IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.     

The USP19 deubiquitinase regulates the stability of c-IAP1 and c-IAP2

The inhibitors of apoptosis (IAPs) are critical regulators of apoptosis and other fundamental cellular processes. Many IAPs are RING domain-containing ubiquitin E3 ligases that control the stability of their interacting proteins. However, how IAP stability is regulated remains unclear. Here we report that USP19, a deubiquitinating enzyme, interacts with cellular IAP 1 (c-IAP1) and c-IAP2. Knockdown of USP19 decreases levels of both c-IAPs, whereas overexpression of USP19 results in a marked increase in c-IAP levels. USP19 effectively removes ubiquitin from c-IAPs in vitro, but it stabilizes c-IAPs in vivo mainly through deubiquitinase-independent mechanisms. The deubiquitinase activity is involved in the stabilization of USP19 itself, which is facilitated by USP19 self-association. Functionally, knockdown of USP19 enhances TNFα-induced caspase activation and apoptosis in a c-IAP1 and 2-dependent manner. These results suggest that the self-ubiquitin ligase activity of c-IAPs is inhibited by USP19 and implicate deubiquitinating enzymes in the regulation of IAP stability.     

Apoptotic sensitivity of murine IAP-deficient cells

Although numerous studies have implicated the IAPs (inhibitor of apoptosis proteins) in the control of apoptotic cell death, analyses of murine Iap-targeted cells have not revealed significant differences in their susceptibility to apoptosis. In the present study, we show that, under defined conditions, murine cells lacking XIAP (X-linked inhibitor of apoptosis) and c-IAP (cellular IAP) 2, but not c-IAP1, exhibit heightened apoptotic sensitivity to both intrinsic and extrinsic apoptotic stimuli.     

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1)

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.     

Smac/DIABLO selectively reduces the levels of c-IAP1 and c-IAP2 but not that of XIAP and livin in HeLa cells

The inhibitor of apoptosis (IAP) proteins bind and inhibit caspases via their baculovirus IAP repeat domains. Some of these IAPs are capable of ubiquitinating themselves and their interacting proteins through the ubiquitin-protein isopeptide ligase activity of their RING domain. The Drosophila IAP antagonists Reaper, Hid, and Grim can accelerate the degradation of Drosophila IAP1 and some mammalian IAPs by promoting their ubiquitin-protein isopeptide ligase activity. Here we show that Smac/DIABLO, a mammalian functional homolog of Reaper/Hid/Grim, selectively causes the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although it efficiently promotes the auto-ubiquitination of them all. Smac binding to c-IAP via its N-terminal IAP-binding motif is the prerequisite for this effect, which is further supported by the findings that Smac N-terminal peptide is sufficient to enhance c-IAP1 ubiquitination, and Smac no longer promotes the ubiquitination of mutant c-IAP1 lacking all three baculovirus IAP repeat domains. In addition, different IAPs require the same ubiquitin-conjugating enzymes UbcH5a and UbcH6 for their ubiquitination. Taken together, Smac may serve as a key molecule in vivo to selectively reduce the protein level of c-IAPs through the ubiquitin/proteasome pathway.     

前列腺凋亡反应蛋白-4抗肿瘤作用研究进展

前列腺凋亡反应基因-4（prostate apoptosis responsegene．4,par-4）是从凋亡的前列腺癌细胞中分离出来的一种基因,该基因编码的产物是前列腺凋亡反应蛋白4（Par-4）。Par-4可通过细胞内、外途径调节各种分子表达,诱导癌细胞凋亡,选择性抑制肿瘤细胞生长,因此,Par-4的表达与肿瘤的发生、发展及预后有密切的联系。Par-4在治疗恶性肿瘤中表现出良好的肿瘤细胞靶向杀伤效应,对正常组织细胞无明显影响,故具有极其重要的应用价值。就Par-4特异性诱导肿瘤细胞凋亡及其潜在抗肿瘤作用的进展进行综述。     

Caspase-3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces cerevisiae and mammalian cells

Using a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of XIAP>c-IAP2>c-IAP1>survivin. The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto-activating caspase-3. The relative anti-apoptotic activities of each IAP depended on the particular death stimulus. For IAP-expressing cells treated with camptothecin, survival correlated with their intrinsic anti-caspase-3 activity. However, c-IAP1-transfected cells were disproportionately resistant to tumor necrosis factor-alpha, suggesting that its anti-apoptotic activities extend beyond caspase-3 or -7 inhibition. Yeast-based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways.     

