins--
DIAP1D20E
was prepared as described previously (43) from GST-TEV-DIAP1D20E
followed by TEV cleavage. pET23a-Reaper-GST was expressed in
BL21(DE3)pLysS by 0.4 mM
isopropyl-1-thio- In Vitro Ubiquitination--
Drosophila embryo
extract was made as follows. 0-5-h-old embryos were collected and aged
for 6 h at 25 °C. The embryos were dechorionated with 50%
bleach, rinsed, suspended in equal volumes of buffer EX (20 mM Tris, pH 7.5, 100 mM NaCl, 5 mM
ATP, 2.5 mM MgCl2, 1 mM
dithiothreitol, 0.25 M sucrose) and homogenized. The supernatant was collected after centrifugation at 12,000 × g. The concentration of the extract was ~10 µg/µl. The
ubiquitination assay was carried out as follows. Hid-His6,
Reaper-GST, or Grim-His6 protein (100 ng each) was
preincubated with 1 µl of embryo extract at room temperature for 10 min. Subsequently, DIAP1D20E (400 ng) and His-ubiquitin (Calbiochem; 3 µg total) were added in buffer UR (25 mM Tris, pH 7.5, 0.5 mM dithiothreitol, 2 mM ATP, 5 mM MgCl2). The reaction was incubated at
37 °C for 40 min and stopped by adding SDS sample buffer. The
ubiquitination of each protein was visualized after separation of
proteins by SDS-PAGE, transfer to polyvinylidene difluoride, and
immunoblotting with the appropriate antibody.
Generation of Reaper Antibody--
Anti-Reaper serum was
obtained by standard immunization techniques using New Zealand White
rabbits and a synthetic Reaper C-terminal peptide conjugated to keyhole
limpet hemocyanin (Research Genetics). The sequence used for peptide
synthesis was CHPKTGRKSGKYRKPSQ.
Generation of ReaperKR--
Reaper was cloned into pcDNA3 by
standard techniques. Further work required removal of a vector
MscI site; thus Reaper/pc3 was digested with
PvuII and recircularized. The oligonucleotides (GATCCATGGCAGTGGCATTCTACATACCCGATCAGGCGACTCTGTTGCGGGAGGCGGAGCAGAGGGAGCAGCAGATTCTCCGCTTGCGGGAGTCACAGTGGAGATTCCTGG; CCAGGAATCTCCACTGTGACTCCCGCAAGCGGAGAATCTGCTGCTCCCTCTGCTCCGCCTCCCGCAACAGAGTCGCCTGATCGGGTATGTAGAATGCCACTGCCATG; CCACCGTCGTCCTGGAAACCCTGCGCCAGTACACTTCATGTCATCCGAGGACCGGAAGAAGGTCCGGCAGATATCGCAGGCCATCGCAAT; and
CTAGATTGCGATGGCCTGCGATATCTGCCGGACCTTCTTCCGGTCCTCGGATGACATGAAGTGTACTGGCGCAGGGTTTCCAGGACGACGGTGG) were hybridized and cloned separately into
Reaper-pcDNA3 In Vitro Translation--
A variant of pcDNA3 was generated
in which a c-Myc epitope tag was cloned downstream of the MCS
XbaI site. The Reaper open reading frame minus its stop
codon was cloned in frame with the Myc tag of pcDNA3-myc using
standard techniques to generate Reaper-Myc. A variant of pSP64T, an
in vitro SP6 expression vector with flanking 5' and 3'
Cell Culture, Transfections, Immunoblotting, Pulse-Chase
Analysis, and Apoptosis Assay--
All of the cell culture reagents
were obtained from Invitrogen unless otherwise specified. Details of
cell culture, vector constructs, immunoblotting, affinity
precipitations, and pulse-chase analysis were as previously described
(14), with the following exceptions. HEK 293T cells were plated at a
density of 1 × 106 cells/10-cm dish for
immunoblotting experiments and 200,000 cells/well in 6-well dishes for
pulse-chase analysis and apoptosis assay. The cells were transfected
24 h after plating using a standard protocol of calcium phosphate
and HEPES-buffered saline. 10-cm dishes were transfected with a total
of 10 µg of DNA, and 6-well plates were transfected with a total of
1.6 µg of DNA/well. Where indicated, the proteasome inhibitor LLnL
(ALLN, Calbiochem) was added to a final concentration of 20 µM for 45 min prior to harvesting cells. When
appropriate, the cells were harvested by rinsing once with
phosphate-buffered saline and lysing the cells on ice with buffer IP
(50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 2.5 mM MgCl2, and 1%
Nonidet P-40, plus 1× Complete protease inhibitor (Roche Molecular)).
