Distinct roles for the cellular inhibitors of apoptosis proteins 1 and 2.
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ABSTRACT: cIAPs (cellular inhibitor of apoptosis proteins) 1 and 2 are able to regulate apoptosis when ectopically expressed in recipient cells and probably also in vivo. Previous work suggested that this is at least partially due to direct caspase inhibition, mediated by two of the three baculovirus IAP repeat (BIR) domains that are contained in these proteins. In support of this we show that the BIR domains 2 and 3 of the two cIAPs are able to bind caspases-7 and -9. However, we demonstrate that neither of these BIR domains is able to inhibit caspases because of critical substitutions in the regions that target caspase inhibition in the X-linked IAP, a tight binding caspase inhibitor. The cIAP BIR domains can be converted to tight binding caspase inhibitors by substituting these critical residues with XIAP residues. Thus, cIAPs maintain protein scaffolds suitable for direct caspase inhibition but have lost or never acquired specific caspase inhibitory interaction sites. Consequently, although the binding function of the cIAP BIRs may be important for their physiologic function, caspase inhibition is not.Journal of Biological Chemistry 03/2006; 281(6):3254-60. · 4.65 Impact Factor
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ABSTRACT: The cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2) proteins have been implicated in the activation of NF-kappaB by TNFalpha; however, genetic deletion of either cIAP1 or 2 did not support a physiologically relevant role, perhaps because of functional redundancy. To address this, we used combined genetic and siRNA knockdown approaches and report that cIAP1 and 2 are indeed critical, yet redundant, regulators of NF-kappaB activation upon TNFalpha treatment. Whereas NF-kappaB was properly activated by TNFalpha in cultured and primary cells deficient in either cIAP1 or 2, removal of both cIAPs severely blunted its activation. After treatment with TNFalpha, cIAP1 and 2 were rapidly recruited to the TNF receptor 1, along with the adapter protein TNF receptor associated factor 2. Importantly, either cIAP1 or 2 was required for proper TNF receptor 1 signalosome function. In their combined absence, polyubiquitination of receptor interacting protein 1, an upstream event necessary for NF-kappaB signaling, was attenuated. As a result, phosphorylation of the inhibitor of kappaB kinase beta was diminished, and signal transduction was severely blunted. Consequently, cells missing both cIAP1 and 2 were sensitized to TNFalpha-mediated apoptosis. Collectively, these data demonstrate that either cIAP1 or 2 is required for proper Rip1 polyubiquitination and NF-kappaB activation upon TNFalpha treatment.Proceedings of the National Academy of Sciences 09/2008; 105(33):11778-83. · 9.74 Impact Factor
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ABSTRACT: Nuclear factor kappaB (NF-kappaB) transcription factors have a key role in many physiological processes such as innate and adaptive immune responses, cell proliferation, cell death, and inflammation. It has become clear that aberrant regulation of NF-kappaB and the signalling pathways that control its activity are involved in cancer development and progression, as well as in resistance to chemotherapy and radiotherapy. This article discusses recent evidence from cancer genetics and cancer genome studies that support the involvement of NF-kappaB in human cancer, particularly in multiple myeloma. The therapeutic potential and benefit of targeting NF-kappaB in cancer, and the possible complications and pitfalls of such an approach, are explored.dressNature Reviews Drug Discovery 02/2009; 8(1):33-40. · 33.08 Impact Factor
News and Commentary
Distinct roles for the cellular inhibitors of apoptosis
proteins 1 and 2
TE Graber1,2and M Holcik*,1
Cell Death and Disease (2011) 2, e135; doi:10.1038/cddis.2011.20; published online 24 March 2011
The cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and
cIAP2) are members of a highly conserved and critically
important family of inhibitor of apoptosis proteins (IAPs) that
function to mitigate both intrinsic and extrinsic death signal-
ling. Their conserved mechanism of action in vitro relies on
competitive inhibition of caspase activity. In recent years,
however, ubiquitin ligase activity within cIAP1/2 has been
shown to have a central role in regulating NF-kB signalling
and programmed cell death through the ubiquitylation of key
components of TNF receptor complexes. Owing to the
importance of NF-kB signalling in a variety of pathologies,
cIAP1/2 have become desirable drug targets and there is thus
a strong rationale for studying how cIAP1/2 expression is
regulated in these contexts. Although these paralogous
proteins function in a redundant manner, their context-
dependent differential expression provide for non-redundant
roles. Here, we will focus on evaluating possible distinct
functions for cIAP1 in specific cellular settings where it acts as
the dominant player and review recent advances in our
understanding of how cIAP1 expression can be regulated at
the post-transcriptional level.