Apoptotic effect of fludarabine is independent of expression of IAPs in B-cell chronic lymphocytic leukemia

Despite the efficiency of fludarabine in the induction of clinical responses in B-cell chronic lymphocytic leukemia (B-CLL) patients, resistance to this drug has been documented. The present study tested whether resistance to fludarabine is related to the expression of inhibitor of apoptosis proteins (IAPs) family members. We analyzed the expression of c-IAP1, c-IAP2 and XIAP, by immunocytochemistry, in 30 blood samples from B-CLL patients and correlated protein expression to fludarabine-induced apoptosis estimated by an annexin-V assay. Expression of c-IAP1, c-IAP2 and XIAP were found predominantly in the cytoplasm, and a wide range of staining intensities was observed among distinct samples. No correlation was found between the levels of IAPs expression and prognostic factors such as age, gender, lymphocyte doubling time, white blood cell count or previous treatment. The expression of IAPs also failed to predict the sensitivity to fludarabine-induced apoptosis. Alternative pathways of cell death may explain the independence of fludarabine-induced apoptosis from the high expression of IAPs.     

Inhibitor of Apoptosis (IAP) Proteins–Modulators of Cell Death and Inflammation

Misregulated innate immune signaling and cell death form the basis of much human disease pathogenesis. Inhibitor of apoptosis (IAP) protein family members are frequently overexpressed in cancer and contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Recent research into IAP biology has unearthed unexpected roles for this group of proteins. In addition, the advances in our understanding of the molecular mechanisms that IAPs use to regulate cell death and innate immune responses have provided new insights into disease states and suggested novel intervention strategies. Here we review the functions assigned to those IAP proteins that act at the intersection of cell death regulation and inflammatory signaling.Apoptosis represents a fundamental biological process that relies on the activation of caspases. Inhibitor of apoptosis (IAP) proteins represent a group of negative regulators of both caspases and cell death. Although best known for their ability to regulate caspases and cell death, it is now clear that they function as arbiters of diverse biological processes (Gyrd-Hansen and Meier 2010). Most prominently, IAPs control ubiquitin (Ub)-dependent signaling events that regulate activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways that in turn drive expression of genes important for inflammation, immunity, cell migration, and cell survival. IAPs thereby function as E3 Ub ligases, mediating the transfer of Ub from E2s to target substrates. This in turn modulates the signaling process through regulating protein stability as well as via nondegradative means (see below for details). Many of the cellular processes controlled by IAPs are frequently deregulated in cancer and, directly or indirectly, contribute to disease initiation, tumor maintenance, and/or progression, making IAPs obvious targets for anticancer therapy (LaCasse et al. 2008). Accordingly, small pharmacological inhibitors of IAPs, frequently referred to as Smac-mimetics (SM), were developed and are currently undergoing clinical trials for the treatment of cancer (LaCasse et al. 2008). The use of SMs in preclinical tumor models and clinical trials has provided compelling evidence for the therapeutic benefit of IAP inhibition.     

Cytoprotective effects of IAPs revealed by a small molecule antagonist

Deregulated expression of members of the IAP (inhibitor of apoptosis) family has been identified in a wide variety of neoplastic cells, and synthetic IAP antagonists represent a promising novel class of chemotherapeutic agents. Early work focused on the ability of these compounds to block the caspase-inhibitory function of XIAP (X-linked IAP). However, recent studies have shown that IAP antagonists, although primarily designed to target XIAP, trigger ubiquitin-mediated degradation of two related proteins, c-IAP (cellular IAP) 1 and c-IAP2, and through this process potentiates the death of tumour cells via autocrine cellular-signalling pathways. In this context, the relative contribution of XIAP as a target of this class of compounds is unclear. In the present study, we examine the involvement of XIAP using a recently described synthetic IAP antagonist, AEG40730, and through comparison of a human XIAP-depleted tumour cell line with its isogenic wild-type control line. Treatment with nanomolar concentrations of AEG40730 resulted in the loss of both XIAP and c-IAP1 proteins, albeit with different kinetics. Although XIAP-deficient HCT116 cells retained some sensitivity to external apoptotic stimuli, the results suggest that IAP antagonists, such as AEG40730, exert their apoptosis-enhancing effects through XIAP in addition to the c-IAPs. These results indicate that IAP antagonists can target multiple IAPs to augment distinct pro-apoptotic signalling pathways, thereby revealing the potential for these compounds in cancer therapy and underscoring the promise of IAP-targeted therapies.     