Bead-bound material following affinity precipitation was washed three
times with buffer IP prior to analysis by SDS-PAGE and immunoblotting
or autoradiography. The apoptosis-inducing ability of Reaper and
ReaperKR was assayed by co-transfecting pEGFP-C1
(Clontech) with the vectors indicated. After
48 h, live cells (as determined by forward and side
scatter) were analyzed for GFP fluorescence by flow cytometry.
Each transfection was performed in duplicate, with and without 50 µM zVAD-fmk (Biomol) to inhibit caspase activation, for a
total of four transfections/construct. The percentage of live GFP+
cells for each construct in the absence of zVAD-fmk was normalized to
the percentage of live GFP+ cells in the presence of zVAD-fmk such that
the results shown indicate caspase-dependent loss of GFP+
cells while correcting for any differences in transfection efficiency
between constructs.
SL2 Cell Culture--
All of the cell culture reagents were
obtained from Invitrogen unless otherwise specified. SL2 cells were
obtained from the ATCC via the Duke Cell Culture Facility and were
maintained in Schneider's Drosophila medium supplemented
with 10% heat-inactivated fetal bovine serum (HyClone). For
transfection, 3 × 106 cells (at 5 × 105 cells/ml) were seeded in T-25 flasks. 24 h later,
DNA was prepared for transfection by mixing 20-30 µg of appropriate
constructs with 62 µl of 2 M CaCl2 and 438 µl of sterile water. 500 µl of 2× HEPES buffered saline was
bubbled in to each sample over 1 min. DNA mixtures were allowed to sit
for 30 min at room temperature and were then added to cells for 16-24
h, after which cells were pelleted and resuspended in fresh medium.
SL2 Killing Assay--
Enhanced GFP was subcloned into
the EcoRI and XbaI sites of pCasper3, downstream
of the ubiquitin promoter, using standard techniques. Wild type Reaper
and ReaperKR were cloned into the EcoRI and BamHI
sites of pMT, downstream of the metallothionine promoter, using
standard techniques. SL2 cells were co-transfected with 2 µg of GFP
and 20 µg of pMT vector, Reaper, or ReaperKR. After 24 h, the
cells were pelleted at 1,000 × g for 5 min and then
resuspended in fresh medium. Following an additional 24 h, Reaper
or ReaperKR was induced with 70 nM CuSO4, and
the cells were incubated at 25 °C for 3 days. FACS analysis was then
performed. Transfection efficiency was controlled for by normalizing
each transfection to its percentage of GFP (+) in the presence of
zVAD-fmk. Each transformation was analyzed in triplicate (for a total
of 18 samples: nine with zVAD-fmk and nine without). Forward and side
scatter were used to gate viable cells, with the same gate settings
used for all samples. 100,000 gated cells/sample were counted and then
analyzed for GFP fluorescence using FACS ANALYZER (BD). Cells with
>102 GFP signal were taken as positive. The percentage of
survival for sample A was calculated as [%GFP(+)A IAPs Can Ubiquitinate Reaper, HID, and Grim--
Our previous work
and that of others have shown that the interaction between IAPs and
Reaper (and Grim and HID) lowers cellular IAP levels by stimulating
ubiquitin-mediated degradation of the IAPs (12-17). In the course of
these experiments involving Reaper, HID, and DIAP1, we noticed that
overexpression of Reaper in Drosophila imaginal discs led
not only to lower DIAP1 levels but also to elevated levels of HID as
detected by immunofluorescence (Fig. 1,
A-F). The reciprocal experiment examining Reaper protein
levels in the presence of HID overexpression was uninformative because of the inability of our Reaper antibodies to detect Reaper in situ. To eliminate the possibility that Reaper expression was affecting HID transcription (Fig. 1F), we analyzed the
amount of HID mRNA by in situ hybridization and found
that there was no increase in the amount of HID message (Fig.
