Drosophila IAP1-mediated ubiquitylation controls activation of the initiator caspase DRONC independent of protein degradation.

Abstract

Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition ("undead" cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in "undead" cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.

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Drosophila IAP1-Mediated Ubiquitylation Controls
Activation of the Initiator Caspase DRONC Independent
of Protein Degradation
Tom V. Lee1., Yun Fan1., Shiuan Wang1,2, Mayank Srivastava1, Meike Broemer3, Pascal Meier3, Andreas
Bergmann1,2¤*
1Department of Biochemistry and Molecular Biology, Genes and Development Graduate Program, The University of Texas MD Anderson Cancer Center, Houston, Texas,
United States of America, 2Graduate Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, United States of America, 3The Breakthrough Toby
Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom
Abstract
Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological
processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the
inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC
protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (‘‘undead’’ cells), it is thought that DIAP1-
mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to
this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length
DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and
activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is
processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in ‘‘undead’’ cells
due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.
Citation: Lee TV, Fan Y, Wang S, Srivastava M, Broemer M, et al. (2011) Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase
DRONC Independent of Protein Degradation. PLoS Genet 7(9): e1002261. doi:10.1371/journal.pgen.1002261
Editor: Bingwei Lu, Stanford University School of Medicine, United States of America
Received April 29, 2011; Accepted July 6, 2011; Published September 1, 2011
Copyright: ? 2011 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by NIH grants (R01s GM068016, GM081543) and by an anonymous donor to AB. MB is supported by a fellowship of the
Deutsche Forschungsgemeinschaft (DFG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: andreas.bergmann@umassmed.edu
. These authors contributed equally to this work.
¤ Current address: Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
Introduction
Ubiquitylation describes the covalent attachment of ubiquitin, a
76 amino acid polypeptide, to proteins which occurs as a multi-
step process (reviewed in [1,2]). E1-activating enzymes activate
ubiquitin and transfer it to E2-conjugating enzymes. E3-ubiquitin
ligases mediate the conjugation of ubiquitin from the E2 to the
target protein. Repeated ubiquitylation cycles lead to the
formation of polyubiquitin chains attached on target proteins.
Polyubiquitylated proteins are delivered to the 26S proteasome for
degradation. However, non-proteolytic roles of ubiquitylation
have also been described (reviewed in [3,4]). From E1 to E3, there
is increasing complexity. For example, the Drosophila genome
encodes only one E1 enzyme, termed UBA1, which is required for
all ubiquitin-dependent reactions in the cell [5]. In contrast, there
are hundreds of E3-ubiquitin ligases which are needed to confer
substrate specificity.
Programmed cell death or apoptosis is an essential physiological
process for normal development and maintenance of tissue
homeostasis in both vertebrates and invertebrates (reviewed in
[6]). A highly specialized class of proteases, termed caspases, are
central components of the apoptotic pathway (reviewed in [7]).
The full-length form (zymogen) of caspases is catalytically inactive
and consists of a prodomain, a large and a small subunit.
Activation of caspases occurs through dimerization and proteolytic
cleavage, separating the large and small subunits. Based on the
length of the prodomain, caspases are divided into initiator (also
known as apical or upstream) and effector (also known as
executioner or downstream) caspases [7]. The long prodomains
of initiator caspases harbor regulatory motifs such as the caspase
activation and recruitment domain (CARD) in CASPASE-9.
Through homotypic CARD/CARD interactions with the adapter
protein APAF-1, CASPASE-9 is recruited into the apoptosome, a
large multi-subunit complex, where it dimerizes and auto-
processes leading to its activation [8,9]. Activated CASPASE-9
cleaves and activates effector caspases (CASPASE-3, -6, and –7),
which are characterized by short prodomains. Effector caspases
execute the cell death process by cleaving a large number of
cellular proteins [10].
In Drosophila, the initiator caspase DRONC and the effector
caspases DrICE and DCP-1 are essential for apoptosis [11–18].
Like human CASPASE-9, DRONC carries a CARD motif in its
prodomain [19]. Consistently, DRONC interacts with ARK, the
APAF-1 ortholog in Drosophila (also known as DARK, HAC-1 or
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D-APAF-1) [20–22] for recruitment into an apoptosome-like
complex which is required for DRONC activation [20,23–31].
After recruitment into the ARK apoptosome, DRONC cleaves
and activates the effector caspases DrICE and DCP-1 [25,31–34].
Caspases are subject to negative regulation by inhibitor of
apoptosis proteins (IAPs) (reviewed in [35,36]). For example,
DRONC is negatively regulated by Drosophila IAP1 (DIAP1)
[37,38]. diap1 mutations cause a dramatic cell death phenotype, in
which nearly every mutant cell is apoptotic, suggesting an essential
genetic role of diap1 for cellular survival [39–41]. DIAP1 is
characterized by two tandem repeats known as the Baculovirus
IAP Repeat (BIR), and one C-terminally located RING domain
[42]. The BIR domains are required for binding to caspases
[37,38,43]. The RING domain provides DIAP1 with E3-ubiquitin
ligase activity, required for ubiquitylation of target proteins
[35,36]. Importantly, the BIR domains can bind to caspases
independently of the RING domain [37,43].
Usually, IAPs bind to and inhibit activated, i.e. processed
caspases, including CASPASE-3, CASPASE-7 and CASPASE-9
as well as the Drosophila caspases DrICE and DCP-1 (reviewed in
[35,36]). However, a notable exception to this rule is DRONC.
DIAP1 binds to the prodomain of full-length DRONC [37,38,43].
This unusual behavior suggests an important mechanism for the
control of DRONC activation. Indeed, it has been shown that the
RING domain of DIAP1 ubiquitylates full-length DRONC in vitro
[38,44]. It has also been proposed that DIAP1 ubiquitylates auto-
processed DRONC [33]. These ubiquitylation events are critical
for the control of apoptosis, as homozygous diap1 mutants which
lack a functional RING domain (diap1DRING) are highly apoptotic
[41]. Because the BIR domains are intact in diap1DRINGmutants,
binding of DIAP1 to DRONC is not sufficient for inhibition of
DRONC under physiological conditions, and ubiquitylation is the
critical event for DRONC inhibition.
Although the importance of DIAP1-mediated ubiquitylation of
DRONC is well established, it is still unclear how this
ubiquitylation event leads to inactivation of DRONC and of
caspases in general. Because DRONC protein accumulates in
diap1 mutant cells that are kept alive by expression of the effector
caspase inhibitor P35, generating so-called ‘undead’ cells, it has
been proposed that DIAP1-mediated ubiquitylation triggers
proteasomal degradation of full-length DRONC in living cells,
thus protecting them from apoptosis [33,38,45,46]. However,
degradation of full-length DRONC in living cells has never been
observed and non-degradative models have also been proposed
[44]. Furthermore, ubiquitylation of mammalian CASPASE-3 and
CASPASE-7 has been demonstrated in vitro [47–49]. However,
evidence for proteasome-dependent degradation of these caspases
in vivo, i.e. in the context of a living animal, is lacking. In fact, a
non-degradative mechanism has been demonstrated for the
effector caspase DrICE in Drosophila [50].
Here, we further characterize the role of ubiquitylation for the
control of DRONC activation. Consistent with a previous report
[44], we find that ubiquitylation of DRONC by DIAP1 is critical
for inhibition of DRONC’s pro-apoptotic activity. Using loss and
gain of diap1 function, we provide genetic evidence that DIAP1-
mediated ubiquitylation of full-length DRONC regulates this
initiator caspase through a non-degradative mechanism. We find
that the conjugation of ubiquitin suppresses DRONC processing
and activation. Interestingly, once DRONC is processed and
activated, it has reduced protein stability. Finally, we show that
dronc transcripts accumulate in P35-expressing ‘undead’ cells,
accounting for increased DRONC protein levels in these cells.
These data refine the current model of caspase regulation by IAPs.
Results
Overexpression of DIAP1 fails to suppress apoptosis of
Uba1 mutant cells
It has previously been shown that complete loss of ubiquityla-
tion due to mutations of the E1 enzyme Uba1 causes apoptosis in
eye imaginal discs as detected by an antibody that recognizes
cleaved, i.e. activated, CASPASE-3 (CAS3*) [5,51,52] (see also
Figure 1A). Because ubiquitylation of DRONC does not occur in
Uba1 mutants, we hypothesized that inappropriate activation of
DRONC accounts for the apoptotic phenotype of Uba1 mutants.
To test this possibility, we targeted dronc by RNA interference
(RNAi) in Uba1 mutant cells in eye imaginal discs using the
MARCM system and labeled for apoptosis using CAS3* antibody.
