Regulators of IAP function: coming to grips with the grim reaper

Regulators of IAP function: coming to grips with the grim reaperAndreas Bergmann�, Amy Yi-Pei Yang and Mayank Srivastava

Inhibitor of apoptosis proteins (IAPs) are a conserved class of

proteins that control apoptosis in both vertebrates and

invertebrates. They exert their anti-apoptotic function through

inhibition of caspases, the principal executioners of apoptotic

cell death. Recent advances in vertebrates and Drosophila have

demonstrated that IAPs use ubiquitin conjugation to control the

stability, and thus the activity, of select target proteins. The

Drosophila IAP1 gene is an instructive example: it employs at

least two distinct ubiquitin-dependent mechanisms of protein

destruction. The apoptosis-inducing genes grim, reaper and hid

modulate these mechanisms, and determine the outcome.

AddressesThe University of Texas MD Anderson Cancer Center, Department of

Biochemistry & Molecular Biology, Unit 117, 1515 Holcombe Blvd,

Houston, TX 77030 USA�e-mail: [email protected]

Current Opinion in Cell Biology 2003, 15:717–724

This review comes from a themed issue on

Cell division, growth and death

Edited by Jonathon Pines and Sally Kornbluth

0955-0674/$ – see front matter

� 2003 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.ceb.2003.10.002

AbbreviationsBIR baculovirus IAP repeat

DIAP1 Drosophila inhibitor of apoptosis protein 1

GMR glass-multimer reporter

IAP inhibitor of apoptosis protein

RHG reaper, hid and grim

RING really interesting new gene

UBC ubiquitin conjugating

XIAP X-linked IAP

IntroductionApoptosis is a physiological cell-suicide process that plays

an important role during the development of multicellular

organisms and is critical for the maintenance of tissue

homeostasis. Caspases, a highly specialized class of cell-

death proteases, are the main executioners of apoptosis

[1,2]. They are synthesized as inactive zymogen precur-

sors and require proteolytic cleavage for activation. In this

process, the prodomain is cleaved off, and a large and

small subunit are generated [1,2]. Activation of upstream

caspases such as Caspase-9 is regulated by cytochrome cand Apaf-1. Upstream caspases activate downstream cas-

pases in an amplifying cascade, cleaving one another in

sequence. These modes of caspase activation have been

extensively reviewed [1,2]. However, recent advances in

Drosophila have indicated that the inhibition of caspases is

a highly dynamic process involving protein–protein inter-

actions and proteolytic degradation. The Drosophila inhi-

bitor of apoptosis protein 1 (Diap1) plays a central role in

this regulation, and is the focus of this review.

The apoptotic machinery is conserved between verte-

brates and Drosophila, and there are fly homologs of

caspases, Bcl-2 family members, Apaf-1 and IAPs [3].

Genetic analysis in Drosophila has identified three addi-

tional genes that are essential for embryonic cell death in

this species. These genes are reaper, hid and grim, and are

collectively referred to as the RHG genes [4–6]. Two

more genes, sickle and jafrac2, with similar characteristics

to the reaper, hid and grim genes, have recently been

identified [7–10]. Chromosomal deletions removing the

RHG genes completely block apoptosis during embry-

ogenesis, and cause embryonic lethality [4], demonstrat-

ing the importance of the RHG genes for apoptosis in

Drosophila.

The RHG genes encode novel proteins without signif-

icant homology to other proteins in the database. How-

ever, they share a common motif at the N terminus

(Figure 1b,c). This motif, referred to as the RHG motif

[11], is essential for the ability of the RHG proteins to

induce apoptosis: truncation of the motif results in partial

or complete loss of the apoptosis-inducing activity of the

RHG proteins. Recently, two mammalian factors, Smac/

Diablo and Omi/HtrA2, have been identified that behave

functionally as homologs of the RHG genes and possess

the RHG motif (Figure 1c) [12–14]. Interestingly, the

RHG motif has to be present at the extreme N terminus

of the proteins. Drosophila Reaper, Hid, Grim and Sickle

carry the RHG motif immediately following the initiator

methionine [4–9] (Figure 1c). However, the RHG motif

of Smac/Diablo, Omi/HtrA2 and Drosophila Jafrac2 is

not located at the N terminus of the precursor forms of

these proteins, and requires proteolytic processing for N-

terminal exposure. Strikingly, because they are mitochon-

drial proteins, but encoded in the nucleus, the RHG

motif is revealed at the N terminus after their mitochon-

drial import when the signal sequence is cleaved off

[10,12–14]. A third mammalian protein containing a

RHG motif is Caspase-9 (Figure 1c). Again, after proteo-

lytic processing of Caspase-9, the small subunit exposes a

tetrapeptide motif at the N terminus that functions as a

RHG [15].