The inhibitor of apoptosis protein fusion c-IAP2.MALT1 stimulates NF-kappaB activation independently of TRAF1 AND TRAF2

The inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All members of the IAP family have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. The t(11, 18)(q21;q21) translocation fuses the BIR domains of c-IAP2 with the paracaspase/MALT1 (mucosa-associated lymphoid tissue) protein, a critical mediator of T cell receptor-stimulated activation of NF-kappaB. The c-IAP2.MALT1 fusion protein constitutively activates the NF-kappaB pathway, and this is considered critical to malignant B cell transformation and lymphoma progression. The BIR domains of c-IAP1 and c-IAP2 interact with tumor necrosis factor receptor-associated factors 1 and 2 (TRAF1 and TRAF2). Here we investigated the importance of TRAF1 and TRAF2 for c-IAP2.MALT1-stimulated NF-kappaB activation. We identified a novel epitope within the BIR1 domains of c-IAP1 and c-IAP2 that is crucial for their physical interaction with TRAF1 and TRAF2. The c-IAP2.MALT1 fusion protein associates with TRAF1 and TRAF2 using the same binding site. We explored the functional relevance of this interaction and established that binding to TRAF1 and TRAF2 is not required for c-IAP2.MALT1-stimulated NF-kappaB activation. Furthermore, gene ablation of TRAF2 or combined down-regulation of TRAF1 and TRAF2 did not affect c-IAP2.MALT1-stimulated signaling. However, TRAF1/2-binding mutants of c-IAP2.MALT1 still oligomerize and activate NF-kappaB, suggesting that oligomerization might be important for signaling of the fusion protein. Therefore, the t(11, 18)(q21;q21) translocation creating the c-IAP2.MALT1 fusion protein activates NF-kappaB and contributes to human malignancy in the absence of signaling adaptors that might otherwise regulate its activity.     

IAPs: guardians of RIPK1

Deregulation of innate immune signalling and cell death form the basis of most human disease pathogenesis. Inhibitor of APoptosis (IAP) protein-family members are frequently overexpressed in cancer and contribute to tumour cell survival, chemo-resistance, disease progression and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin-dependent pathways that modulate innate immune signalling by activation of NF-κB. Recent advances in our understanding of the molecular mechanisms through which IAPs influence cell death and innate immune responses have provided new insights into novel strategies for treatment of cancer. In this review we discuss our current understanding of IAP-mediated NF-κB signalling, as well as elaborate on unexpected insights into the involvement of IAPs in regulating the 'Ripoptosome', a novel intrinsic cell death-inducing platform. We propose an evolutionarily conserved concept whereby IAPs function as guardians of killer platforms such as the apoptosome in Drosophila and the Ripoptosome in mammals.     

Anti-apoptotic potential of insect cellular and viral IAPs in mammalian cells

IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.     

Inhibitor of apoptosis proteins and apoptosis

Apoptosis is a physiological cell death process that plays a critical role in development, homeostasis, and immune defense of multicellular animals. Inhibitor of apoptosis proteins (IAPs) constitute a family of proteins that possess between one and three baculovirus IAP repeats. Some of them also have a really interesting new gene finger domain, and can prevent cell death by binding and inhibiting active caspases, but are regulated by IAP antagonists. Some evidence also indicates that IAP can modulate the cell cycle and signal transduction. The three main factors, IAPs, IAP antagonists, and caspases, are involved in regulating the progress of apoptosis in many species. Many studies and assumptions have been focused on the anfractuous interactions between these three main factors to explore their real functional model in order to develop potential anticancer drugs. In this review, we describe the classification, molecular structures, and properties of IAPs and discuss the mechanisms of apoptosis. We also discuss the promising significance of clinical applications of IAPs in the diagnosis and treatment of malignancy.     