The Emerging Importance of cIAP1/2 in Modulating
NF-jB Signalling and Programmed Cell Death
cIAP1, cIAP2 and the X chromosome-linked IAP (XIAP) are
members of the family of inhibitor of apoptosis proteins (IAPs)
that were initially reported to function as competitive inhibitors
of caspases through their baculoviral IAP repeat (BIR)
domains.1Although all three proteins directly bind and inhibit
caspases, cIAP1/2 were found to only weakly exhibit this
activity in vivo.2Instead, cIAP1/2 have been found to
demonstrate functional redundancy by virtue of E3 ubiquitin
ligase activity within their RING domains that targets specific
substrates for proteasomal degradation (K48-polyubiquitin
linkages), or for participation in specific signalling pathways
Defining the gamut of cIAP1/2 ubiquitin ligase substrates
has uncovered a critical role for IAP ubiquitin ligase activity in
modulating inflammatory signalling pathways. The cellular
response to the inflammatory cytokine TNF-a is the best
understood of these pathways and serves to signal cell
survival through activation of NF-kB transcription factors, or
cell death through apoptosis or programmed necrosis
(necroptosis). Upon binding of TNF-a to the TNF receptor I,
the adaptor proteins TRADD, TRAF2 and/or TRAF 5 are
recruited along with cIAP1 and/or cIAP2 and the RIP1 kinase
(Figure 1a). RIP1 serves as a substrate for cIAP1 or cIAP2-
mediated K63-polyubiquitylation that signals recruitment
of the TAK/TAB complex and subsequent activation of
NF-kB. The cIAP1/2-mediated polyubiquitylation of RIP1
in the TNF-a-stimulated cell also prevents internalisation of
complex I that would lead to the formation of complex II,
activation of caspase-8 and death by apoptosis. If caspase 8
activity is limiting, or if the activity of RIP1 or its binding
partner RIP3 is high, complex III forms and leads to
programmed necrosis (necroptosis) (Figure 1a). Thus,
the absence of cIAP1/2 renders cells sensitive to TNF-a-
induced death. Paradoxically, removal of either cIAP1 or
cIAP2 in unstimulated cells enhances constitutive NF-kB
signalling, as the result of NIK stabilisation and induction of
both the classical and alternative arms of the NF-kB
Specialised Roles for cIAP1?
As the activities of cIAP1 and cIAP2 appear to be redundant,
one may ask in which cellular contexts could these proteins
have distinct roles? Confounding this question is the
observation that some cell types exhibit a strong compensa-
tory mechanism, whereby reducing cIAP1 levels leads to an
increase in cIAP2 expression. This phenomenon was first
observed in spleen and thymus isolated from cIAP1 knockout
animals and is attributed to the ability of cIAP1 to target cIAP2
forubiquitin-mediated degradation.5Surprisingly, cIAP1isnot
a target for degradation by cIAP2, as cIAP2 knockout animals
show unchanged expression of cIAP1.6This compensatory
mechanism does not appear to be a universal event. For
example, reduction in the levels of cIAP1 by RNAi enhances
1Apoptosis Research Centre, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON K1H 8L1, Canada
*Corresponding author: M Holcik, Apoptosis Research Centre, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON K1H 8L1, Canada.
Tel: þ(613) 738 3207; Fax: þ(613) 738 4833; E-mail: email@example.com
2Current address: Montreal Neurological Institute, Room BT110, 3801 Rue University, Montreal, QC H3A 2B4, Canada.
Citation: Cell Death and Disease (2011) 2, e135; doi:10.1038/cddis.2011.20
& 2011 Macmillan Publishers Limited All rights reserved 2041-4889/11
inembryonickidney cells(HEK293T).Further,the generallack
of correlation between levels of cIAP1 and cIAP2 across a
panel of cancer cell lines7implies that the oft-cited compensa-
rule. Here, we will highlight two recent studies that have
uncovered a starring role for cIAP1 inspecific cellular contexts.