1H). These results implied that the regulation of Reaper and
HID levels were somehow linked at a post-transcriptional step. This
seemed particularly significant in light of the fact that the Reaper,
HID, and Grim proteins all play a key role in developmental and
radiation-induced apoptosis in Drosophila, but there have
been no published reports on post-transcriptional mechanisms
controlling the abundances of these proteins.
Although an effect on translational regulation remained a possible
explanation for the elevated HID levels, this seemed unlikely because
Reaper has been shown to suppress rather than enhance translation (14,
17). However, we had also noted that Reaper immunoprecipitates from
cells transfected with both Reaper and a human IAP (XIAP) contained a
prominent 18-kDa species recognized by anti-Reaper immunoblotting.
Given the established link between the RHG proteins, IAPs, and
ubiquitin-mediated degradation, we strongly suspected that this species
was monoubiquitinated Reaper. Indeed, the 18-kDa band evident in the
Reaper immunoblot of the anti-Reaper immunoprecipitate was also
recognized by anti-ubiquitin antibody (Fig.
2A). Given that
Drosophila IAP-1 (DIAP1) is the physiologically relevant IAP
with regard to Reaper, we repeated these experiments with Reaper and
DIAP1 with similar results (Fig. 2A).
We then transfected cells with Reaper and either XIAP or DIAP1 and
looked for Reaper-ubiquitin conjugates in the IAP immunoprecipitate. We
detected multiple ubiquitinated species at 9-kDa intervals above the
9-kDa nonubiquitinated Reaper (Fig. 2B, black
arrowheads). Importantly, these protein species were absent when
cells were transfected with Reaper and an XIAP RING finger point mutant
(H467A) that lacks ubiquitin ligase activity. Note also that only the 18- and 27-kDa species could be detected in the anti-Reaper immunoblot because the Reaper antibody was prepared against an extreme C-terminal peptide from Reaper, where four of five ubiquitin-modifiable lysines are located (see Fig. 5A). As such, it is likely that
multiubiquitination of Reaper was interfering with antibody binding.
The presence of ubiquitinated Reaper species in the IAP co-precipitate
suggested that Reaper might be a substrate of IAP ubiquitin-ligase activity. Additionally, we suspected that the RHG protein HID might
also serve as a substrate for IAP-mediated ubiquitination because our
initial experiment showed that HID levels increased as DIAP1 levels
were lowered by Reaper (Fig. 1). To prove that Reaper and HID could in
fact serve as substrates for IAP-mediated ubiquitination, we performed
in vitro ubiquitination reactions with recombinant forms of
these proteins. These results show clearly that Reaper and HID are
substrates for DIAP1-stimulated ubiquitination (Fig. 2C).
Furthermore, we found that Grim is also ubiquitinated in
vitro (Fig. 2C), suggesting that all three of these
Drosophila RHG proteins may be regulated at the level of
protein stability.
Reaper Is Stabilized by Inhibiting the Proteasome--
To further
elucidate the machinery involved with this phenomenon, we focused on
the regulation of Reaper stability and asked whether or not the
proteasome was involved in the degradation of ubiquitinated Reaper.
When reticulocyte lysates were used to transcribe and translate Reaper,
the addition of LLnL (also known as ALLN) to inhibit proteasomal
degradation resulted in much higher levels of Reaper production (Fig.
3A). Transcription and
translation of two unrelated control proteins (cdc25 and Grp94)
demonstrated that the effect of LLnL was not a nonspecific increase in
protein production (Fig. 3A). Extending these results to the
more complex milieu of transfected 293T cells, we found that the
addition of LLnL for 45 min significantly increased the amount of
Reaper detected by immunoprecipitation and immunoblotting (Fig.
3B). Both experiments suggest that Reaper is targeted for
proteasomal degradation.