Overexpression of the Drosophila RHG genes in flies or in

mammalian cell culture is sufficient to induce apoptosis

[5,6,16,17]. For example, transgenes that place the

717

www.current-opinion.com Current Opinion in Cell Biology 2003, 15:717–724

cDNAs of the RHG genes under control of the eye-

specific glass-multimer reporter (GMR) promoter

(GMR-hid, GMR-reaper) induce ablation of eye structures

due to excessive apoptosis (Figures 2b,c) [5,6,16]. This

eye ablation phenotype is restored to normal if the uni-

versal caspase inhibitor P35 is coexpressed in the devel-

oping fly eye (GMR-p35; Figure 2d,e) [5,6,16]. This

finding suggests that the RHG genes induce apoptosis

through activation of a caspase program. However,

because the RHG genes do not directly interact with

caspases, additional components must be involved.

Drosophila inhibitor of apoptosis protein 1To identify these components, the eye ablation pheno-

types of GMR-hid and GMR-reaper were used in genetic

modifier screens. The first gene to be discovered in these

screens was diap1 [18]. Heterozygous mutations of diap1were found to be strong enhancers of GMR-hid- and

GMR-reaper-induced eye phenotypes (Figures 2f,g)

[18]. Furthermore, coexpression of diap1 (GMR-diap1)

with GMR-hid or GMR-reaper partially suppresses the

eye phenotype (Figures 2h,i) [18]. The importance of

IAPs in the regulation of apoptosis was made obvious by

phenotypic analysis of homozygous diap1 mutants. Loss

of diap1 leads to uncontrolled caspase activation and

apoptosis [19–21]. These findings suggest that diap1encodes an important anti-apoptotic function.

IAPs are a highly conserved class of proteins that were

initially discovered in baculovirus, but were soon after

found in metazoan organisms such as Drosophila and

humans [18,22–27]. They are distinguished by the pre-

sence of between one and three BIR (baculovirus IAP

repeat) motifs, and some have a C-terminal RING (really

interesting new gene) domain (Figure 3). As discussed

later, the RING domain encodes an E3-ubiquitin ligase

that is required for ubiquitin-mediated degradation.

The BIR motifs of IAPs are essential for their anti-

apoptotic function. They mediate the binding of IAPs

to caspases and are responsible for inhibiting the caspases

[28,29]. However, although initial reports stated that

IAPs form complexes with the zymogen form of caspases

[30,31], recent data suggest that IAP binding requires —

paradoxically — cleavage and activation of caspases

[15,32–35,36�]. Thus, the activity of caspases is regulated

at two levels. Although zymogen processing is an impor-

tant element in the control of caspase activation, inhibi-

tion of activated caspases by IAPs provides a second level

of regulation.

Interestingly, the individual BIR motifs of a given IAP

have different substrate specificity. For example, human

X-linked IAP (XIAP) inhibits the activity of Caspase-3,

-7 and -9. However, inhibition of Caspase-3 and -7 by

Figure 1

Reaper M AVAFYIPDQATLLGrim M AIAYFIPDQAQLLHid M AVPFYLPEGGADDSickle M AIPFFEEEHAPKS

Smac/DIABLO - AVPIJafrac2 - AKPE

Omi/HtrA2 - AVPShCaspase-9 - ATPF

droncdiap1

p35

reaperhid

grimsickle

Cell-death-inducingstimuli

(c)

(a)

(b)

D-Apaf-1 (dark)

?

Reaper

Sickle

Grim

Hid

65 aa

138 aa

410 aa

108 aa

drICEdcp-1

Celldeath

Current Opinion in Cell Biology

The Drosophila cell death pathway. (a) During Drosophila development, the RHG genes reaper, hid, grim and sickle are central for the induction

of apoptosis; they activate the initiator caspase Dronc through inhibition of Diap1. Dronc in turn processes and activates the executioner caspases

DrICE and Dcp-1. The RHG proteins integrate a large number of cell-death-inducing stimuli including Ecdysone, p53, developmental signals and

cell–cell interactions. Shown is also the Drosophila Apaf-1 homolog, dark, which activates the initiator caspase Dronc. The baculoviral cell death

inhibitor P35 is known to inhibit apoptosis by directly binding to caspases, most notably DrICE and Dcp-1. (b) Schematic outline of the Drosophila

RHG proteins. The N termini of the RHG proteins contain a conserved motif, the RHG motif, indicated in blue (see [c]). In addition to the RHG

motif, they share partial similarity in blocks of over 30 residues each (red boxes; four in Hid), which was designated as the Trp-block [11]. The function

of the Trp-block is currently unknown. Not drawn to scale. (c) The RHG motif. Conserved and partially conserved residues of the RHG motif are

highlighted in bold. For efficient binding to IAPs, the RHG motif has to be present at the extreme N terminus. In case of the Drosophila RHG proteins, it

is located immediately following the initiator Met, which is presumably removed by a methionine amino peptidase [64]. The mammalian

RHG-containing proteins undergo proteolytic processing to expose the RHG motif at the N terminus. The tetrapeptides shown are sufficient for

binding to XIAP [36�,65,66].