In this system, Uba1 mutant cells expressing dronc RNAi are
positively marked by GFP. Consistent with our hypothesis, knock-
down of dronc strongly reduces apoptosis in Uba1 mutant clones
(Figure 1B). Furthermore, we tested clones doubly mutant for
Uba1 and ark, the Drosophila ortholog of APAF-1 that is required for
DRONC activation (see Introduction). Apoptosis induced in Uba1
mutant clones is strongly suppressed if ark function is removed
(Figure S1). These observations suggest that the apoptotic
phenotype in Uba1 clones is caused by inappropriate activation
of DRONC, presumably due to lack of ubiquitylation.
However, the protein levels of DIAP1 are increased in Uba1
mutant clones [5,52]. There are two possibilities to explain the
apoptotic phenotype in Uba1 mutants despite increased DIAP1
levels. First, the DIAP1 levels may not be sufficiently increased to
inhibit DRONC. Alternatively, binding of DIAP1 to DRONC
alone may not be sufficient for inhibition of DRONC; instead,
ubiquitylation by DIAP1 is required to block DRONC activation,
as previously suggested [44]. To distinguish between these two
possibilities, we strongly overexpressed diap1 in Uba1 mutant
clones in eye discs using the MARCM system and imaged for
apoptosis by CAS3* labeling. Surprisingly, despite massive
expression of diap1 (.20 fold over wild-type levels; Figure 1C90),
apoptosis still proceeds in Uba1 mutant clones (Figure 1C9), even
though expression of the same transgene can block strong
apoptotic phenotypes in several apoptotic paradigms (Figure S2).
Apparently, overexpression of DIAP1 is not sufficient to inhibit
DRONC and to protect Uba1 mutant cells from apoptosis.
Author Summary
The Drosophila inhibitor of apoptosis 1 (DIAP1) readily
promotes ubiquitylation of the CASPASE-9–like initiator
caspase DRONC in vitro and in vivo. Because DRONC
protein accumulates in diap1 mutant cells that are kept
alive by effector caspase inhibition—producing so-called
‘‘undead’’ cells—it has been proposed that DIAP1-mediat-
ed ubiquitylation would target full-length DRONC for
proteasomal degradation, ensuring survival of normal cells.
However, this has never been tested rigorously in vivo. By
examining loss and gain of diap1 function, we show that
DIAP1-mediated ubiquitylation does not trigger degrada-
tion of full-length DRONC. Our analysis demonstrates that
DIAP1-mediated ubiquitylation controls DRONC process-
ing and activation in a non-proteolytic manner. Interest-
ingly, once DRONC is processed and activated, it has
reduced protein stability. We also demonstrate that
‘‘undead’’ cells induce transcription of dronc, explaining
increased protein levels of DRONC in these cells. This study
re-defines the mechanism by which IAP-mediated ubiqui-
tylation regulates caspase activity.
Non-Degradative Ubiquitylation of DRONC
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Because DIAP1 is the key regulator of DRONC and because
DRONC is required for the apoptotic phenotype of Uba1 mutant
cells, as evidenced by knock-down of dronc (Figure 1B), our data
provide genetic evidence that binding of DIAP1 is not sufficient for
DRONC inhibition in Uba1 mutant cells.
Consistent with this view, it has previously been shown that
DIAP1 does ubiquitylate full-length DRONC in vitro [33,38,44].
We tested whether DIAP1 can also ubiquitylate DRONC in vivo.
Because the available DRONC antibodies failed to immunopre-
cipitate endogenous DRONC, we transfected DRONC-V5 along
Figure 1. Apoptosis in Uba1 mutant clones is dependent on DRONC and cannot be inhibited by expression of DIAP1. Shown are eye-
antennal imaginal discs from third instar larvae. Posterior is to the right. In each panel, arrows highlight two representative clones. (A) Uba1 mosaic
eye-antennal discs labeled for cleaved CASPASE-3 (a-CAS3*) antibody (red). These discs were incubated at 30uC 12 hours before dissection (see
Material and Methods). Presence of GFP marks the location of Uba1 clones (see arrow). (B) TUNEL labeling of Uba1 mosaic eye-antennal imaginal discs
expressing an RNAi transgene targeting dronc (UAS-droncIR (inverted repeat)) using the MARCM technique (see Material and Methods). Clones are
positively marked by GFP. TUNEL-positive cell death is largely blocked by dronc knockdown (B9 and B0). (C) Strong overexpression of diap1 in Uba1
clones (magenta in C90) fails to rescue the apoptotic phenotype, as visualized by CAS3* labeling (red in C9). Uba1 clones are marked by GFP due to the
MARCM technique. Please note that diap1 is so strongly overexpressed in the clones that we had to adjust the settings in such a way that
endogenous DIAP1 in wild-type tissue is below the detection limit (C90). Genotypes: (A) hs-FLP UAS-GFP; FRT42D Uba1D6/FRT42D tub-Gal80; tub-GAL4.
(B) hs-FLP UAS-GFP; FRT42D Uba1D6/FRT42D tub-Gal80; tub-GAL4/UAS-droncIR. (C) hs-FLP UAS-GFP/UAS-diap1; FRT42D Uba1D6/FRT42D tub-Gal80; tub-
GAL4.
doi:10.1371/journal.pgen.1002261.g001
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with DIAP1+or DIAP1DRINGmutants (CD6, lacking the last six
C-terminal residues, and F437A changing a critical Phe residue in
the RING domain to Ala [53]) and His-tagged Ubiquitin into
Drosophila S2 cells. Ubiquitylated proteins were affinity purified
under denaturing conditions using Ni columns. The eluates were
subsequently examined by immunoblotting with anti-V5 antibod-
ies to detect ubiquitylated forms of DRONC. Under these
conditions, DIAP1+readily ubiquitylates full-length DRONC in
S2 cells (Figure 2), whereas the RING mutants DIAP1CD6and
DIAP1F437A
were significantly impaired in their ability to
ubiquitylate DRONC (Figure 2). These results indicate that
DIAP1 ubiquitylates full-length DRONC in a RING-dependent
manner in cultured cells.
Overexpression of DIAP1 does not induce degradation of
DRONC
Because DIAP1 readily ubiquitylates DRONC, it has been
postulated that DIAP1-mediated ubiquitylation leads to proteaso-
mal degradation of DRONC [33,38,45]. However, this has never
been rigorously tested in vivo. Therefore, we examined, whether
overexpression of diap1 in wild-type animals can influence
DRONC protein levels in vivo. To this end, we generated clones
overexpressing diap1 (marked by absence of GFP) in eye discs, and
analyzed the protein abundance of DRONC. Interestingly, despite
high expression of diap1 (Figure 3A90), the levels of DRONC
remained unchanged and were not influenced by DIAP1
(Figure 3A9). The anti-DRONC antibody used in this assay is
specific for DRONC (Figure S3). Importantly, the diap1 transgene
used produces a functional DIAP1 protein that is able to inhibit
apoptosis in several paradigms (Figure S2). Therefore, these data
suggest that overexpressed DIAP1 does not target DRONC for
degradation in living cells.
REAPER-induced loss of DIAP1 does not increase DRONC
protein levels
Because of the surprising observation that overexpressed DIAP1
does not cause degradation of DRONC, we tested whether
removal of DIAP1 changes DRONC protein levels. Expression of
the IAP antagonist reaper (rpr) induces DIAP1 degradation and
apoptosis [54–58]. Therefore, we examined whether RPR-
induced degradation of DIAP1 changes DRONC protein levels.
If DIAP1 targets DRONC for degradation, we would expect that
DRONC protein levels would accumulate in response to rpr
expression. Expression of rpr in eye imaginal discs posterior to the
morphogenetic furrow (MF) using the GMR promoter (GMR-rpr) is
well suited for this analysis. The MF is a dynamic structure that
initiates at the posterior edge of the eye disc and moves towards
the anterior during 3rdinstar larval stage [59,60] (Figure 4A,
arrow). Expression of rpr by GMR is induced in all cells posterior to
the MF [61] (red in Figure 4A). Therefore, GMR-rpr eye discs
provide a continuum of all developmental stages in which cells
close to the MF have only recently induced rpr expression, while
cells towards the posterior edge of the disc have been exposed to
rpr progressively longer. Therefore, if DRONC accumulates
during any of these stages, we should be able to detect it. In
wild-type eye discs, DRONC protein is homogenously distributed
throughout the disc. Only in the MF, higher levels of DRONC are
detectable (arrowhead in Figure 4B0). This high expression of
DRONC in the MF serves as an orientation mark. DIAP1 protein
levels are low anterior to the MF, but increase in the MF
(arrowhead) and posterior to it in wild-type discs (Figure 4B9). In
GMR-rpr eye discs, overall DIAP1 levels are reduced in the rpr-
expressing domain posterior to the MF (Figure 4C9), but
particularlystronglyreduced
(Figure 4C9, D9, arrow) consistent with previous reports [54–58].