Death by any other name. TNF-a signalling normally leads
to transcriptional activation of cIAP2 in a bid to subvert
apoptosis. However, Vanlangenakker et al. found that the L929
mouse fibrosarcoma cell line sustains programmed necrosis
(rather than apoptosis) following treatment with TNF-a
and unexpectedly expresses very low levels of cIAP2.8The
cIAP2 levels were also unaffected in TNF-a-activated cells
transfected with cIAP1 siRNA. This is in stark contrast to other
cell types where compensation by cIAP2 protein in a cIAP1-
null background has been demonstrated.5Reducing cIAP1
expression using the small-molecule cIAP1/2 antagonist BV6
or by cIAP1 RNAi renders these cells sensitive to TNF-a-
mediated necrosis.8The increased sensitivity was found to be
due to enhanced activity of RIP1 kinase in cIAP1-depleted
cells and thus an increase in the levels of the ‘necrosome’ – a
necroptosis-specific complex (complex III in Figure 1a) that is
analogous to the caspase-8-activating complex II that forms
during TNF-a-mediated apoptosis. An additional surprise in this
study was that cIAP1 is able to inhibit production of necrosis-
inducing mitochondrial reactive oxygen species (ROS) in
a RIP-dependent manner suggesting a unique role for
cIAP1 in mitochondrial metabolism. The authors found that
RNAi-mediated downregulation of the mitochondrial redox
carrier complex I (a component of the mitochondrial electron
transport chain) in the absence of cIAP1 prevented RIP1-
dependent ROS production. Interestingly, this did not involve
translocation of either RIP1 or RIP3 to mitochondria suggesting
the involvement of an unknown factor in this pathway
(Figure 1a). Thus, in this specific context, cIAP1 expression
is necessary and sufficient to keep the brakes on RIP-
dependent necroptosis owing to the lack of compensatory
cIAP1 is in the kNOw. In examining cIAP1-null mice
and their response to intra-tracheal bacterial infection,
Prakash et al.9found the expected cIAP2 compensation
present in lung tissue. However, examination of peritoneal
macrophages from these same mice revealed no upregulation
of cIAP2. The lack of cIAP activity in these macrophages
illustrates that mechanisms preventing compensation by
upregulation of cIAP2 exist in vivo, although we do not yet
have any insight into their nature. Perhaps not surprisingly, a
defect in proliferative activity and inflammatory response of
cIAP1-null peritoneal macrophages rendered the mice resis-
tant to LPS-induced endotoxin shock. This finding is similar to
that observed in cIAP2-null mice,6but cIAP1-null macrophages
were also unable to produce and release nitric oxide (NO). This
striking finding, due to impairment in their ability to express
has a critical role inactivating the NF-kB pathway through K63-linked polyubiquitylation of RIP1 kinase and/orTRAF2 or 5. The propagation of the NF-kB signalinto the nucleus
induces expression of a number of pro-survival genes including cIAP2 and inducible nitric oxide synthetase (iNOS). The lack of cIAP1 ubiquitin ligase activity leads to receptor
internalisation and the formation of death-inducing cytosolic complexes mediated by deubiquitylated RIP1. Specifically, the RIP1- and RIP3-containing Complex II can form to
activate caspase 8, leading to cleavage and inactivation of RIP1 and RIP3 and the initiation of an apoptotic cascade. Alternatively, in the absence of caspase-8 or in a context
where deubiquitylated RIP1 or RIP3 activity is high, the ‘necrosome’ (complex III) can form, in which mutual and auto-phosphorylation of RIP1 and RIP3 (labelled ‘P’ in this
diagram) leads topropagation ofnecroticsignals and programmed necroticcell death.Reactiveoxygen species (ROS) are animportant mediatorofnecroptosisand cIAP1limits
factor (labelled ‘X’ in the diagram) must be responsible for increased mitochondrial redox activity. (b) cIAP1 mRNA sequence features and post-transcriptional regulatory nodes.