718 Cell division, growth and death

Current Opinion in Cell Biology 2003, 15:717–724 www.current-opinion.com

XIAP requires the BIR2 domain and a small segment

N-terminal to BIR2 [32–35,37], whereas BIR3 inhibits

Caspase-9 [15,36�]. Thus, the presence of several BIR

motifs increases the flexibility with which an IAP inhibits

caspases.

IAPs do not only interact with caspases. There is compel-

ling genetic and biochemical evidence that the RHG

proteins Reaper, Hid and Grim induce apoptosis through

interaction with, and antagonism of, Diap1, thus liberat-

ing caspases (Figure 1a). This interaction requires the

BIR motifs of Diap1 and the RHG motif of Reaper, Hid

and Grim [38]. Deletion of the RHG motif abolishes the

interaction with Diap1, demonstrating the biochemical

function of the RHG motif. Similarly, the mammalian

factors Smac/DIABLO and Omi/HtrA2 use their RHG

motif to oppose caspase inhibition by IAPs [12–14,39,40].

Taken together, these data suggest that the RHG pro-

teins induce apoptosis through inhibition of Diap1, result-

ing in the release of caspases.

Recent data provide evidence that the functional out-

comes of the protein–protein interactions of Diap1 and

various binding partners are determined by protein degra-

dation, mediated by ubiquitination (the covalent attach-

ment of ubiquitin, a 76-amino-acid protein, to target

proteins for degradation). Two distinct mechanisms of

ubiquitin-mediated degradation have been uncovered for

Diap1. These will be discussed below.

N-end rule pathway: co-degradation of Diap1and associated caspases promotes survivalRecently, it was reported that the protein stability of

Diap1 is controlled by the N-end rule pathway [41��].According to the N-end rule, the identity of the N-

terminal residue determines the half-life of the protein

[42]. A destabilizing residue such as Asn at the N terminus

induces rapid degradation of the protein by the ubiquitin-

conjugating pathway. Full-length Diap1 does not contain

a destabilizing residue at the N terminus. However, there

is a caspase cleavage site at residue 20 that is cleaved by

Drosophila caspases DrICE and DCP-1 (Figure 4) [41��].The resulting fragment, Diap121-438, bears a destabilizing

Asn residue at the N terminus, and is rapidly degraded by

the N-end rule pathway [41��]. Thus, this mode of

stability control requires active caspases.

To investigate the functional significance of N-end-rule

degradation of Diap1, mutants were constructed; in some

of these mutants caspase cleavage exposes a different,

stabilizing residue according to the N-end rule, whereas

others were resistant to caspase cleavage in the first place.

Surprisingly, these mutants, which encode more stable

Figure 2

Wild-type

GMR-hid+

GMR-hidGMR-p35

GMR-hiddiap1–

GMR-hidGMR-diap1

GMR-reaper+

GMR-reaperGMR-p35

GMR-reaperdiap1–

GMR-reaperGMR-diap1

(a)

(b)

(d)

(f)

(h)

(c)

(e)

(g)

(i)


Genetic interactions of GMR-hid and GMR-reaper with apoptosis

inhibitors. (a) A normal eye of a wild-type fly. (b) and (c) Expression

of hid (b) and reaper (c) under control of the eye-specific GMR promoter

gives rise to strong eye ablation phenotypes. (d) and (e) Co-expression

of the caspase inhibitor p35 (see Figure 1a) completely rescues the

GMR-hid- (d) and GMR-reaper- (e) induced eye phenotypes. This

analysis suggests that Hid and Reaper induce apoptosis through

activation of a caspase program. (f) and (g) Removing one genomic

copy of the diap1 gene strongly enhances the GMR-hid- (f) and GMR-

reaper- (g) induced eye phenotypes. Note that the eyes in (f) and (g) are

much smaller (enhanced) than the unmodified eyes in (b) and (c),suggesting that Diap1 provides an essential anti-apoptotic function.

(h) and (i) Co-expression of Diap1 with GMR-hid (h) andGMR-reaper (i) partially rescues the eye phenotypes, consistent with

the notion that Diap1 acts as apoptosis inhibitor.

Regulators of IAP function Bergmann, Yang and Srivastava 719


Diap1 proteins, protect less efficiently against Reaper-

induced apoptosis than does wild-type Diap1, suggesting

that degradation of Diap1 actually promotes its anti-

apoptotic function [41��]. This finding is surprising, as

it was expected that loss of Diap1 would decrease the

apoptotic threshold, resulting in apoptosis. Even more

paradoxical is the requirement for active caspases in N-

end-rule-dependent degradation of Diap1. Somehow

these caspases must be inactivated in the process of

Diap1 degradation in order for Diap1 to exert its anti-

apoptotic function. This was not addressed by Ditzel et al.(2003) [41��], but it seems reasonable to assume that

associated active caspases are degraded simultaneously

with Diap1 (Figure 4). Thus, Diap1 serves as a safeguard

by inducing the degradation of associated active caspases

that either form spontaneously or are produced by weak

apoptotic signals.