However, accumulation of DRONC is not observed (Figure 4C0,
D0). In contrast, it appears that DRONC levels are also reduced.
They are still high in the MF (Figure 4C0, arrowhead), but drop
immediately thereafter.
We also related DRONC levels to caspase activation. In the
MF, where CAS3* activity is not detectable, DRONC is still high
(Figure 4D9, D0; arrowhead), but in the CAS3*-positive area,
DRONC levels are reduced (Figure 4D9, D0; arrow). These data
indicate that loss of DIAP1 does not cause accumulation of
DRONC protein implying that DIAP1 does not induce degrada-
tion of DRONC. In contrast, it appears that DIAP1 stabilizes
DRONC at least under these conditions (see Discussion).
in the CAS3*-positivearea
‘‘Undead’’ diap1 mutant cells induce transcription of
dronc
Finally, we analyzed DRONC protein levels in diap1DRING
mutants which cannot ubiquitylate DRONC [44]. It has previously
been shown that clones of the RING mutant diap122-8saccumulate
DRONC protein [45,46] implying that ubiquitylation by the RING
domain of DIAP1 causes degradation of DRONC. We repeated
these experiments and indeed confirmed that DRONC levels are
increased in diap122-8smutant clones (Figure S4). Thus, these results
appear inconsistent withthe data presented inFigure 3 and Figure 4
Figure 2. DIAP1 ubiquitylates DRONC in S2 cells. DRONC C.A–
V5 was coexpressed with His-Ub and the indicated DIAP1 constructs in
S2 cells. Ubiquitylated proteins were purified and analyzed by
immunoblot for ubiquitylated DRONC with V5 antibodies. Co-expres-
sion of DIAP1wtleads to higher molecular weight modification of
DRONC, while the RING-ligase inactive mutants (CD6, F437A) cannot
ubiquitylate DRONC. * marks non-modified DRONC that is due to
unspecific DRONC:matrix association.
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in which manipulating DIAP1 levels did not provide evidence for
DIAP1-mediated degradataion of DRONC. However, one caveat
with the diap122-8sexperiment was the use of the caspase inhibitor
P35whichkept diap122-8smutant cellsinan‘undead’ condition[45].
It has been pointed out that the ‘undead’ state may change the
properties of the affected cells (reviewed by [62]) and in fact
abnormal induction of transcription in ‘undead’ cells has been
reported [45,63–66]. Thus, to explain the conflicting results
between the diap122-8sdata [45] and our data shown here, we
hypothesized that p35-expressing ‘undead’ diap122-8sclones induce
dronc transcription, leading to accumulationof DRONC protein. To
test this hypothesis, we used a transcriptional lacZ reporter
containing 1.33 kb of regulatory genomic sequences upstream of
the transcriptional start site of the dronc gene fused to lacZ (dronc1.33-
lacZ) [67,68]. Compared to controls (Figure 5A, 5A9) and consistent
with the hypothesis, dronc1.33-lacZ reporter activity is increased in
p35-expressing ‘undead’ diap122-8scells in wing imaginal discs and
matches the increased DRONC protein pattern (Figure 5B9-5B90).
We also produced ‘undead’ cells in eye imaginal discs by co-
expression of the IAP-antagonist hid and the caspase inhibitor p35 in
the dorsal half of the eye disc using a dorsal eye- (DE-) GAL4 driver
(Figure 5C). Similar to wing discs, dronc reporter activity is increased
in ‘undead’ cells in the dorsal half of the eye (Figure 5D). Expression
of p35 alone does not trigger transcription of dronc (Figure 5E)
suggesting it is not the mere presence of P35 which causes dronc
transcription, but the ‘undead’ nature of the affected cells.
These observations may explain why DRONC protein
accumulates in ‘undead’ diap122-8smutant cells, but they still do
not rule out the possibility that DRONC protein accumulates in
diap122-8s
mutants due to lack of ubiquitylation and thus
degradation. To clarify this issue we examined dronc1.33-lacZ
and DRONC levels in diap122-8smutant clones without simulta-
neous p35 expression. Without the inhibition of apoptosis by P35,
diap122-8sclones die rapidly. Nevertheless, we were able to recover
wing discs which contained small diap122-8smutant clones. In these
clones, neither dronc1.33-lacZ nor DRONC levels are detectably
Figure 3. Overexpression of diap1 does not trigger degradation of DRONC. Shown is an eye imaginal disc from a third instar larva. Posterior
is to the right. diap1-overexpressing clones are marked by absence of GFP and can be detected using anti-DIAP1 antibodies in magenta (A90). The
boundary between diap1-expressing clones and normal tissue is indicated by a white stippled line in (A9). DRONC levels are unchanged (A9). (A) and
(A0) are merged images. Genotype: UAS-diap1/hs-FLP; tub.GFP.GAL4.
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increased (Figure 5F). Notably, these clones are located in the wing
pouch in which we observed accumulation of dronc reporter
activityandDRONC protein
(Figure 5B0). Thus, the ‘undead’ condition of p35-expressing
diap122-8smutant cells causes accumulation of DRONC protein
due to induction of dronc transcription, explaining the observations
of Ryoo et al. (2004) [45]. In the absence of p35 expression,
transcription of dronc and accumulation of DRONC protein are
not observed, providing additional evidence that ubiquitylation of
DRONC by the RING domain of DIAP1 does not trigger
degradation of DRONC.
under‘undead’ conditions
Ubiquitylation controls processing and thus activation of
DRONC in vivo
Our in vivo analysis implies that DIAP1-mediated ubiquitylation
does not trigger proteasomal degradation of DRONC. To identify
the role of ubiquitylation for regulation of DRONC activity, we
analyzed the fate of DRONC protein in RING mutants of diap1.
Of note, these mutants retain their ability to bind to DRONC,
because DRONC binding is not mediated by the RING domain,
but by the BIR2 domain [37,38,43]. The RING mutant diap133-1s
is especially suitable for this analysis because a premature stop
codon results in deletion of the entire RING domain but leaves the
BIR domains intact [44] (Figure 6A), thus abrogating its E3
activity, but not caspase binding. Importantly, diap133-1sis
characterized by a strong apoptotic phenotype, suggesting
inappropriate caspase activation [41,45]. Indeed, we showed
previously that diap1DRINGmutant phenotypes are dependent upon
DRONC, because dronc mutants suppress diap1DRINGphenotypes
[11]. Therefore, ubiquitylation of DRONC by DIAP1 is critical to
maintain cell survival.
We examined the cause of the diap133-1sapoptotic phenotype.
First, as a control, we determined whether the diap133-1sgene
produces a stable protein in vivo. We chose to analyze stage 6–9
embryos, because normal developmental cell death starts at stage
11 [69]; therefore, stage 6–9 diap133-1smutant embryos allow
analysis of DIAP1 in the absence of upstream apoptotic signals. In
immunoblots of embryonic extracts obtained from a cross of
heterozygous diap133-1smales and females, the DIAP133-1sprotein
is easily distinguished from wild-type DIAP1+protein due to its
faster electrophoretic mobility (Figure 6A, top panel). The
presence of the DIAP133-1sprotein suggests that the apoptotic
phenotype in diap133-1smutant embryos is not caused by instability
of the mutant protein. Interestingly, the protein levels of DIAP1+
and RING-deleted DIAP133-1sare similar (Figure 6A, top panel)
suggesting that loss of the RING domain does not influence the
protein stability of DIAP1 in the absence of pro-apoptotic signals.
Next, we analyzed DRONC protein in extracts from diap133-1s
mutant embryos. Consistent with the data in Figure 4 and Figure 5,
we do not detect a significant increase in the protein levels of
DRONC in these embryos (Figure 6A, middle panel). However, a
significant amount of DRONC is present in a processed form in
extracts of stage 6–9 diap133-1smutant embryos which is absent in
control extracts from wild-type embryos (Figure 6A, middle panel).
Therefore, DRONC processing, and thus activation, occurs in
RING-depleted diap133-1smutant embryos despite the fact that the
BIR domains of DIAP1 are unaffected. The processed form of
DRONC in this mutant of MW ,36 kDa corresponds to the
major apoptotic form of DRONC composed of the large subunit
and the prodomain of DRONC [70]. This finding, and the one
presented in Figure 1, confirms that binding of DIAP1 to DRONC
is not sufficient to prevent processing and activation of DRONC.
Instead, the RING domain is required to control DRONC
processing. Because the RING domain contains an E3-ubiquitin
ligase activity [45,55–58] and because ubiquitylation of full-length
DRONC does not trigger proteasomal degradation (Figure 3,
Figure 4, and Figure 5), we conclude that ubiquitylation of
DRONC by the RING domain of DIAP1 is necessary to inhibit
DRONC processing and thus activation.