Inhibitor of Apoptosis Proteins (IAPs) and Their Antagonists Regulate Spontaneous and Tumor Necrosis Factor (TNF)-induced Proinflammatory Cytokine and Chemokine Production

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.     

Distinctive effects of the cellular inhibitor of apoptosis protein c-IAP2 through stabilization by XIAP in glioblastoma multiforme cells

Inhibitor of apoptosis proteins (IAPs) are extensively involved in NFκB signaling pathways. Regulation of c-IAP2 turnover by other proteins was investigated in glioblastoma multiforme (GBM) cells in the present study. When overexpressed, X-linked IAP (XIAP) enhanced expression of ectopic c-IAP2, but not c-IAP1, and endogenous c-IAP2 levels were reduced once XIAP expression was silenced. TNFα stimulation substantially increased c-IAP2 expression, and this upregulation was impaired by suppression of XIAP. Similarly, when XIAP was limiting due to severe hypoxic conditions, c-IAP2 levels were downregulated. These data together indicate that XIAP is an important regulator responsible for stabilization of c-IAP2 levels under different conditions. Protein interactions occur through binding of BIR2 and BIR3 domains of c-IAP2 with the RING finger of XIAP. XIAP inhibition of c-IAP2 auto-degradation was dependent on this physical interaction, and it was independent of XIAP E3 ligase activity. Global c-IAP2 ubiquitination was not affected by XIAP, although c-IAP2 levels were significantly increased. A CARD-RING-containing fragment of c-IAP2 was found to target XIAP for proteasome-independent degradation, but it was unable to sensitize GBM cells to chemo-reagents. The XIAP-stabilized c-IAP2 was found to enhance IκB-α phosphorylation on serines 32 and 36, and to antagonize XIAP-induced increase in mature Smac and Bcl10. Taken together, our data identify a distinctive role of c-IAP2 as stabilizer of XIAP, which is likely involved in regulation of NFκB activation and apoptosis in GBM cells.     

The inhibitor of apoptosis protein family and its antagonists in acute leukemias

The Inhibitor of Apoptosis Protein family (IAP) functions as inhibitors of apoptotic pathways, both death receptor- and mitochondrial mediated. We detail the current body of knowledge for the IAP family with regard to their structure and function, their expression in normal and leukemic cells, and their prognostic importance in acute leukemia. Although there is some evidence that IAPs play an important role in the chemoresistance of leukemia cell lines, little is known about their influence on this phenomenon in acute leukemia cells of human origin. IAPs are also explored as a specific target for new antitumor strategies, including antisense oligonucleotides of XIAP (X-chromosome-linked IAP) or survivin and small molecules of polyphenylurea-based XIAP inhibitors. Several proteins negatively regulate the function of the IAP family. One of those antagonists is Smac/DIABLO. Short peptides of Smac were found to enhanced apoptosis, induced by chemo- or immunotherapy, in the leukemic cells in vitro. Moreover, small-molecule agents, resembling Smac/DIABLO in function, were shown to potentiate cytotoxicity of chemotherapy in different malignancies. IAPs, exhibiting downstream influence on both external and intrinsic pathways as well as on some caspase-independent mechanisms of apoptosis, are potentially attractive target for anti-tumor therapy, although their role in the pathology and prognosis of acute leukemia has to be further elucidated.     

Enhanced Cytoprotective Effects of the Inhibitor of Apoptosis Protein Cellular IAP1 through Stabilization with TRAF2