Degradation of Diap1 and associated active caspases by

the anti-apoptotic N-end-rule pathway is reminiscent of

the role of IAPs in vertebrates. Here, it was shown that

IAPs preferentially inhibit active caspases. For instance,

human XIAP binds to the processed form of Caspase-3

and -7 and blocks the access of potential substrates

[32–35]. Interestingly, XIAP is also a target of caspase

cleavage, and one of the resulting fragments, BIR3-

RING, is a potent inhibitor of caspases [43]. This fragment

bears a destabilizing Ala residue at the N terminus; how-

ever, it has not been addressed whether this fragment and

its associated caspases are subject to N-end-rule-depen-

dent degradation. Nevertheless, vertebrate and insect IAPs

appear to have developed similar mechanisms to accom-

plish the same task — the inhibition of active caspases.

RING-dependent degradation: selectivedegradation of Diap1 induces apoptosisIn addition to the BIR domains, some IAPs contain a

RING domain (Figure 3). The RING domain is a Zn-

binding fold of �70 residues that does not appear to be

required for the interaction of IAPs with either caspases or

RHG proteins [29,31,44�]. Recent data suggest that the

RING domain encodes an E3-ubiquitin ligase activity

[45,46], implying that it plays a role in protein degradation

via the ubiquitin-conjugating pathway. Ubiquitination of

target proteins occurs through the sequential transfer of

ubiquitin from E1-activating enzymes to E2-conjugating

enzymes and finally, mediated by the E3-ubiquitin ligase,

to target proteins. Ubiquitin-tagged proteins are poly-

ubiquitinated and degraded by the 26S proteasome.

Similar to N-end rule degradation, the E3-ligase of the

Figure 3

X-IAP

c-IAP1

c-IAP2

NAIP

ML-IAP

Survivin

D-IAP 1 RING

RING

RING

RING

RING

RING

RING

BIR1 BIR2

BIR1 BIR2 BIR3D-IAP 2

dBRUCE

BIRDeterin

BIR1 BIR2 BIR3

BIR1 BIR2 BIR3

BIR1 BIR2 BIR3

BIR1 BIR2 BIR3

BIR

BRUCE

BIR

497

604

618

1403

4845

142

438

498

280

4876

153

Human

Drosophila

BIR 236TS-IAP

CARD

CARD

UBC

UBC

BIR

BIR


Domain structure of Drosophila and human IAPs. The Drosophila genome encodes four IAP genes; the human genome contains eight. IAPs

are characterized by the presence of at least one BIR motif, and some contain an E3-RING domain which is invariantly located at the extreme

C terminus. The Bruce genes contain a UBC domain instead of a RING domain. Human c-IAP1 and c-IAP2 also have a CARD (caspase recruitment

domain); however, the role of this domain for IAP function has not been investigated. Not drawn to scale.



RING domain stimulates the ubiquitination and proteo-

lysis of IAPs [46]; however, in contrast to degradation by

the N-end rule, RING-dependent degradation of IAPs is

pro-apoptotic, and occurs in cells committed to die.

The E3-ligase activity of the RING domain is stimulated

or modulated by binding of the pro-apoptotic RHG

proteins Reaper, Hid and Grim to Diap1. This interac-

tion induces auto-ubiquitination of Diap1 for self

destruction in vivo (Figure 4) [47��–51��]. As a result

of RING-dependent degradation of Diap1, caspases are

no longer inhibited.

How does release of caspases occur after RING-mediated

degradation of Diap1? There are two possibilities. The

first possibility is that RHG proteins induce the specific

degradation of Diap1, but not the caspase, in the pre-

formed Diap1/caspase complex. Alternatively, RING-

dependent degradation might serve to remove all free,

caspase-unbound Diap1, such that newly synthesized

caspase molecules are no longer subject to Diap1-

mediated inhibition. This latter possibility is also sup-

ported by the finding that Reaper and Grim repress total

protein translation, with the result that the amount of

newly synthesized Diap1 (and other proteins) is even

further reduced [48��,51��]. Because caspases have a

much longer half-life than Diap1 [51��], an excess of free

caspases will be generated in this way, which can be

further activated by Apaf-1/Dark and additional proteins.

Candidate E2-ubiquitin-conjugating enzymes that are

involved in mediating RING-dependent activity have

also been genetically identified: ubcD1 [49��] and morgue[47��,50��]. UbcD1 is a classical E2-conjugating enzyme,

whereas morgue encodes an ubiquitin-conjugating en-

zyme variant that lacks a critical Cys residue present in

all classical E2s. Morgue also contains a F-box, a domain

characteristic of E3-ubiquitin ligases. Both genes were

identified as genetic modifiers of GMR-reaper-induced

eye phenotypes, and the proteins interact with Diap1

[47��,49��,50��]. Thus, UbcD1 and Morgue probably act

in concert to ubiquitinate Diap1. Another potential E2-

conjugating enzyme is encoded by dBruce, an unusual

member of the IAP family that encodes a gigantic protein

of 4876 residues with a single BIR motif and an E2-ubc

domain (Figure 3) [52]. Its exact function is currently

unknown, but it appears to specifically regulate the activity

of reaper and grim, but not of hid [52]. Interestingly, also,

Scythe, a Reaper-interacting protein, bears at the N ter-

minus a domain with similarity to ubiquitin [53].