To further characterize the role of ubiquitylation in the
regulation of DRONC processing, we performed an immunoblot
analysis with extracts from wild-type and Uba1 mosaic imaginal
discs, which, under our experimental conditions, are about half
mutant for Uba1 and half wild-type. Immunoblot analysis
demonstrated that a significant amount of DRONC is processed
in Uba1 mosaic discs (Figure 6B). Thus, these data further support
the notion that ubiquitylation of full-length DRONC is necessary
for inhibition of DRONC processing.
Discussion
In this paper, we provide three take-home messages. First, we
provide genetic evidence that binding of DIAP1 to DRONC is not
sufficient for inhibition of DRONC. Instead, ubiquitylation of
DRONC controls its apoptotic activity, consistent with the
apoptotic phenotype of diap1DRINGmutants, that retain caspase
binding abilities. Second, DIAP1-mediated ubiquitylation of full-
length DRONC does not lead to its proteasomal degradation;
rather, ubiquitylation directly controls processing and activation of
DRONC. Interestingly, processed and active DRONC shows
reduced protein stability. Third, ‘undead’ cells accumulate dronc
transcripts.
Binding of DIAP1 is not sufficient for Dronc inhibition
Based on biochemical studies in vitro and overexpression studies
in cultured cells, many of cancerous origin, it was initially
proposed that binding of IAPs to caspases through their BIR
domains is sufficient to inhibit caspases [71–80]. However, when
ubiquitylation of caspases by IAPs was described [38,44,47,48], it
was unclear what role ubiquitylation would play for control of
caspase activity, especially since for none of them, ubiquitin-
mediated degradation has been observed (see below). Because the
RING domain is also required for auto-ubiquitylation of DIAP1
[54–58], mutations of the RING domain would be expected to
increase DIAP1 protein levels and protect cells from apoptosis.
However, the opposite phenotype, massive apoptosis, was
observed [41]. Nevertheless, despite the biochemical studies
showing that the BIR domains of DIAP1 are sufficient for
interaction with DRONC [37,38,43], one could argue that
Figure 4. Loss of DIAP1 in GMR-rpr eye discs does not alter DRONC protein levels. (A) Schematic illustration of the GMR-reaper (GMR-rpr)
eye imaginal disc from 3rdinstar larvae. The morphogenetic furrow (MF, arrowhead) initiates at the posterior (P) edge of the disc and moves towards
the anterior (A) (arrow). The GMR enhancer is active posterior to the MF (bracket) and thus expresses rpr posterior to the MF (red area). (B-B0) Eye disc
showing normal protein distribution of DIAP1 (B9) and DRONC (B0). Both DIAP1 and DRONC levels are increased in the MF (arrowhead). (B) is the
merged image of DIAP1 and DRONC labeling. (C–C0) Eye discs expressing two copies of GMR-rpr eye disc labeled for DIAP1 (C9) and DRONC (C0).
Arrowheads mark the MF. DIAP1 levels are markedly reduced posterior to the MF (C9, arrow). Surprisingly, DRONC protein levels are also reduced (C0,
arrow). The brackets indicate the extent of GMR expression. (D–D0) 26GMR-rpr eye disc labeled for cleaved CASPASE 3 (CAS3*) (D9) and DRONC (D0).
DRONC protein levels are reduced in the CAS3*-positive area (arrow). Arrowheads mark the MF. The brackets indicate the extent of GMR expression.
doi:10.1371/journal.pgen.1002261.g004
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Figure 5. ‘‘Undead’’ diap1 mutant cells trigger transcription of dronc. Shown are 3rdinstar larval wing (A,B,F) and eye imaginal discs (C,D,E)
labeled for DRONC protein levels (blue) and dronc transcriptional activity (red) using the dronc1.33-lacZ reporter (ß-GAL labeling). (A,A9) Co-labeling
for DRONC protein (A) and dronc reporter activity (A9) of a wild-type wing disc expressing the dronc1.33-lacZ transgene. (B-B90) A diap122-8smosaic
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DIAP1DRINGmutants have lost the ability to interact with
DRONC in vivo. While we cannot exclude this possibility due to
the inability of our antibodies to immunoprecipitate endogenous
proteins, another experiment supports the notion that ubiquityla-
tion is necessary for DRONC inhibition: when wild-type diap1 was
strongly overexpressed in an ubiquitylation-deficient Uba1 mutant
background, DRONC-dependent apoptosis was not inhibited
(Figure 1C), suggesting that binding of DIAP1 is not sufficient for
inhibition of DRONC. Instead, ubiquitylation is critical for
inhibition of DRONC activity.
DIAP1 does not control protein levels of full-length
DRONC
The current model holds that DIAP1-mediated ubiquitylation
leads to proteasomal degradation of full-length DRONC in living
cells [33,38,45]. However, our data do not support this model in
vivo. This model is based on biochemical ubiquitylation studies
without in vivo validation and does not take into account that
ubiquitylation often serves non-proteolytic functions [1,3,4]. Both
overexpression and loss of diap1 does not cause a detectable
alteration of the protein levels of DRONC (Figure 3, Figure 4,
Figure 5), arguing against a degradation model. The only example
where DRONC accumulation has been observed is in P35-
expressing ‘undead’ diap1DRINGmutant cells [45,46], and we
showed here that the ‘undead’ nature of these cells causes
transcriptional induction of dronc (Figure 5). Together, these
observations argue against a degradation model of full-length
DRONC and favor a non-traditional (non-proteolytic) role of
ubiquitylation regarding control of DRONC activity. Similarly,
DIAP1-mediated ubiquitylation of the effector caspase DrICE
Figure 6. Ubiquitylation controls processing of DRONC. (A) Top: schematic outline of the domain structure of DIAP1+(wild-type) and RING-
deleted DIAP133-1s. Not drawn to scale. Immunoblots of embryonic extracts of stage 6–9 wild-type (wt) and heterozygous diap133-1smutants were
probed with anti-DIAP1 (upper panel) and anti-DRONC antibodies (middle panel). The RING-depleted diap133-1sallele produces a stable protein
(DIAP133-1s) that is detectable by its faster electrophoretic mobility (upper panel). In RING-depleted diap133-1sembryos a significant portion of
processed DRONC is present (middle panel) which likely accounts for the apoptotic phenotype of diap133-1sembryos [41]. These extracts were
obtained from a cross of heterozygous males and females. Thus, only one quarter of the embryos is homozygous mutant for diap133-1s; yet, processed
DRONC is easily detectable. The anti-DRONC antibody is specific for the large subunit of DRONC. Lower panel: loading control. (B) Extracts of imaginal
discs from wild-type (wt) and mosaic Uba1 imaginal discs were analyzed by immunoblotting using an antibody raised against the small subunit of
DRONC. Clones of the temperature sensitive allele Uba1D6were induced at the permissive temperature in first larval instar and then shifted to the
non-permissive temperature (30uC) during third larval instar 12 hours before dissection (see Material and Methods). This treatment ensures that
approximately 50% of the mosaic disc is mutant for Uba1. Although only 50% of the disc tissue is mutant for Uba1, processed DRONC is easily
detectable. Lower panel: loading control.
doi:10.1371/journal.pgen.1002261.g006
wing disc expressing p35 under nub-GAL4 control in a dronc1.33-lacZ background. A mutant clone in the wing pouch is highlighted by an arrow in the
GFP-only channel (B). DRONC protein (B9) and ß-GAL immunoreactivity as readout of dronc1.33-lacZ activity (B0) are increased in the same cells and
overlap (B90). Please note that the dronc1.33-lacZ reporter produces nuclear ß-GAL, while DRONC protein appears cytoplasmic. (C) GFP expression in the
eye imaginaldisc indicates the dorsal expression domain (arrow)of the dorsal eye (DE)-GAL4 driver[95]. (D) Increased dronc reporter activity in the dorsal
half of the eye imaginal disc (arrow) in undead cells obtained by co-expression of hid and p35 using DE-GAL4. (E) Expression of p35 alone by DE-GAL4
does not induce dronc reporter activity. (F-F0) A diap122-8smosaic wing disc in a dronc1.33-lacZ background which does not express p35. diap122-8s
mutantclonesaremarkedbytheabsenceofGFP(F).Anarrowpointstoarepresentativediap122-8scloneinthewingpouch.Inthesameposition,neither
DRONC protein (F9) nor dronc reporter activity (F0) are increased. Note, that this clone is present in the wing pouch which has the capacity to upregulate
DRONCanddronctranscription inthe‘undead’,p35-expressingcondition (seepanelB0).Genotypes:(A)dronc1.33-lacZ/+.(B)ubx-FLP;nub-GAL4UAS-p35/
dronc1.33-lacZ; diap122-8sFRT80/ubi-GFP FRT80. (C) DE-GAL4 UAS-GFP/+. (D) UAS-p35 UAS-hid/dronc1.33-lacZ; DE-GAL4. (E) UAS-p35/dronc1.33-lacZ; DE-
GAL4. (F) ubx-FLP; nub-GAL4/dronc1.33-lacZ; diap122-8sFRT80/ubi-GFP FRT80.
doi:10.1371/journal.pgen.1002261.g005
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inactivates this effector caspase through a non-degradative
mechanism [50].