A diagram of the capped (m7G)and polyadenylated cIAP1mRNAillustratingthe size(in kilobases,kb)and location ofthe 50and 30untranslated regions(UTRs).Thisdiagramis
not to scale. RNA binding proteins have been found to alter cIAP1 mRNA stability and translation. Specifically, ultraviolet radiation induces nucleocytoplasmic translocation of
hnRNPA1 and binding to AU-rich elements in the 30UTR, leading to decreased cIAP1 mRNA stability. An IRES that enhances translation of cIAP1 mRNA following endoplasmic
reticulum(ER)stress is150bp longand islocatedproximal tothe initiationcodon (AUG)ofthe mainopenreading frame (ORF).AnupstreamORF(uORF)whose CUGinitiation
codon bisects the IRES codes for a non-functional peptide of 21 amino acids and acts to repress cIAP1 mRNA translation under normal physiological conditions. Several RNA
binding proteins or IRES trans-acting factors (ITAFs) have been implicated in the regulation of cIAP1 translation including p86 nuclear factors 90 and 45 (NF90 and NF45,
respectively), insulin-like growth factor 2 binding protein 1 (IGF2BP1), and RNA helicase A (RHA)
(a) The central role of cIAP1 in NF-kB signalling and programmed cell death. Binding of tumour necrosis factor alpha (TNF-a) to its plasma membrane receptor
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Cell Death and Disease
inducible NO synthetase (iNOS), establishes a new and
specialised role for cIAP1 in the innate immune response.
These examples of distinct regulation and specialised roles
for cIAP1 in pathological contexts highlight its potential as a
target for the development of drug therapies that modulate its
expression. In addition, the key role that cIAP1 has in cell
survival, tumourogenesis and cancer progression coupled with
frequent cancer-associated mutations in IAP alleles gives a
strong rationale for studying the regulation of cIAP1 and cIAP2
expression. Although post-transcriptional regulation of cIAP2
has been described,10its expression is primarily controlled at
the level of the proteasome by cIAP1.5In contrast, as outlined
below, expression of cIAP1 is tightly controlled through
modulation of stability and translation of its mRNA.
cIAP1: The Paradigm of Post-transcriptional Control
Regulation of cIAP1 mRNA stability. cIAP1 mRNA half-
life is significantly reduced following exposure to ultraviolet
radiation (UVR). The resultant decrease in cIAP1 expression
leads to a significant increase in NF-kB activity.11We found
that the stability of cIAP1 mRNA is mediated by AU-rich
elements (ARE) in its 30UTR. AREs are cis-acting elements
containing an AUUUA pentamer (or similar sequences), and
generally target the mRNA for degradation through the
exosome.12Although the exact mechanism through which
cIAP1 mRNA is degraded remains unknown, we identified
hnRNP A1 as a protein factor that is responsible for cIAP1
mRNA instability (Figure 1b). hnRNP A1 is known as a
modulator of splicing13and IRES-dependent translation.14
Like many RNA binding proteins, hnRNP A1 is primarily a
nuclear protein although it accumulates in the cytoplasm
following specific environmental cues. For example, osmotic
stress causes hnRNPA1 to accumulate in the cytoplasm
where it represses translation of specific mRNAs14or
associates with stress granules.15UVR was found to
induce a similar cytoplasmic accumulation of hnRNPA1,
resulting in increased association of hnRNP A1 with cIAP1
mRNA and its subsequent degradation.11
Regulation of cIAP1 mRNA translation. cIAP1 protein
expression is also repressed as the result of both a long
(1.2kb) 50UTR that hinders ribosomal movement, and a small
upstream open reading frame (uORF) that strongly attenu-
ates initiation of cIAP1 translation at the authentic AUG start
codon (Figure 1b; Warnakulasuriyarachchi et al.16). In
response to stress, however, translation of cIAP1 is
mediated by the internal ribosome entry site (IRES), which
is active following induction of endoplasmic reticulum (ER)
stress or arsenite treatment and results in enhanced
expression of cIAP1.16–18cIAP1 IRES activity is regulated
by specific trans-acting protein factors including p86, a
protein formed by cleavage of p97/DAP5 during ER stress.19
Using RNA affinity chromatography, additional cIAP1 IRES
binding proteins NF45, NF90, IGF2BP1 and RHA were
identified (Figure 1b; Graber et al.20). NF45 was further
shown to be required for induction of cIAP1 during the ER
stress response. This data suggests the existence of an
auxiliary translation initiation complex that is active during
times when the cell has reduced general protein synthesis
allowing for continued translation of this important regulator
of apoptosis and NF-kB signalling.
cIAP1 post-transcriptional regulatory nodes therefore
represent druggable targets. Although we do not yet under-
stand the cellular mechanism that prevents compensation by
cIAP2 in the cell contexts explored herein, targeting cIAP1
expression in such situations represents a tractable strategy
for the development of drug therapies that modulate pro-
grammed cell death and/or the innate immune response.