The Reaper N Terminus Is Required for IAP-mediated
Degradation--
Given the ability of the RHG proteins to interact
physically with IAPs, we hypothesized that this direct binding would be necessary for IAPs to promote Reaper, HID, and Grim ubiquitination and
degradation. To verify this, we tested a deletion mutant of Reaper that
lacked the first 15 amino acids and was therefore missing the canonical
RHG IAP-binding motif (Reaper
To demonstrate the biological significance of the Reaper-IAP
interaction in modulating Reaper protein levels, we examined the
relative stability of Reaper and Reaper Ubiquitination-resistant Reaper Is Not Destabilized by
IAPs--
To fully address the biological significance of this novel
mechanism for regulating Reaper levels, we wanted to ask whether or not
the ubiquitination and degradation of Reaper per se affects its abundance and biological activity. This question precluded the use
of Reaper IAP Ligase Activity Contributes to Reaper Instability--
If IAPs
were in fact mediating the degradation of Reaper, we reasoned that
ReaperKR should be resistant to the effects of IAP overproduction. We
therefore performed pulse-chase analysis on Reaper and ReaperKR in 293T
cells that had also been transfected with XIAP. The results from this
assay confirmed that ReaperKR was significantly more stable than the
wild type protein (Fig. 5F). In contrast, when the
pulse-chase analysis was repeated using the XIAP H467A ubiquitin ligase
mutant, wild type Reaper was stable, implying that the destabilization
of Reaper was in fact specific to IAP ubiquitin-ligase activity (Fig.
5F).
Regulation of Reaper Stability Affects Its Ability to Induce
Apoptosis--
Finally, because Reaper is a potent pro-apoptotic
protein, we wanted to assay the killing ability of the
degradation-resistant ReaperKR with respect to wild type Reaper. We
reasoned that if the regulation of Reaper levels by IAP-stimulated
ubiquitination was biologically significant, then the
degradation-resistant ReaperKR would be an even more potent killer than
wild type Reaper. To test this hypothesis, we first compared the
killing activities of Reaper and ReaperKR in transfected human cells.
As shown in Fig. 6A, ReaperKR
was a substantially better inducer of apoptosis than Reaper. Finally,
to assay the biological function of Reaper in the context of its native
species, we examined the relative killing activities of Reaper and
ReaperKR in Drosophila SL2 cells. Once again, the
degradation-resistant ReaperKR was a much better inducer of
caspase-dependent cell death than wild type Reaper (Fig.
6B). Collectively, these experiments demonstrate that
regulation of Reaper by IAP-mediated ubiquitination and degradation has
a significant impact on the ability of Reaper to initiate apoptosis.
Our results implicate the ubiquitin-proteasome pathway in the
regulation of Reaper stability and biological activity. In
vitro ubiquitination assays coupled with overexpression and mutant
studies strongly suggest that IAPs such as XIAP and DIAP1 can serve as ubiquitin ligases for Reaper, HID, and Grim. Furthermore, the regulation of Reaper stability has a significant effect on the ability
of Reaper to initiate apoptosis. As such, the work reported here
ascribes a new anti-apoptotic function to the IAP RING finger domain in
that it promotes the degradation of RHG family members.
The findings reported here suggest that IAP proteins can ubiquitinate
Reaper and its relatives and that this requires a stable interaction
between the RHG protein and the IAP. We and several other groups
recently reported that the interaction between Reaper, HID, and Grim
and the IAPs can stimulate IAP auto-ubiquitination and degradation,
thereby facilitating caspase activation and cell death (12-17). Given
the ability of Reaper to stimulate IAP degradation and vice versa, it
is not entirely clear how the outcome of the Reaper-IAP battle is
determined. Because Reaper is also transcriptionally regulated
(35-39), the balance between Reaper-mediated death and IAP-mediated
survival may be partially determined by the strength of Reaper
induction following a particular apoptotic stimulus. Similarly, it is
likely that cells with different levels and types of IAPs will differ
in their susceptibility to Reaper. Almost certainly, other modulatory
factors also help to determine the outcome of the Reaper-IAP
interaction. One such candidate factor is Morgue, a newly identified
protein related to variant ubiquitin-conjugases that was
isolated in a screen for modifiers of Reaper and Grim cell death (13,
16). This protein may assist Reaper- and Grim-mediated IAP degradation
in some way, helping to shift the balance toward death when Morgue is
present. Also, the ability of Reaper to suppress translation may assist
in shifting the IAP-Reaper balance toward cell death.
Our data demonstrating that Reaper, HID, and Grim are all subject to
IAP-stimulated ubiquitination may help to explain previous reports that
have noted a cooperative apoptosis-inducing effect when more than one
RHG protein is present (40, 41). Although it has been thought that this
effect might be due to slightly different biological functions, the
data presented here suggest that these proteins may cooperate in
vivo by indirectly modulating each other's abundance; that is, as
the RHG proteins successfully stimulate ubiquitin-mediated destruction
of the IAPs, their own half-lives are extended, and they are able to
accumulate to higher levels. This would explain the rise in HID levels
following Reaper overexpression (Fig. 1).