Interestingly, in GMR-rpr eye imaginal discs, DRONC protein
levels appear to be reduced in apoptotic cells compared to living
cells (Figure 4C0, 4D0). Due to the apoptotic activity of REAPER,
DRONC is present in its processed and activated form. Reduced
protein stability of DRONC has also been reported after
incorporation into the ARK apoptosome where it is processed
and activated [46]. Combined, these observations suggest that
while DIAP1-mediated ubiquitylation of full-length DRONC does
not trigger its degradation, processed and activated DRONC has
reduced protein stability and may indeed be degraded. It is
unclear whether degradation of activated DRONC is mediated by
DIAP1, as proposed previously [33]. In GMR-rpr eye imaginal
discs, reduced DRONC levels correlate with a reduction of DIAP1
protein (Figure 4C9, 4D9). This correlation indicates that DIAP1
may actually stabilize DRONC protein, at least in part.
Alternatively, because DRONC and DIAP1 form a complex
[37], REAPER-induced degradation of DIAP1 may result in co-
degradation of complexed DRONC in the process. Further studies
are needed to determine the cause of decreased DRONC stability
in apoptotic cells.
In addition to Drosophila DRONC, mammalian CASPASE-3
and CASPASE-7 have been reported to be ubiquitylated in vitro
[47,48]. However, proteasome-mediated degradation of these
caspases in vivo has not been reported. Although a decrease of
CASPASE-3 levels has been noted upon overexpression of XIAP,
this was shown for an artificial CASPASE-3 mutant, in which the
order of the subunits was reversed and the Cys residue in the
active site changed to Ser [47]. This catalytically inactive
CASPASE-3 mutant is not proteolytically processed [47].
Therefore, physiological in vivo evidence for IAP-mediated
degradation of mammalian caspases is lacking.
Moreover, the phenotype of a RING-deleted XIAP mutant
mouse is consistent with our data [49]. The XIAPDRINGmutant,
which was generated by a knock-in approach in the endogenous
XIAP gene, is characterized by increased caspase activity [49].
Intriguingly, the protein levels of CASPASE-3, CASPASE-7 and
CASPASE-9 did not significantly change in the XIAPDRINGmutant
despite the fact that ubiquitylation of CASPASE-3 was reduced.
However, processing of these caspases was easily detectable in
XIAPDRINGmutants [49]. These data are very similar to the ones
presented here for diap133-1s(Figure 6), and together strongly
suggest that non-proteolytic ubiquitylation controls caspase
processing and activity in both vertebrates and invertebrates.
Non-proteolytic roles of ubiquitylation have been described in
recent years and are involved in many processes including signal
transduction, endocytosis, DNA repair, and histone activity
(reviewed in [1,3,4]). Two types of ubiquitylation lead to non-
proteolytic functions. Monoubiquitylation is involved in endocy-
tosis, DNA repair and histone activity. In fact, mammalian
CASPASE-3 and CASPASE-7 have been found to be mono-
ubiquitylated in vitro [48]. However, it is unclear whether DRONC
is monoubiquitylated by DIAP1. The presence of high molecular-
weight ubiquitin conjugates in vitro (Figure 2) suggests that
DRONC may be polyubiquitylated, at least under the experi-
mental conditions [38,44].
Polyubiquitylation serves both proteolytic and non-proteolytic
functions depending on the Lysine (K) residue used for
polyubiquitin chain formation. In general, if polyubiquitylation
occurs via K48, the target protein is subject to proteasome-
mediated degradation. If it occurs on a different Lys residue, such
as K63, non-proteolytic functions are induced [1,3,4]. The best
studied examples of both K48- and K63-linked polyubiquitylation
are in the NF-kB pathway (reviewed in [3,81]). While K48-
polyubiquitylation leads to proteasomal degradation, K63-linked
polyubiquitin chains act as scaffolds to assemble protein complexes
required for NF-kB activation [3,81]. It is unknown what type of
polyubiquitin chain is attached to DRONC, but it is possible that
it is not K48-linked. Interestingly, in this context it has been shown
that auto-ubiquitylation of DIAP1 preferentially involves K63-
linked polyubiquitin chains [82]. By analogy, DIAP1 may
ubiquitylate DRONC through formation of K63-linked poly-
ubiquitin chains. This will be an interesting avenue to explore in
future experiments.
Conjugated monoubiquitin and polyubiquitin chains can serve
as docking sites for factors containing ubiquitin-binding domains
(UBD) [2,4,83]. The UBD-containing factors interpret the
ubiquitylation status of the target protein, and trigger the
appropriate response. For example, K48-linked polyubiquitin
chains are recognized by Rad23 and Drk2 which deliver them to
the proteasome [2]. Other forms of ubiquitin conjugates are
recognized by different UBD-containing factors which control the
activity of the ubiquitylated protein. Therefore, it is possible that
an as yet unknown UBD-containing protein binds to ubiquitylated
DRONC and controls its activity. For example, such an
interaction could block the recruitment of ubiquitylated DRONC
into the ARK apoptosome. Another possibility is that ubiquityla-
tion could block dimerization of DRONC, which is required for
activation of DRONC [34].
‘‘Undead’’ cells trigger dronc transcription
‘Undead’ cells can be obtained by expression of the effector
caspase inhibitor P35 [84]. In these cells, apoptosis has been
induced, but cannot be executed due to effector caspase inhibition.
Nevertheless, the initiator caspase DRONC is active in ‘undead’
cells and can promote non-apoptotic processes [51]. Recent work
has suggested that ‘undead’ cells may alter their cellular behavior.
For example, ‘undead’ cells change their size and shape, and have
some migratory abilities to invade neighboring tissue [62]. They
are also able to promote proliferation of neighboring cells causing
hyperplastic overgrowth [15,45,63–66] (reviewed by [85,86]).
Altered transcription of the TGF-ß/BMP member decapentaplegic
(dpp), the Wnt-homolog wingless (wg), and the p53 ortholog dp53 has
also been reported in ‘undead’ cells [45,64–66]. Intriguingly, while
dpp and wg are usually not expressed in the same cells [87],
‘undead’ cells co-express them ectopically, clearly indicating an
altered transcriptional program.
As part of this altered transcriptional program, we showed that
‘undead’ cells also stimulate transcription of the initiator caspase
dronc (Figure 5). Interestingly, p35 expression in normal cells does
not induce dronc transcription suggesting that it is not the mere
presence of P35 that induces dronc transcription, but instead the
‘undead’ condition of the affected cells. This transcriptional
induction of dronc provides an explanation why DRONC protein
levels are increased in ‘undead’ cells. It may also help to explain
another observation regarding ‘undead’ cells. It has been
demonstrated that although these cells are unable to die, they
maintain the apoptotic machinery indefinitely [62,88]. Therefore,
as part of this maintenance program, ‘undead’ cells stimulate dronc
transcription. This is likely not restricted for dronc. Martin et al.
(2009) [62] also showed that DrICE protein levels remain high in
‘undead’ cells which may also be caused by increased drICE
transcription. It is also possible that the induction of dp53 by
‘undead’ cells [66] is part of this maintenance program, because
we have shown that Dp53 induces expression of hid and rpr [89]
and a positive feedback loop between dp53, hid and dronc exists in
‘undead’ cells [66]. This may all occur at a transcriptional level.
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The mechanism by which ‘undead’ cells stimulate expression of
dpp, wg, dp53, dronc and potentially drICE are currently unknown
and are avenues for future research.
Material and Methods
Drosophila genetics
Fly crosses were conducted using standard procedures at 25uC.
The following mutants and transgenes were used: Uba1D6[5]; arkG8
[26]; diap122-8sand diap133-1s[44]; vps25N55[90]; droncI29[11]; UAS-
droncIR (dronc inverted repeats) [91]; GMR-rpr [92]; dronc1.33-lacZ
[67,68], ubx-FLP [93], nub-GAL4 [94], DE- (dorsal eye-) GAL4 [95],
and UAS-hid [96]. nub-FLP is nub-GAL4 UAS-FLP. UAS-p35 and
UAS-FLP were obtained from Bloomington. Uba1D6is a tempera-
ture sensitive allele which at 25uC is a hypomorphic allele, but at
30uC is a null allele [5]. In the experiments in Figure 1, Figure 6B,
and Figure S1, Uba1D6and Uba1D6arkG8mosaic larvae were
incubated at 25uC; 12 hours before dissection the temperature was
shifted to 30uC. This treatment allows recovery of Uba1D6null
mutant clones, which otherwise are cell lethal.