Inhibitor of apoptosis (IAP) proteins are key regulators of intracellular signaling that interact with tumor necrosis factor (TNF) receptor superfamily members as well as proapoptotic molecules such as Smac/DIABLO and caspases. Whereas the X-linked IAP is an established caspase inhibitor, the protective mechanisms utilized by the cellular IAP (c-IAP) proteins are less clear because c-IAPs bind to but do not inhibit the enzymatic activities of caspases. In this study, c-IAPs are shown to be highly unstable molecules that undergo autoubiquitination. The autoubiquitination of c-IAP1 is blocked upon coexpression with TNF receptor-associated factor (TRAF) 2, and this is achieved by inhibition of the E3 ubiquitin ligase activity intrinsic to the RING of c-IAP1. Consistent with these observations, loss of TRAF2 results in a decrease in c-IAP1 levels. Stabilized c-IAP1 was found to sequester and prevent Smac/DIABLO from antagonizing X-linked IAP and protect against cell death. Therefore, this study describes an intriguing cytoprotective mechanism utilized by c-IAP1 and provides critical insight into how IAP proteins function to alter the apoptotic threshold.The inhibitors of apoptosis (IAPs)² are an evolutionarily conserved gene family described originally as encoding cell death inhibitors. IAP proteins have subsequently been found to participate in a variety of additional intracellular signaling processes (1), and it has become evident that IAP proteins are versatile molecules playing numerous distinct roles within the cell. Although a more complete understanding of these additional functions for IAP proteins is emerging, the distinct mechanisms utilized by some IAP proteins to function in their originally defined roles as cell death inhibitors remain unclear.Members of the IAP family are characterized by the presence of 1–3 tandem repeats of an ∼70-residue baculovirus IAP repeat domain (2). The baculovirus IAP repeat domains of many IAP proteins have been shown to be the region within IAP proteins that associates with caspases and other proapoptotic molecules (3, 4). IAP proteins have remarkably different apoptotic inhibitory abilities. For example, X-linked IAP (XIAP) is a highly potent cell death inhibitor (5) and is thought to be the only mammalian IAP protein that directly inhibits the enzymatic activities of caspases (2–4, 6). Although cellular IAP1 and -2 (c-IAP1 and c-IAP2) are anti-apoptotic proteins that can bind to caspase-7 and -9, they do not inhibit the enzymatic activities of these caspases (2, 6).Many IAP proteins, including c-IAP1 and c-IAP2, contain a carboxyl-terminal RING domain that can function as an E3 ubiquitin ligase (7). The E3 ubiquitin ligase activity of the RING domain in c-IAP1 and c-IAP2 was previously shown to negatively regulate the apoptotic inhibitory properties of c-IAP proteins and to promote autoubiquitination and degradation of c-IAP1 (8, 9), thus hindering attempts to define the cellular properties of this protein.A specialized property of the c-IAP proteins is their involvement in tumor necrosis family (TNF) signaling (10–12). Both c-IAP1 and c-IAP2 were discovered in a biochemical screen for factors associated with the type 2 TNF receptor. This association was found to be indirect and bridged by interactions with TNF receptor-associated factors (TRAFs), most notably TRAF1 and TRAF2 (11). Though the consequences of the association between TRAF2 and c-IAP1 on TNF-mediated signaling have been investigated (12), less is known about the functional significance of the association between TRAF2 and c-IAP1 on cell death inhibition. Because both c-IAP1 and TRAF2 possess E3 ubiquitin ligase activity in their respective RING domains, it seemed that the association between these molecules might impact the protective properties of c-IAP1 and alter the apoptotic threshold.In this study, the role of TRAF2 in c-IAP1 stability and how the association of TRAF2 with c-IAP1 affects the apoptotic inhibitory properties of c-IAP1 were examined. The presence of TRAF2 greatly enhanced the stability of c-IAP1, and these data suggest that the interaction between TRAF2 and c-IAP1 inhibits the E3 ubiquitin ligase activity intrinsic to the RING domain of c-IAP1. Using stabilized c-IAP1, the anti-apoptotic activity of c-IAP1 was characterized, and it was found that c-IAP1 suppresses apoptosis to a degree comparable with XIAP. Furthermore, we show that c-IAP1 functions to prevent the IAP antagonist, Smac/DIABLO (13, 14), from interfering with XIAP inhibition of caspases. Together, this study demonstrates that although c-IAP1 does not directly inhibit caspase activity, stabilized c-IAP1 can sequester Smac/DIABLO, prevent Smac/DIABLO from antagonizing XIAP, and inhibit cell death.     

The IAP family：endogenous caspase inhibitors with multiple biological activities

IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains.These proteins have multiple biological activities that include binding and inhibiting caspases,regulating cell cycle progression,and modulating receptor-mediated signal transduction.Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells,and their degradation appears to be important for T cells to commit to death.In addition to three BIR domains,each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase(E3) activity to IAPs,and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus.Given the fact that IAPs can bind a variety of proteins,such as caspases and TRAFs,it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction,cell cycle,and apoptosis.