Interestingly, the vertebrate IAP antagonist Smac is also a substrate
for IAP-mediated ubiquitination, suggesting that ubiquitination of
IAP-binding partners may be widespread (42). In this regard, it would
be interesting to determine whether the stability of Omi is regulated
by IAP proteins as well. Conversely, the weakly pro-apoptotic proteins
Smac and Omi have not been reported to stimulate IAP degradation. If
Smac and Omi do not, in fact, have this activity, their interaction
with the IAP ubiquitin-ligase would be unidirectional, with the IAP
targeting Smac and Omi for destruction, while the IAP itself
remained stable. This may be the case if Smac and Omi do not
engage the IAPs in precisely the same way as Reaper, HID, and Grim
or if a domain in addition to the RHG motif is also required to
stimulate IAP auto-ubiquitination.
Finally, the interplay that we have described between Reaper and the
IAPs illustrates that the decision to undergo apoptosis (or not) is an
active struggle within the cell. In this particular struggle, the
outcome can be tipped one way or the other by regulating the protein
stability of these antagonistic apoptotic regulators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 4 h and
purified using glutathione-Sepharose 4B (Amersham Biosciences) per the
manufacturer's instructions. HID-His6 and Grim-His6 were purified as described previously (17). All
of the proteins for in vitro ubiquitination assays were
dialyzed against the buffer UD (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 10% glycerol)
before use.
PvuII to generate clones A (N-terminal
mutant) and B (C-terminal tetra-mutant). ReaperKR was generating by
splicing the BamHI/MscI fragment from clone A
into clone B above, and this insert was subcloned into the various
vectors indicated.
-globin untranslated region and a polyadenosine tail was generated
called pSP64BN. The BglII cloning site of pSP64T was
replaced with an oligonucleotide encoding the multiple cloning site of
pEBB, including unique BamHI and NotI sites.
ReaperKR-FLAG, Reaper-FLAG, Reaper, and Reaper
1-15 were subcloned
into pSP64BN by standard techniques. To produce radioactive protein for
half-life assays, Reaper-myc, Reaper, Reaper
1-15, Reaper-FLAG,
ReaperKR-FLAG, Cdc25, and Grp94 templates were added at 20 ng
µl
1 to rabbit reticulocyte lysate (Stratagene)
containing 1 µCi µl
1 of S-35 Translabel (ICN), 1×
((
)-cysteine, (
)-methionine) amino acid mix and other
components per manufacturer's protocol. Translated proteins were
resolved by SDS-PAGE, soaked in 1 M salicylic acid for
1 h, dried, and exposed to Biomax MR film (Kodak). To assay protein stability, 100 µg ml
1 cycloheximide was added
to translated proteins, which were then incubated at 30 °C for an
additional 30 and 60 min, boiled in SDS sample buffer, and processed as above.
zVAD-fmk]/[%GFP(+)A + zVAD-fmk]. The average percentage
of GFP(+) was calculated, and the standard deviations for each sample
were used to determine error.
1-15 versus Wild Type Stability Assay--
Full-length or
1-15 reaper-GFP fusions were generated by overlap PCR using
the following oligonucleotides: 5' reaper,
GAAGGAGGATCCATGGCAGTGGCATTCTACATACCC; 5' overlap,
TATCGCAAGCCATCGCAAAGATCTATGGTGAGCAAGGGCGAG; 3' overlap, CTCGCCCTTGCTCACCATAGATCTTTGCGATGGCTTGCGATA; and 3' GFP,
CCTCCGGATCCCTACTTGTACAGCTCGTCCATGCCGAG. Fusion open reading
frames were subcloned into the BamHI and EcoRI sites of pMT, downstream of the metallothionine promoter, using standard techniques. pIE3-DIAP-HA was a gift from Kristin White (Harvard/MGH). 10 µg of DIAP and 20 µg of full-length or
1-15 reaper-GFP were transfected into SL2 cells as described above. 24 h after resuspension in fresh medium, GFP fusions were induced with 700 nM CuSO4 and placed into 50 µM
zVAD-fmk to prevent cell death. After 16 h of induction, the cells
were pelleted, washed with fresh medium, and resuspended in medium
lacking copper but supplemented with 50 µM zVAD-fmk. The
cells were immediately subjected to FACS analysis as above, with
additional analyses at 16 and 24 h. Each sample was analyzed in
triplicate as above. The averages were calculated for each sample, and
the standard deviations were used to determine error. The percentage of
Reaper remaining in the presence of DIAP1 = [+D]/[no D] where
[+D] = [% GFP(+) with DIAP at time T]/[% GFP(+) with
DIAP at time 0], and [no D] = [% GFP(+) without DIAP at time
T]/[% GFP(+) without DIAP at time 0].