Generation of mutant clones and expression of
transgenes
Mutant clones were induced in eye-antennal imaginal discs
using the FLP/FRT mitotic recombination system [97] using ey-
FLP [98]. In this case, clones are marked by loss of GFP.
Expression of diap1 and dronc RNAi in Uba1D6clones (Figure 1)
was induced from UAS-diap1 or UAS-droncIR transgenes using the
MARCM system [99]. Here, clones are positively marked by GFP.
For induction of diap1-expressing clones in Figure 3, the UAS-diap1
transgene was crossed to hs-FLP; tub,GFP,GAL4 (,=FRT).
Clones are marked by the absence of GFP. MARCM clones and
diap1-overexpressing clones were induced in first instar larvae by
heat-shock for one hour in a 37uC water bath. Expression of UAS-
p35 in diap122-8smosaic discs was accomplished by nub-GAL4.
Immunohistochemistry
Eye-antennal imaginal discs from third instar larvae were dissected
using standard protocols and labeled with antibodies raised against
the following antigens: DIAP1 (a kind gift of Hermann Steller and
Hyung Don Ryoo); cleaved CASPASE-3 (CAS3*) (Cell Signaling
Technology) and anti-ß-GAL (Promega). The DRONC antibody
used for immunofluorescence was raised against the C-terminus of
DRONC in guinea pigs [44]. This antibody is specific for DRONC
(Figure S3). Cy3- and Cy-5 fluorescently-conjugated secondary
antibodies were obtained from Jackson ImmunoResearch. In each
experiment, multiple clones in 10–20 eye and wing imaginal discs
were analyzed, unless otherwise noted. Images were captured using
an Olympus Optical FV500 confocal microscope.
Ubiquitylation assays
Schneider S2 cells were co-transfected with pMT-DRONC
C.A V5, pAcDIAP1 (wt or CD6, F437A, respectively, described
in [50]) and pAc His-HA-Ub at equal ratios. Expression of
DRONC was induced overnight with 350 mM CuSO4. Cells were
lysed under denaturing conditions and ubiquitylated proteins were
purified using Ni2+-NTA agarose beads (QIAGEN). Immunoblot
analysis was performed with a-V5 (Serotec) and a-DIAP1
antibodies [37,43].
Immunoblot analysis
For the immunoblots in Figure 6A, embryos were collected,
decorionated and snap frozen in liquid nitrogen. Embryos were
sonicated in Laemmli SDS loading buffer while being frozen. The
equivalent of 30 lysed embryos was loaded per lane. Immunoblots
were done using standard procedures. The anti-DRONC antibody
used in Figure 6A is a peptide antibody raised against the large
subunit of DRONC. The anti-DRONC antibody used in
Figure 6B was raised against the C-terminus of DRONC in
guinea pigs.
Supporting Information
Figure S1
ark mosaic eye-antennal disc labeled for cleaved CASPASE-3
(CAS3*) antibody (red). These discs were incubated at 30uC
12 hours before dissection (see Material and Methods). Absence of
GFP marks the location of Uba1 ark clones (see arrows). There is
scattered apoptosis detectable. However, this occurs throughout
the disc and does not correlate with the positions of the Uba1 ark
double mutant clones. Genotype: ey-FLP; FRT42D Uba1D6arkG8/
FRT42D ubi-GFP.
(TIF)
Loss of ark suppresses apoptosis in Uba1 clones. Uba1
Figure S2
vps25 mutants. Because the UAS-diap1 transgene failed to suppress
apoptosis in Uba1 clones (Figure 1C), we tested its ability to inhibit
the strong apoptotic phenotype in two other paradigms. (A)
Overexpression of the IAP-antagonist hid specifically in the fly eye
under GMR promoter control gives rise to a strong eye ablation
phenotype due to massive induction of apoptosis [100]. (B)
Coexpression of UAS-diap1 partially suppresses the GMR-hid eye
ablation phenotype [42]. (C) vps25 mutant clones induce a strong
apoptotic phenotype. vps25 encodes an component involved in
endosomal protein sorting [90]. The apoptotic phenotype of vps25
and Uba1 as well as other phenotypes caused by inactivation of
these genes are very similar, and both mutants were obtained in
the same genetic screen [5,90]. The left panel is the merge of GFP
and anti-cleaved CASPASE-3 (CAS3*) labeling, the right panel
(C9) displays only the CAS3* channel. White arrows mark a few
clones as examples. (D) Overexpression of diap1 completely
suppresses the strong apoptotic phenotype of vps25 mutant clones.
The experimental conditions applied here are identical to the
Uba1 experiment in Figure 1C. The left panel is the merge of GFP
and anti-cleaved CASPASE-3 (CAS3*) labeling, the right panel
(D9) displays only the CAS3* channel. Genotype: hs-FLP UAS-
GFP/UAS-diap1; FRT42D vps25N55/FRT42D tub-Gal80; tub-GAL4.
Genotypes: (A) GMR-hid GMR-GAL4. (B) UAS-diap1; GMR-hid
GMR-GAL4. (C) ey-FLP; FRT42D vps25N55/FRT42D P[ubi-GFP].
(D) ey-FLP; FRT42D vps25N55/FRT42D P[ubi-GFP].
(TIF)
UAS-diap1 rescues GMR-hid and apoptosis induced in
Figure S3
specificity of the anti-DRONC antibody used for immunofluores-
cence in Figure 3, Figure 4, and Figure 5 was verified in droncI29
mosaic eye (A) and wing (B) imaginal discs. The droncI29allele
contains a premature STOP codon at position 53 [11]. droncI29
clones were induced using the MARCM system, hence they are
positively marked by GFP (arrows). The anti-DRONC antibody
does not produce labeling signals in the mutant clones (arrows in
A9 and B9, and the merge in A0 and B0), demonstrating that it is
specific for DRONC. Genotype: hs-FLP; droncI29FRT80/ubi-GFP
FRT80.
(TIF)
Specificity of the anti-DRONC antibody. The
Figure S4
protein autonomously. (A, A9) Using MARCM, p35-expressing,
‘undead’ diap122-8smutant clones (green) were induced in eye discs
and labeled for DRONC protein (red). DRONC protein
‘‘Undead’’ diap122-8s
cells accumulate DRONC
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autonomously accumulates in P35-expressing diap122-8sclones
(arrows). Similar results were obtained in wing discs (data not
shown). Genotype: hs-FLP tub-GAL4 UAS-GFP/+; UAS-p35/+;
diap122-8sFRT80/tub-GAL80 FRT80.
(TIF)
Acknowledgments
We would like to thank Hans-Martin Herz for the vps25 data in Figure S2;
Audrey Christiansen for the outline of eye imaginal discs in Figure 4A;
Hugo Bellen, Georg Halder, Sharad Kumar, Hyung Don Ryoo, Hermann
Steller, and Kristin White for antibodies and fly stocks; and the
Bloomington Stock Center for fly stocks.
Author Contributions
Conceived and designed the experiments: AB PM TVL YF. Performed the
experiments: TVL YF SW MS MB. Analyzed the data: AB PM TVL YF
SW MS MB. Contributed reagents/materials/analysis tools: AB PM TVL
YF SW MS MB. Wrote the paper: TVL AB.
References
1. Welchman RL, Gordon C, Mayer RJ (2005) Ubiquitin and ubiquitin-like
proteins as multifunctional signals. Nat Rev Mol Cell Biol 6: 599–609.
2. Hicke L, Schubert HL, Hill CP (2005) Ubiquitin-binding domains. Nat Rev Mol
Cell Biol 6: 610–621.
3. Chen ZJ, Sun LJ (2009) Nonproteolytic functions of ubiquitin in cell signaling.
Mol Cell 33: 275–286.
4. Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of
ubiquitin in endocytosis and signaling. Science 315: 201–205.
5. Lee TV, Ding T, Chen Z, Rajendran V, Scherr H, et al. (2008) The E1
ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autono-
mously and tissue growth non-autonomously. Development 135: 43–52.
6. Degterev A, Yuan J (2008) Expansion and evolution of cell death programmes.
Nat Rev Mol Cell Biol 9: 378–390.
7. Kumar S (2007) Caspase function in programmed cell death. Cell Death Differ
14: 32–43.
8. Bao Q, Shi Y (2007) Apoptosome: a platform for the activation of initiator
caspases. Cell Death Differ 14: 56–65.
9. Riedl SJ, Salvesen GS (2007) The apoptosome: signalling platform of cell death.
Nat Rev Mol Cell Biol 8: 405–413.
10. Timmer JC, Salvesen GS (2007) Caspase substrates. Cell Death Differ 14:
66–72.