Figure 4

REAPERHID

GRIMDronc

Diap1BIR BIR RING

DQVD N21

DrICEDcp-1

Ub

Ub UbUb

Ub

Ub Ubiquitin

N-end-rule-dependentubiquitination

RING-dependentubiquitination


N-end-rule- and RING-dependent ubiquitination of Diap1 and associated proteins. The components of the cell-death pathway in Drosophila are

shown. Genetic interactions are presented by red arrows (see also Figure 1a). The domain structure of Diap1 is depicted. N-end-rule-dependent

degradation is presented in green, RING-dependent ubiquitination in blue. The dashed lines indicate activation or modulation of the corresponding

degradation pathway. Please note that the green and blue arrows illustrate the flow of ubiquitination, but do not indicate genetic interactions.

Activated caspases DrICE and Dcp-1 cleave Diap1 at residue 20 and expose the destabilizing Asn21 for N-end rule-dependent degradation (green

dashed line). This in turn triggers degradation of Diap1 and presumably of associated caspases by ubiquitination (solid green arrows). The RHG

proteins Reaper, Hid and Grim bind to the BIR domains (not drawn) and stimulate or modulate the activity of the RING E3-ligase (dashed blue line).

This activates ubiquitination and degradation of Diap1 and the RHG proteins (blue solid arrows). The RING domain also promotes ubiquitination of

Dronc [44�]. However, it is unknown whether Dronc is degraded in response to ubiquitination.



Other targets of RING-dependentubiquitination: the RHG proteins andcaspasesSurprisingly, the RHG proteins not only induce Diap1

ubiquitination, but are themselves targets of Diap1-

mediated ubiquitination and degradation (Figure 4)

[54��]. Similarly, human Smac was also found to be

subject to IAP-mediated degradation [55��]. Ubiquitina-

tion of the RHG proteins by Diap1 is dependent on a

functional RING domain. This control of RHG protein

stability has a significant effect on their ability to induce

apoptosis. A mutant form of Reaper lacking all Lys

residues, which serve as ubiquitin acceptors, is resistant

to Diap1-mediated degradation and a more potent indu-

cer of apoptosis [54��]. Thus, these studies suggest that

RHG and IAP proteins mutually control their abundance

(Figure 4), and define another anti-apoptotic function of

IAPs. This negative feedback loop provides a balance

between death-inducing and survival-promoting signals.

A slight change in the stability or abundance of these

proteins might shift the balance one way or another.

Furthermore, Diap1 can promote ubiquitination of the

caspase Dronc in a RING-dependent manner in vitro(Figure 4) [44�]. In humans, XIAP and cIAP2 can act

as E3-ligases for Caspase-3 and -7 in vitro [56,57]. How-

ever, it has not been convincingly demonstrated in vivothat RING-dependent ubiquitination of Dronc, Caspase-

3 and -7 results in proteolytic degradation. It should be

noted that ubiquitination does not always lead to degra-

dation. Ubiquitin has some non-traditional roles, such as

membrane trafficking, transcriptional regulation and pro-

tein sorting, which do not involve proteolysis [58]. Thus,

it is possible that RING-dependent ubiquitination of

Dronc, and of caspases in general, modifies their activity

without inducing degradation.

ConclusionsThe activity of caspases is subject to tight genetic control.

Caspase activity needs to reach a certain threshold level

before a cell is committed to die. Diap1 (and IAPs in

general) appear to define this apoptotic threshold, which

is characteristic for each individual cell type. In this

highly dynamic process, Diap1 controls the stability of

itself and associated proteins, and can integrate several

distinct regulatory signals (Figure 4). N-end-rule-

dependent degradation of Diap1 is an important compo-

nent of its anti-apoptotic activity. Although Diap1 is

degraded in this process, this actually increases rather

than decreases the apoptotic threshold, because caspases

are effectively inactivated in this process. However, to

reduce the apoptotic threshold and to liberate caspases

from IAP inhibition in cells committed to die, the E3-

ligase activity of the RING domain needs to be stimu-

lated to selectively degrade Diap1. This pro-apoptotic

component of Diap1 is triggered by the RHG proteins

Reaper, Hid and Grim. Thus, these studies make impor-

tant contributions to our understanding of the control and

significance of the protein stability of Diap1.

IAPs are a highly versatile class of proteins. In this review,

we focused on the role of IAPs as inhibitors of apoptosis.