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Reaper expression lowers DIAP1 levels and
post-transcriptionally increases HID levels. DIAP1 and HID were
detected by immunofluorescence of imaginal discs from
Drosophila embryos overexpressing p35 (A and
D), HID + p35 (B and E), and Reaper + p35 (C and F). Expression of the caspase
inhibitor p35 prevented Reaper- and HID-induced apoptosis. in
situ hybridization was used to detect mRNA expression of HID
(G and H) and Reaper
(I).
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Fig. 2.
Reaper, Hid, and Grim ubiquitination.
A, Rpr and FLAG-tagged XIAP or Reaper and GST-tagged DIAP1
were transfected into HEK 293T cells. Immunoprecipitations were
performed using anti-Reaper serum coupled to protein A (IP:
Rpr). The precipitates were resolved by SDS-PAGE and blotted using
anti-Reaper serum (R) or anti-ubiquitin antibody
(Ub). The Reaper parent species is indicated (gray
arrowhead), as is the 18-kDa monoubiquitinated species
(black arrowhead). B, Rpr and FLAG-XIAP, Rpr and
FLAG-H467A XIAP ligase mutant (H467A), or Rpr and GST-DIAP1 were
transfected into HEK 293T cells. Affinity precipitations were performed
using anti-FLAG antibody coupled to protein G (IP: XIAP) or
glutathione-Sepharose (IP: DIAP). The precipitates were
resolved by SDS-PAGE and blotted using anti-Reaper serum (R)
or anti-ubiquitin antibody (Ub). Reaper parent species is
indicated (gray arrowhead), as are the mono- and
polyubiquitinated species (black arrowheads). C,
left panel, recombinant Reaper-GST. Middle panel,
Hid-His6. Right panel, Grim-His6
were mixed with Drosophila embryo extract for 10 min.
Subsequently, recombinant DIAP1 and His-ubiquitin were added, and the
mixture shifted to 37 °C for 40 min. The samples were resolved by
SDS-PAGE and blotted using indicated antibodies. Note parent species
(gray arrows) and polyubiquitinated species
(brackets).
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Fig. 3.
Proteasome-dependent degradation
of Reaper. A, Myc-tagged Reaper (Rpr-myc), Cdc25, or
Grp94 were translated in the presence of 20 µM LLnL or
Me2SO carrier in rabbit reticulocyte lysate. For Reaper,
equal amounts of lysate were immunoprecipitated using the 9E10 anti-Myc
antibody. For controls, equal volumes of lysate were loaded for
analysis. The samples were resolved by SDS-PAGE and exposed to film.
B, untagged Reaper was transfected into HEK 293T cells that
were treated for 45 min prior to harvesting with 20 µM
LLnL or Me2SO carrier. The cells were harvested and
subjected to immunoprecipitation and immunoblotting using anti-Reaper
serum.
1-15). In a previous report, Reaper
1-15 failed to co-precipitate with cellular IAP (24), and we have
shown that Reaper
1-15 was unable to stimulate XIAP degradation
(14). As expected, Reaper
1-15 failed to bind DIAP1 (Fig.
4A), whereas full-length
Reaper co-precipitated with DIAP1 quite well (Fig. 2B). Both
wild type and Reaper
1-15 were then transcribed and translated in
rabbit reticulocyte lysates, and the results were analyzed by
autoradiography. Our results showed that the Reaper
1-15 protein
was considerably more abundant than the wild type, consistent with the
mutant being more stable (Fig. 4B). When cycloheximide was
added to the reticulocyte lysates to stop translation, the Reaper
1-15 protein was markedly more stable than wild type Reaper over a
60-min time course (Fig. 4C).