11. Xu D, Li Y, Arcaro M, Lackey M, Bergmann A (2005) The CARD-carrying
caspase Dronc is essential for most, but not all, developmental cell death in
Drosophila. Development 132: 2125–2134.
12. Xu D, Wang Y, Willecke R, Chen Z, Ding T, et al. (2006) The effector caspases
drICE and dcp-1 have partially overlapping functions in the apoptotic pathway
in Drosophila. Cell Death Differ 13: 1697–1706.
13. Chew SK, Akdemir F, Chen P, Lu WJ, Mills K, et al. (2004) The apical caspase
dronc governs programmed and unprogrammed cell death in Drosophila. Dev
Cell 7: 897–907.
14. Daish TJ, Mills K, Kumar S (2004) Drosophila caspase DRONC is required for
specific developmental cell death pathways and stress-induced apoptosis. Dev
Cell 7: 909–915.
15. Kondo S, Senoo-Matsuda N, Hiromi Y, Miura M (2006) DRONC coordinates
cell death and compensatory proliferation. Mol Cell Biol 26: 7258–7268.
16. Waldhuber M, Emoto K, Petritsch C (2005) The Drosophila caspase DRONC is
required for metamorphosis and cell death in response to irradiation and
developmental signals. Mech Dev 122: 914–927.
17. Kilpatrick ZE, Cakouros D, Kumar S (2005) Ecdysone-mediated up-regulation
of the effector caspase DRICE is required for hormone-dependent apoptosis in
Drosophila cells. J Biol Chem 280: 11981–11986.
18. Muro I, Berry DL, Huh JR, Chen CH, Huang H, et al. (2006) The Drosophila
caspase Ice is important for many apoptotic cell deaths and for spermatid
individualization, a nonapoptotic process. Development 133: 3305–3315.
19. Dorstyn L, Colussi PA, Quinn LM, Richardson H, Kumar S (1999) DRONC,
an ecdysone-inducible Drosophila caspase. Proc Natl Acad Sci U S A 96:
4307–4312.
20. Kanuka H, Sawamoto K, Inohara N, Matsuno K, Okano H, et al. (1999)
Control of the cell death pathway by Dapaf-1, a Drosophila Apaf-1/CED-4-
related caspase activator. Mol Cell 4: 757–769.
21. Rodriguez A, Oliver H, Zou H, Chen P, Wang X, et al. (1999) Dark is a
Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily
conserved death pathway. Nat Cell Biol 1: 272–279.
22. Zhou L, Song Z, Tittel J, Steller H (1999) HAC-1, a Drosophila homolog of
APAF-1 and CED-4 functions in developmental and radiation-induced
apoptosis. Mol Cell 4: 745–755.
23. Quinn LM, Dorstyn L, Mills K, Colussi PA, Chen P, et al. (2000) An essential
role for the caspase dronc in developmentally programmed cell death in
Drosophila. J Biol Chem 275: 40416–40424.
24. Dorstyn L, Read S, Cakouros D, Huh JR, Hay BA, et al. (2002) The role of
cytochrome c in caspase activation in Drosophila melanogaster cells. J Cell Biol
156: 1089–1098.
25. Dorstyn L, Kumar S (2008) A biochemical analysis of the activation of the
Drosophila caspase DRONC. Cell Death Differ 15: 461–470.
26. Srivastava M, Scherr H, Lackey M, Xu D, Chen Z, et al. (2007) ARK, the Apaf-
1 related killer in Drosophila, requires diverse domains for its apoptotic activity.
Cell Death Differ 14: 92–102.
27. Mills K, Daish T, Harvey KF, Pfleger CM, Hariharan IK, et al. (2006) The
Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not
all, programmed cell death. J Cell Biol 172: 809–815.
28. Akdemir F, Farkas R, Chen P, Juhasz G, Medved’ova L, et al. (2006) Autophagy
occurs upstream or parallel to the apoptosome during histolytic cell death.
Development 133: 1457–1465.
29. Mendes CS, Arama E, Brown S, Scherr H, Srivastava M, et al. (2006)
Cytochrome c–d regulates developmental apoptosis in the Drosophila retina.
EMBO Rep 7: 933–939.
30. Yu X, Wang L, Acehan D, Wang X, Akey CW (2006) Three-dimensional
structure of a double apoptosome formed by the Drosophila Apaf-1 related
killer. J Mol Biol 355: 577–589.
31. Yuan S, Yu X, Topf M, Dorstyn L, Kumar S, et al. (2011) Structure of the
Drosophila apoptosome at 6.9 a resolution. Structure 19: 128–140.
32. Hawkins CJ, Yoo SJ, Peterson EP, Wang SL, Vernooy SY, et al. (2000) The
Drosophila caspase DRONC cleaves following glutamate or aspartate and is
regulated by DIAP1, HID, and GRIM. J Biol Chem 275: 27084–27093.
33. Muro I, Hay BA, Clem RJ (2002) The Drosophila DIAP1 protein is required to
prevent accumulation of a continuously generated, processed form of the apical
caspase DRONC. J Biol Chem 277: 49644–49650.
34. Snipas SJ, Drag M, Stennicke HR, Salvesen GS (2008) Activation mechanism
and substrate specificity of the Drosophila initiator caspase DRONC. Cell Death
Differ 15: 938–945.
35. O’Riordan MX, Bauler LD, Scott FL, Duckett CS (2008) Inhibitor of apoptosis
proteins in eukaryotic evolution and development: a model of thematic
conservation. Dev Cell 15: 497–508.
36. Vaux DL, Silke J (2005) IAPs, RINGs and ubiquitylation. Nat Rev Mol Cell Biol
6: 287–297.
37. Meier P, Silke J, Leevers SJ, Evan GI (2000) The Drosophila caspase DRONC is
regulated by DIAP1. Embo J 19: 598–611.
38. Chai J, Yan N, Huh JR, Wu JW, Li W, et al. (2003) Molecular mechanism of
Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquiti-
nation. Nat Struct Biol 10: 892–898.
39. Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA (1999) The Drosophila
caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated
by HID. Cell 98: 453–463.
40. Goyal L, McCall K, Agapite J, Hartwieg E, Steller H (2000) Induction of
apoptosis by Drosophila reaper, hid and grim through inhibition of IAP
function. EMBO J 19: 589–597.
41. Lisi S, Mazzon I, White K (2000) Diverse domains of THREAD/DIAP1 are
required to inhibit apoptosis induced by REAPER and HID in Drosophila.
Genetics 154: 669–678.
42. Hay BA, Wassarman DA, Rubin GM (1995) Drosophila homologs of
baculovirus inhibitor of apoptosis proteins function to block cell death. Cell
83: 1253–1262.
43. Zachariou A, Tenev T, Goyal L, Agapite J, Steller H, et al. (2003) IAP-
antagonists exhibit non-redundant modes of action through differential DIAP1
binding. Embo J 22: 6642–6652.
44. Wilson R, Goyal L, Ditzel M, Zachariou A, Baker DA, et al. (2002) The DIAP1
RING finger mediates ubiquitination of Dronc and is indispensable for
regulating apoptosis. Nat Cell Biol 4: 445–450.
45. Ryoo HD, Gorenc T, Steller H (2004) Apoptotic cells can induce compensatory
cell proliferation through the JNK and the Wingless signaling pathways. Dev
Cell 7: 491–501.
46. Shapiro PJ, Hsu HH, Jung H, Robbins ES, Ryoo HD (2008) Regulation of the
Drosophila apoptosome through feedback inhibition. Nat Cell Biol 10:
1440–1446.
47. Suzuki Y, Nakabayashi Y, Takahashi R (2001) Ubiquitin-protein ligase activity
of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of
caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc
Natl Acad Sci U S A 98: 8662–8667.
48. Huang H, Joazeiro CA, Bonfoco E, Kamada S, Leverson JD, et al. (2000) The
inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and
promotes in vitro monoubiquitination of caspases 3 and 7. J Biol Chem 275:
26661–26664.
49. Schile AJ, Garcia-Fernandez M, Steller H (2008) Regulation of apoptosis by
XIAP ubiquitin-ligase activity. Genes Dev 22: 2256–2266.
Non-Degradative Ubiquitylation of DRONC
PLoS Genetics | www.plosgenetics.org 12 September 2011 | Volume 7 | Issue 9 | e1002261

Page 13

50. Ditzel M, Broemer M, Tenev T, Bolduc C, Lee TV, et al. (2008) Inactivation of
Effector Caspases through non-degradative Polyubiquitylation. Molecular
Cell;in press.
51. Fan Y, Bergmann A (2010) The cleaved-Caspase-3 antibody is a marker of
Caspase-9-like DRONC activity in Drosophila. Cell Death Differ 17: 534–539.
52. Pfleger CM, Harvey KF, Yan H, Hariharan IK (2007) Mutation of the Gene
Encoding the Ubiquitin Activating Enzyme Uba1 Causes Tissue Overgrowth in
Drosophila. Fly 1: 95–105.