However, they are also found as components of TNF-

receptor signaling [24] and TGF-b signaling [59,60], and

some of them are involved in cytokinesis during mitosis

[61]. Again, in TNF-receptor signaling they have been

shown to induce degradation of TNF-receptor-associated

factors [62,63]. Thus, a common theme in IAP function

appears to be the control of their own stability as well as

the stability of associated proteins. It will be exciting to

determine whether this holds true for other cellular

functions of IAPs.

AcknowledgementsWe would like to thank Georg Halder and Mary Ellen Lane for criticalreading of the manuscript. We are grateful for support by the Robert A WelchFoundation, the MD Anderson Research Trust, the March of DimesFoundation and NIH grant 1R01GM068016-01A1.

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17. Haining WN, Carboy-Newcomb C, Wei CL, Steller H: Thepro-apoptotic function of Drosophila hid is conserved inmammalian cells. Proc Natl Acad Sci USA 1999, 96:4936-4941.

18. Hay BA, Wassarman DA, Rubin GM: Drosophila homologs ofbaculovirus inhibitor of apoptosis proteins function to blockcell death. Cell 1995, 83:1253-1262.

19. Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA: The Drosophilacaspase inhibitor DIAP1 is essential for cell survival and isnegatively regulated by HID. Cell 1999, 98:453-463.

20. Goyal L, McCall K, Agapite J, Hartwieg E, Steller H: Induction ofapoptosis by Drosophila reaper, hid and grim through inhibitionof IAP function. EMBO J 2000, 19:589-597.

21. Lisi S, Mazzon I, White K: Diverse domains of THREAD/DIAP1 arerequired to inhibit apoptosis induced by REAPER and HID inDrosophila. Genetics 2000, 154:669-678.

22. Birnbaum MJ, Clem RJ, Miller LK: An apoptosis-inhibiting genefrom nuclear polyhedrosis virus encoding a polypeptide withCys/His sequence motifs. J Virol 1994, 68:2521-2528.

23. Roy N, Mahadevan MS, McLean M, Shutler G, Yaraghi Z,Farahani R, Baird S, Besner-Johnston A, Lefebvre C, Kang X et al.:The gene for neuronal apoptosis inhibitory protein is partiallydeleted in individuals with spinal muscular atrophy. Cell 1995,80:167-178.

24. Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV: The TNFR2–TRAF signaling complex contains two novel proteins relatedto baculoviral inhibitor of apoptosis proteins. Cell 1995,83:1243-1252.

25. Liston P, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvant G,Farahani R, McLean M, Ikeda JE, MacKenzie A, Korneluk RG:Suppression of apoptosis in mammalian cells by NAIP and arelated family of IAP genes. Nature 1996, 379:349-353.

26. Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL: Cloning andexpression of apoptosis inhibitory protein homologs thatfunction to inhibit apoptosis and/or bind tumor-necrosis-factor-receptor-associated factors. Proc Natl Acad Sci U S A1996, 93:4974-4978.

27. Duckett CS, Nava VE, Gedrich RW, Clem RJ, Van Dongen JL,Gilfillan C, Shiels H, Hardwick JM, Thompson CB: A conservedfamily of cellular genes related to the baculovirus iap gene andencoding apoptosis inhibitors. EMBO J 1996, 15:2685-2694.

28. Deveraux QL, Takahashi R, Salvesen GS, Reed JC: X-linked IAPis a direct inhibitor of cell-death proteases. Nature 1997,388:300-304.

29. Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC:The cIAP-1 and cIAP-2 proteins are direct inhibitors of specificcaspases. EMBO J 1997, 16:6914-6925.

30. Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q,Srinivasula SM, Alnemri ES, Salvesen GS, Reed JC: IAPs blockapoptotic events induced by caspase-8 and cytochrome cby direct inhibition of distinct caspases. EMBO J 1998,17:2215-2223.

31. Meier P, Silke J, Leevers SJ, Evan GI: The Drosophila caspaseDRONC is regulated by DIAP1. EMBO J 2000, 19:598-611.

32. Huang Y, Park YC, Rich RL, Segal D, Myszka DG, Wu H: Structuralbasis of caspase inhibition by XIAP: differential roles of thelinker versus the BIR domain. Cell 2001, 104:781-790.

33. Riedl SJ, Renatus M, Schwarzenbacher R, Zhou Q, Sun C,Fesik SW, Liddington RC, Salvesen GS: Structural basis for theinhibition of caspase-3 by XIAP. Cell 2001, 104:791-800.

34. Chai J, Shiozaki E, Srinivasula SM, Wu Q, Datta P, Alnemri ES,Shi Y, Dataa P: Structural basis of caspase-7 inhibition by XIAP.Cell 2001, 104:769-780.

35. Suzuki Y, Nakabayashi Y, Nakata K, Reed JC, Takahashi R:X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes. J Biol Chem 2001, 276:27058-27063.

36.�

Shiozaki EN, Chai J, Rigotti DJ, Riedl SJ, Li P, Srinivasula SM,Alnemri ES, Fairman R, Shi Y: Mechanism of XIAP-mediatedinhibition of caspase-9. Mol Cell 2003, 11:519-527.