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Fig. 4.
Reaper 1-15 is not
an IAP substrate. A, HEK 293T cells were co-transfected
with GST-DIAP1 and
1-15 Reaper (
1-15). Co-precipitations were
performed using either anti-Reaper serum (
-Rpr) or
glutathione-Sepharose (GSH). The samples were resolved by
SDS-PAGE and blotted using anti-Reaper serum. Note that Fig.
2B demonstrates co-immunoprecipitation of full-length Reaper
and DIAP1. B, untagged Reaper and Reaper
1-15 were
translated in rabbit reticulocyte lysate. Equal amounts of lysate were
subject to immunoprecipitation using anti-Reaper serum. The samples
were resolved by SDS-PAGE and exposed to film. C, reaper or
Reaper
1-15 were produced in reticulocyte lysates, cycloheximide
was added, and the proteins were incubated for a further 30 or 60 min
at 30 °C. Equal volumes of reticulocyte lysate were
immunoprecipitated with anti-Reaper serum and processed as above. The
precipitates were resolved by SDS-PAGE and exposed to film. The results
were quantified by ImageJ application (NIH). D,
Drosophila SL2 cells were transfected with either
copper-inducible Reaper-GFP or
1-15 Reaper-GFP in the presence or
absence of constitutively expressed DIAP1. At time 0, copper was
removed, and the cells were harvested at 0, 16, and 24 h and
subjected to FACS analysis to determine the percentage of GFP-positive
cells. The percentage of Reaper-GFP in the presence of DIAP1 was
determined by comparing the percentage of GFP to that at time 0 in
cells containing DIAP1 relative to the percentage of GFP in cells
lacking DIAP1. The samples were processed and analyzed in triplicate,
and the standard deviations were used to determine error.
IP, immunoprecipitation; WT, wild type.
1-15 in
Drosophila SL2 cells. Because SL2 cells have a low
transfection efficiency, it was difficult to follow Reaper protein
levels by immunoblotting or radiolabeling. Therefore, we generated
Reaper-GFP and Reaper
1-15-GFP constructs under the control of a
metallothionine promoter to perform a fluorescence-based protein
stability assay. Each Reaper construct was transfected into SL2 cells,
with or without DIAP1 that was driven by the constitutive baculovirus
IE1 promoter. After 16 h of induction with copper, the copper
containing medium was replaced with fresh (copper-free) medium, thereby
inactivating the Reaper promoter. SL2 cells were then immediately
analyzed for GFP fluorescence (and then analyzed again at 16 and
24 h). The results of this experiment confirmed our hypothesis
that wild type Reaper was subject to DIAP1-stimulated degradation, but
Reaper
1-15 was not (Fig. 4D). An identical experiment
in which cycloheximide was used in place of copper-removal gave similar
results (data not shown). It is worth noting that Reaper
1-15
retains all five of the lysines in Reaper (Fig.
5A), so the enhanced stability of the Reaper deletion mutant most likely stems from its inability to
interact with IAPs and not from a lack of potential ubiquitin conjugation sites.
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Fig. 5.
Lysine-deficient Reaper exhibits increased
stability. A, schematic of the Reaper protein sequence,
showing the number and relative distribution of lysines. The amino acid
positions are indicated. B, HEK 293T cells were
co-transfected with GST or GST-DIAP1 and lysine-deficient Reaper
(ReaperKR-FLAG). Co-precipitations were performed using either
anti-FLAG antibody or glutathione-Sepharose (GSH). The
samples were resolved by SDS-PAGE and blotted using anti-FLAG antibody.
C, FLAG-tagged Rpr and lysine-deficient Reaper
(RprKR) were translated in rabbit reticulocyte lysate. Equal
volumes of lysate were immunoprecipitated with anti-FLAG antibody. The
samples were resolved by SDS-PAGE and exposed to film. D,
HEK 293T cells were transfected with FLAG-tagged Rpr and RprKR. 45 min
prior to harvest, the cells were treated with 20 µM LLnL
(or not). The cells were harvested, and the Reapers were precipitated
using anti-FLAG antibody. The samples were resolved by SDS-PAGE and
blotted using anti-FLAG antibody. E, HEK 293T cells were
transfected with Rpr or RprKR. At the indicated times, the cell lysates
were subjected to immunoprecipitation using anti-FLAG antibody to
precipitate Rpr and RprKR. The samples were resolved by SDS-PAGE and
exposed to film, and the results were quantified using the ImageJ
application (NIH). F, HEK 293T cells were transfected with
Rpr or RprKR and either GST-XIAP or GST-XIAP H467A. The samples were
processed as in E.