53. Silke J, Kratina T, Chu D, Ekert PG, Day CL, et al. (2005) Determination of cell
survival by RING-mediated regulation of inhibitor of apoptosis (IAP) protein
abundance. Proc Natl Acad Sci U S A 102: 16182–16187.
54. Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H (2002) Regulation
of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat Cell
Biol 4: 432–438.
55. Hays R, Wickline L, Cagan R (2002) Morgue mediates apoptosis in the
Drosophila melanogaster retina by promoting degradation of DIAP1. Nat Cell
Biol 4: 425–431.
56. Holley CL, Olson MR, Colon-Ramos DA, Kornbluth S (2002) Reaper
eliminates IAP proteins through stimulated IAP degradation and generalized
translational inhibition. Nat Cell Biol 4: 439–444.
57. Wing JP, Schreader BA, Yokokura T, Wang Y, Andrews PS, et al. (2002)
Drosophila Morgue is an F box/ubiquitin conjugase domain protein important
for grim-reaper mediated apoptosis. Nat Cell Biol 4: 451–456.
58. Yoo SJ, Huh JR, Muro I, Yu H, Wang L, et al. (2002) Hid, Rpr and Grim
negatively regulate DIAP1 levels through distinct mechanisms. Nat Cell Biol 4:
416–424.
59. Wolff T, Ready DF (1991) The beginning of pattern formation in the Drosophila
compound eye: the morphogenetic furrow and the second mitotic wave.
Development 113: 841–850.
60. Cagan RL, Ready DF (1989) The emergence of order in the Drosophila pupal
retina. Dev Biol 136: 346–362.
61. Ellis MC, O’Neill EM, Rubin GM (1993) Expression of Drosophila glass protein
and evidence for negative regulation of its activity in non-neuronal cells by
another DNA-binding protein. Development 119: 855–865.
62. Martin FA, Perez-Garijo A, Morata G (2009) Apoptosis in Drosophila:
compensatory proliferation and undead cells. Int J Dev Biol 53: 1341–1347.
63. Huh JR, Guo M, Hay BA (2004) Compensatory proliferation induced by cell
death in the Drosophila wing disc requires activity of the apical cell death
caspase Dronc in a nonapoptotic role. Curr Biol 14: 1262–1266.
64. Perez-Garijo A, Martin FA, Morata G (2004) Caspase inhibition during
apoptosis causes abnormal signalling and developmental aberrations in
Drosophila. Development 131: 5591–5598.
65. Perez-Garijo A, Shlevkov E, Morata G (2009) The role of Dpp and Wg in
compensatory proliferation and in the formation of hyperplastic overgrowths
caused by apoptotic cells in the Drosophila wing disc. Development 136:
1169–1177.
66. Wells BS, Yoshida E, Johnston LA (2006) Compensatory proliferation in
Drosophila imaginal discs requires Dronc-dependent p53 activity. Curr Biol 16:
1606–1615.
67. Daish TJ, Cakouros D, Kumar S (2003) Distinct promoter regions regulate
spatial and temporal expression of the Drosophila caspase dronc. Cell Death
Differ 10: 1348–1356.
68. Cakouros D, Daish TJ, Kumar S (2004) Ecdysone receptor directly binds the
promoter of the Drosophila caspase dronc, regulating its expression in specific
tissues. J Cell Biol 165: 631–640.
69. Abrams JM, White K, Fessler LI, Steller H (1993) Programmed cell death during
Drosophila embryogenesis. Development 117: 29–43.
70. Muro I, Monser K, Clem RJ (2004) Mechanism of Dronc activation in
Drosophila cells. J Cell Sci 117: 5035–5041.
71. Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) X-linked IAP is a
direct inhibitor of cell-death proteases. Nature 388: 300–304.
72. Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) The c-IAP-1
and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J 16:
6914–6925.
73. Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, et al. (1998) A
single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem 273:
7787–7790.
74. Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, et al. (1999) NMR
structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature
401: 818–822.
75. Sun C, Cai M, Meadows RP, Xu N, Gunasekera AH, et al. (2000) NMR
structure and mutagenesis of the third Bir domain of the inhibitor of apoptosis
protein XIAP. J Biol Chem 275: 33777–33781.
76. Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, et al. (2001) A
conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates
caspase activity and apoptosis. Nature 410: 112–116.
77. Shiozaki EN, Chai J, Rigotti DJ, Riedl SJ, Li P, et al. (2003) Mechanism of
XIAP-mediated inhibition of caspase-9. Mol Cell 11: 519–527.
78. Riedl SJ, Renatus M, Schwarzenbacher R, Zhou Q, Sun C, et al. (2001)
Structural basis for the inhibition of caspase-3 by XIAP. Cell 104: 791–800.
79. Chai J, Shiozaki E, Srinivasula SM, Wu Q, Datta P, et al. (2001) Structural basis
of caspase-7 inhibition by XIAP. Cell 104: 769–780.
80. Silke J, Hawkins CJ, Ekert PG, Chew J, Day CL, et al. (2002) The anti-apoptotic
activity of XIAP is retained upon mutation of both the caspase 3- and caspase 9-
interacting sites. J Cell Biol 157: 115–124.
81. Wertz IE, Dixit VM (2010) Signaling to NF-kappaB: regulation by ubiquitina-
tion. Cold Spring Harb Perspect Biol 2: a003350.
82. Herman-Bachinsky Y, Ryoo HD, Ciechanover A, Gonen H (2007) Regulation
of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct
ubiquitin system pathways. Cell Death Differ 14: 861–871.
83. Adhikari A, Xu M, Chen ZJ (2007) Ubiquitin-mediated activation of TAK1 and
IKK. Oncogene 26: 3214–3226.
84. Hay BA, Wolff T, Rubin GM (1994) Expression of baculovirus P35 prevents cell
death in Drosophila. Development 120: 2121–2129.
85. Bergmann A, Steller H (2010) Apoptosis, stem cells, and tissue regeneration. Sci
Signal 3: re8.
86. Fan Y, Bergmann A (2008) Apoptosis-induced compensatory proliferation. The
Cell is dead. Long live the Cell! Trends Cell Biol 18: 467–473.
87. Tabata T (2001) Genetics of morphogen gradients. Nat Rev Genet 2: 620–630.
88. Yu SY, Yoo SJ, Yang L, Zapata C, Srinivasan A, et al. (2002) A pathway of
signals regulating effector and initiator caspases in the developing Drosophila
eye. Development 129: 3269–3278.
89. Fan Y, Lee TV, Xu D, Chen Z, Lamblin AF, et al. (2010) Dual roles of
Drosophila p53 in cell death and cell differentiation. Cell Death Differ 17:
912–921.
90. Herz HM, Chen Z, Scherr H, Lackey M, Bolduc C, et al. (2006) vps25 mosaics
display non-autonomous cell survival and overgrowth, and autonomous
apoptosis. Development 133: 1871–1880.
91. Leulier F, Ribeiro PS, Palmer E, Tenev T, Takahashi K, et al. (2006) Systematic
in vivo RNAi analysis of putative components of the Drosophila cell death
machinery. Cell Death Differ 13: 1663–1674.
92. White K, Tahaoglu E, Steller H (1996) Cell killing by the Drosophila gene
reaper. Science 271: 805–807.
93. Emery G, Hutterer A, Berdnik D, Mayer B, Wirtz-Peitz F, et al. (2005)
Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in
the Drosophila nervous system. Cell 122: 763–773.
94. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering
cell fates and generating dominant phenotypes. Development 118: 401–415.
95. Morrison CM, Halder G (2010) Characterization of a dorsal-eye Gal4 Line in
Drosophila. Genesis 48: 3–7.
96. Zhou L, Schnitzler A, Agapite J, Schwartz LM, Steller H, et al. (1997)
Cooperative functions of the reaper and head involution defective genes in the
programmed cell death of Drosophila central nervous system midline cells. Proc
Natl Acad Sci U S A 94: 5131–5136.
97. Xu T, Rubin GM (1993) Analysis of genetic mosaics in developing and adult
Drosophila tissues. Development 117: 1223–1237.
98. Newsome TP, Asling B, Dickson BJ (2000) Analysis of Drosophila photoreceptor
axon guidance in eye-specific mosaics. Development 127: 851–860.
99. Lee T, Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM)
for Drosophila neural development. Trends Neurosci 24: 251–254.
100. Grether ME, Abrams JM, Agapite J, White K, Steller H (1995) The head
involution defective gene of Drosophila melanogaster functions in programmed
cell death. Genes Dev 9: 1694–1708.
Non-Degradative Ubiquitylation of DRONC
PLoS Genetics | www.plosgenetics.org13 September 2011 | Volume 7 | Issue 9 | e1002261