Using a structural approach, these authors confirm the findings of [15],and show that binding of XIAP to processed caspase-9 prevents it fromundergoing dimerization, which is necessary for its activation.

37. Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N,Salvesen GS, Reed JC: A single BIR domain of XIAP sufficient forinhibiting caspases. J Biol Chem 1998, 273:7787-7790.

38. Vucic D, Kaiser WJ, Harvey AJ, Miller LK: IAPs physically interactwith and block apoptosis induced by Drosophila proteins HIDand GRIM. Mol Cell Biol 1998, 18:3300-3309.

39. Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H,Connolly LM, Day CL, Tikoo A, Burke R, Wrobel C et al.: HtrA2promotes cell death through its serine protease activity and itsability to antagonize inhibitor of apoptosis proteins. J Biol Chem2002, 277:445-454.

40. Martins LM, Iaccarino I, Tenev T, Gschmeissner S, Totty NF,Lemoine NR, Savopoulos J, Gray CW, Creasy CL, Dingwall C,Downward J: The serine protease Omi/HtrA2 regulatesapoptosis by binding XIAP through a reaper-like motif. J BiolChem 2002, 277:439-444.

41.��

Ditzel M, Wilson R, Tenev T, Zachariou A, Paul A, Deas E,Meier P: Degradation of DIAP1 by the N-end rule pathwayis essential for regulating apoptosis. Nat Cell Biol 2003,5:467-473.

This is a remarkable paper describing co-degradation of Diap1 andassociated active caspases by the N-end rule pathway as a mechanismto protect cells from apoptosis. Caspase cleavage at position 20 convertsDiap1 into a target of N-end-rule-dependent degradation. Furthermore,the authors show that Diap1 instability mediated by the N-end rule isessential for suppression of apoptosis.

42. Varshavsky A: The N-end rule and regulation of apoptosis.Nat Cell Biol 2003, 5:373-376.

43. Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS,Reed JC: Cleavage of human inhibitor of apoptosis protein XIAPresults in fragments with distinct specificities for caspases.EMBO J 1999, 18:5242-5251.

44.�

Wilson R, Goyal L, Ditzel M, Zachariou A, Baker DA, Agapite J,Steller H, Meier P: The DIAP1 RING finger mediatesubiquitination of Dronc and is indispensable for regulatingapoptosis. Nat Cell Biol 2002, 4:445-450.

The authors provide genetic and biochemical evidence that Diap1 sup-presses apoptosis by RING-dependent ubiquitination of Dronc. However,the ubiquitination of Dronc does not appear to induce its degradation.

45. Joazeiro CA, Weissman AM: RING finger proteins: mediators ofubiquitin ligase activity. Cell 2000, 102:549-552.

46. Yang Y, Fang S, Jensen JP, Weissman AM, Ashwell JD: Ubiquitinprotein ligase activity of IAPs and their degradation inproteasomes in response to apoptotic stimuli. Science 2000,288:874-877.

47.��

Hays R, Wickline L, Cagan R: Morgue mediates apoptosis in theDrosophila melanogaster retina by promoting degradation ofDIAP1. Nat Cell Biol 2002, 4:425-431.

This and the following references [48��–51��] demonstrate — with mod-ifications – that the RHG proteins induce Diap1 degradation in a RING-dependent manner. In addition, this paper and [50��] report the geneticidentification of morgue as a component of the cell death machinery inDrosophila. Morgue interacts with Diap1, and induces its degradation.



48.��

Holley CL, Olson MR, Colon-Ramos DA, Kornbluth S:Reaper eliminates IAP proteins through stimulated IAPdegradation and generalized translational inhibition. Nat CellBiol 2002, 4:439-444.

In addition to induced degradation of Diap1, another function of Reaper isuncovered in this paper and in [51��]. Reaper and Grim, but not Hid, havethe remarkable activity to suppress total protein translation. This activityis independent of the RHG domain of Reaper. This process furtherreduces the amount of Diap1, lowering the apoptotic threshold. Theexact mechanism of translational repression is unknown.

49.��

Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H:Regulation of Drosophila IAP1 degradation and apoptosis byreaper and ubcD1. Nat Cell Biol 2002, 4:432-438.

These authors perform a comprehensive genetic and biochemical anal-ysis of the function of the RING domain in Diap1 degradation. Mutationalinactivation of the RING domain blocks Diap1 auto-ubiquitination anddegradation both in vitro and in vivo. Furthermore, the E2-conjugatingenzyme ubcD1 is identified as an important factor for Reaper-inducedDiap1 ubiquitination and degradation.

50.��

Wing JP, Schreader BA, Yokokura T, Wang Y, Andrews PS,Huseinovic N, Dong CK, Ogdahl JL, Schwartz LM, White K,Nambu JR: Drosophila Morgue is an F box/ubiquitin conjugasedomain protein important for grim-reaper mediated apoptosis.Nat Cell Biol 2002, 4:451-456.