1-15 because that mutant lacks the IAP interaction domain
and is therefore unable to inhibit IAP activity or stimulate IAP
degradation. We therefore chose to construct an additional Reaper
mutant that would interact normally with the IAPs but would itself be
impervious to ubiquitination. Accordingly, we mutated all of the
lysines in Reaper to arginines (ReaperKR) to inhibit ubiquitin
conjugation (Fig. 5A). As expected, ReaperKR still
interacted quite stably with DIAP1 (Fig. 5B). When ReaperKR
was produced in vitro using reticulocyte lysates, we
observed that much more of the mutant was made than wild type Reaper,
suggesting that the lysine mutations were in fact stabilizing the
protein (Fig. 5C). We then expressed Reaper and ReaperKR by
transient transfection of 293T cells and observed that the ReaperKR
produced to much higher steady state levels than wild type Reaper (Fig.
5D). Furthermore, although inhibition of the proteasome with
LLnL increased Reaper levels, it had no effect on ReaperKR levels (Fig.
5D). We then performed a pulse-chase assay in 293T cells to
directly examine the intrinsic half-lives of Reaper versus
ReaperKR. As expected, the lysine mutant had a markedly increased
half-life relative to wild type Reaper (Fig. 5E). These
experiments supported our hypothesis that the ubiquitin-proteasome
pathway is important for the stability of the Reaper protein and that
mutation of the lysines in Reaper makes it resistant to degradation.
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Fig. 6.
Lysine-deficient Reaper is a more potent
inducer of apoptosis. A, HEK 293T cells were co-transfected
with GFP and vector alone (control), Reaper, or
lysine-deficient ReaperKR in the presence or absence of the
irreversible caspase inhibitor zVAD-fmk. After 48 h, the cells
were harvested and subjected to FACS analysis to determine the
percentage of GFP-positive cells. The percentage of survival was
calculated by the percentage of GFP-positive cells without zVAD-fmk
relative to the percentage of GFP-positive cells with zVAD-fmk. The
samples were processed in duplicate, and the standard deviations were
used to determine error. B, Drosophila SL2 cells
were co-transfected with constitutively expressed GFP and
metallothionine-driven Reaper, ReaperKR, or vector control. Reaper was
induced 16 h after transfection (to allow for GFP expression), and
the cells were harvested after a further 48 h of incubation. The
cells were subjected to FACS analysis as above. The percentage of
survival was calculated by dividing the percentage of GFP-positive
induced cells by the percentage of GFP-positive induced cells treated
with zVAD-fmk. The samples were processed in triplicate, and the
standard deviations were used to determine error.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Kristin White (Harvard/MGH) for providing the pIE3/DIAP1 clone. We are grateful to Dr. Rick Fehon and Rima Kulikauskas for assistance and space for insect cell culture.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 GM61919 (to S. K.) and RO1 GM57422 (to B. H.).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.
§ Recipients of Medical Scientist Training Program grants and predoctoral fellows of the US Army Materiel Research Command Breast Cancer Research Program DAMD17-01-1-0232. These authors contributed equally to this work.
Scholar of the Leukemia and Lymphoma Society. To whom
correspondence should be addressed: Box 3813, Rm. C370 LSRC, Research Dr., Dept. of Pharmacology and Cancer Biology, Duke University Medical
Center, Durham, NC 27710. Tel.: 919-613-8624; Fax: 919-681-1005; E-mail: kornb001@mc.duke.edu.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M209734200
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ABBREVIATIONS |
---|
The abbreviations used are: RHG, Reaper/HID/Grim; LLnL and ALLN, N-acetyl-Leu-Leu-Nle-CHO; Rpr, Reaper; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone; GST, glutathione S-transferase; HID, head involution defective, XIAP, X-linked IAP; DIAP, Drosophila IAP; GFP, green fluorescent protein; FACS, fluorescence activated cell sorting.
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