Conflict of interest
The authors declare no conflict of interest.
1. Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC. The c-IAP-1 and c-IAP-2
proteins are direct inhibitors of specific caspases. Embo J 1997; 16: 6914–6925.
2. Eckelman BP, Salvesen GS. The human anti-apoptotic proteins cIAP1 and cIAP2 bind but
do not inhibit caspases. J Biol Chem 2006; 281: 3254–3260.
3. Mahoney DJ, Cheung HH, Mrad RL, Plenchette S, Simard C, Enwere E et al. Both cIAP1
and cIAP2 regulate TNFalpha-mediated NF-kappaB activation. Proc Natl Acad Sci USA
2008; 105: 11778–11783.
4. Baud V, Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat
Rev Drug Discov 2009; 8: 33–40.
5. Conze DB, Albert L, Ferrick DA, Goeddel DV, Yeh WC, Mak T et al. Posttranscriptional
downregulation of c-IAP2 by the ubiquitin protein ligase c-IAP1 in vivo. Mol Cell Biol 2005;
6. Conte D, Holcik M, Lefebvre CA, Lacasse E, Picketts DJ, Wright KE et al. Inhibitor of
apoptosis protein cIAP2 is essential for lipopolysaccharide-induced macrophage survival.
Mol Cell Biol 2006; 26: 699–708.
7. Cheung HH, Mahoney DJ, Lacasse EC, Korneluk RG. Down-regulation of c-FLIP Enhances
death of cancer cells by smac mimetic compound. Cancer Res 2009; 69: 7729–7738.
8. Vanlangenakker N, Vanden Berghe T, Bogaert P, Laukens B, Zobel K, Deshayes K et al.
cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-
dependent reactive oxygen species production. Cell Death Differ 2011; 18: 656–665.
9. Prakash H, Becker D, Bo ¨hme L, Albert L, Witzenrath M, Rosseau S et al. cIAP-1 controls
innate immunity to C. pneumoniae pulmonary infection. PLoS One 2009; 4: e6519.
10. Sherrill KW, Lloyd RE. Translation of cIAP2 mRNA is mediated exclusively by a stress-
modulated ribosome shunt. Mol Cell Biol 2008; 28: 2011–2022.
11. Zhao TT, Graber TE, Jordan LE, Cloutier M, Lewis SM, Goulet I et al. hnRNP A1 regulates
UV-induced NF-kappaB signalling through destabilization of cIAP1 mRNA. Cell Death
Differ 2009; 16: 244–252.
12. Barreau C, Paillard L, Osborne HB. AU-rich elements and associated factors: are there
unifying principles? Nucleic Acids Res 2005; 33: 7138–7150.
13. Pinol-Roma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and
cytoplasm. Nature 1992; 355: 730–732.
14. Lewis SM, Veyrier A, Hosszu Ungureanu N, Bonnal S, Vagner S, Holcik M. Subcellular
relocalization of a trans-acting factor regulates XIAP IRES-dependent translation. Mol Biol
Cell 2007; 18: 1302–1311.
15. vanderHouvenvanOordtW,Diaz-MecoMT,LozanoJ,KrainerAR,MoscatJ,Ca ´ceresJF.
The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and
modulates alternative splicing regulation. Cell Biol 2000; 149: 307–316.
HIAP2 is regulated by an upstream open reading frame. Cell Death Differ 2003; 10: 899–904.
17. Warnakulasuriyarachchi D, Cerquozzi S, Cheung HH, Holcı ´k M. Translational induction of
death and is mediated via an inducible internal ribosome entry site element. J Biol Chem
2004; 279: 17148–17157.
1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA 2004; 10: 469–481.
19. Lewis SM, Cerquozzi S, Graber TE, Ungureanu NH, Andrews M, Holcik M. The eIF4G
homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic
reticulum stress. Nucleic Acids Res 2008; 36: 168–178.
20. Graber TE, Baird SD, Kao PN, Mathews MB, Holcik M. NF45 functions as an IRES trans-
acting factor that is required for translation of cIAP1 during the unfolded protein response.
Cell Death Differ 2010; 17: 719–729.
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