Similar to [47��], these authors identify the gene morgue. Morgue expres-sion is sufficient to induce degradation of Diap1. It encodes an unusualprotein that combines an E2-conjugase domain and an E3 F-box. Theassociation of Morgue with Diap1 is dependent on the E2-conjugasedomain, but independent of the F-box.

51.��

Yoo SJ, Huh JR, Muro I, Yu H, Wang L, Wang SL, Feldman RM,Clem RJ, Muller HA, Hay BA: Hid, Rpr and Grim negativelyregulate DIAP1 levels through distinct mechanisms.Nat Cell Biol 2002, 4:416-424.

This is the only paper out of the series [47��–51��] that showed that Hid,but not Reaper and Grim, can promote Diap1 auto-ubiquitination anddegradation in a RING-dependent manner. Reaper and Grim are alsoable to reduce the protein levels of Diap1; however, according to thisstudy, this is due to generalized translational inhibition, as also dis-cussed in [48��], and not to RING-dependent degradation. How thesedifferences between this study and [47��–51��] come about is currentlyunknown.

52. Vernooy SY, Chow V, Su J, Verbrugghe K, Yang J, Cole S,Olson MR, Hay BA: Drosophila Bruce can potently suppressRpr- and grim-dependent but not hid-dependent cell death.Curr Biol 2002, 12:1164-1168.

53. Thress K, Henzel W, Shillinglaw W, Kornbluth S: Scythe: a novelreaper-binding apoptotic regulator. EMBO J 1998,17:6135-6143.

54.��

Olson MR, Holley CL, Yoo SJ, Huh JR, Hay BA, Kornbluth S:Reaper is regulated by IAP-mediated ubiquitination.J Biol Chem 2003, 278:4028-4034.

The RHG proteins themselves, most notably Reaper, are identified astargets of Diap1/RING-dependent ubiquitination and degradation in vivo,which significantly controls the biological activity of Reaper. This study

suggests that the RHG proteins and Diap1 mutually control their stability,and defines another anti-apoptotic role of Diap1.

55.��

MacFarlane M, Merrison W, Bratton SB, Cohen GM: Proteasome-mediated degradation of Smac during apoptosis: XIAPpromotes Smac ubiquitination in vitro. J Biol Chem 2002,277:36611-36616.

This is the first study that demonstrates that an IAP, XIAP, can ubiquitinatea RHG protein, Smac, in vitro in a RING-dependent manner. Furthermore,Smac abundance in vivo is dependent on the proteasome.

56. Huang H, Joazeiro CA, Bonfoco E, Kamada S, Leverson JD,Hunter T: The inhibitor of apoptosis, cIAP2, functions as aubiquitin-protein ligase and promotes in vitromonoubiquitination of caspases 3 and 7. J Biol Chem 2000,275:26661-26664.

57. Suzuki Y, Nakabayashi Y, Takahashi R: Ubiquitin-protein ligaseactivity of X-linked inhibitor of apoptosis protein promotesproteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad SciU S A 2001, 98:8662-8667.

58. Wilkinson CR: New tricks for ubiquitin and friends. Trends CellBiol 2002, 12:545-546.

59. Oeda E, Oka Y, Miyazono K, Kawabata M: Interaction ofDrosophila inhibitors of apoptosis with thick veins, a type Iserine/threonine kinase receptor for decapentaplegic. J BiolChem 1998, 273:9353-9356.

60. Birkey Reffey S, Wurthner JU, Parks WT, Roberts AB, Duckett CS:X-linked inhibitor of apoptosis protein functions as a cofactorin transforming growth factor b signaling. J Biol Chem 2001,276:26542-26549.

61. Silke J, Vaux DL: Two kinds of BIR-containing proteins —inhibitors of apoptosis, or required for mitosis. J Cell Sci 2001,114:1821-1827.

62. Li X, Yang Y, Ashwell JD: TNF-RII and c-IAP1 mediateubiquitination and degradation of TRAF2. Nature 2002,416:345-347.

63. Kuranaga E, Kanuka H, Igaki T, Sawamoto K, Ichijo H, Okano H,Miura M: Reaper-mediated inhibition of DIAP1-inducedDTRAF1 degradation results in activation of JNK in Drosophila.Nat Cell Biol 2002, 4:705-710.

64. Wright CW, Clem RJ: Sequence requirements for Hid bindingand apoptosis regulation in the baculovirus inhibitor ofapoptosis Op-IAP. Hid binds Op-IAP in a manner similar toSmac binding of XIAP. J Biol Chem 2002, 277:2454-2462.

65. Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y: Structuralbasis of IAP recognition by Smac/DIABLO. Nature 2000,408:1008-1012.

66. Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T,Herrmann J, Wu JC, Fesik SW: Structural basis for bindingof Smac/DIABLO to the XIAP BIR3 domain. Nature 2000,408:1004-1008.



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Regulators of IAP function: coming to grips with the grim reaper