c-IAP1 cooperates with Myc by acting as a ubiquitin ligase for Mad1

c-IAP1, a member of the inhibitor of apoptosis protein (IAP) family and a RING finger ubiquitin ligase (E3), has been proposed to be an important oncogene. In many types of cancers, the levels of c-IAP1 are upregulated, which contributes positively to tumorigenesis. However, the mechanism by which c-IAP1 promotes tumorigenesis has proven elusive. Although proteins in the IAP family may function as caspase inhibitors, c-IAP1 was shown to be a poor inhibitor of caspases. Here we show that c-IAP1 catalyzes ubiquitination of Max-dimerization protein-1 (Mad1), a cellular antagonist of Myc. Ubiquitination of Mad1 by c-IAP1 accelerates its degradation by the 26S proteasome pathway, and this reduction of the Mad1 levels cooperates with Myc to promote cell proliferation. Our results demonstrate that c-IAP1 exerts its oncogenic functions by promoting the degradation of an important negative regulator in the Myc pathway.     

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.     

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.     

Smac/Diablo antagonizes ubiquitin ligase activity of inhibitor of apoptosis proteins

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activities.     

Suppression of MEK/ERK signalling by Myc: role of Bin-1

We report for the first time that over-expression of Myc suppresses mitogen-activated ERK kinase (MEK)/extracellular regulated kinase (ERK) signalling in chick embryo fibroblasts (CEF). Myc does not interfere with individual components of the signalling cascade, since efficient signal propagation via MEK and ERK in Myc-infected CEF can be seen. However, using the Myc-binding domain (MBD) of Bin-1, which binds to and negatively regulates the activity of Myc, we selectively suppressed Myc-induced apoptosis, without affecting its transforming properties. This was accompanied by a restoration in MEK/ERK signalling, suggesting a critical role for this pathway in regulating apoptosis in these cells. This was also confirmed using a specific pharmacological inhibitor of MEK. Experiments with conditioned media suggest that over-expression of Myc may inhibit autocrine growth factor production, which can be restored by co-expression of MBD. Although the identity of the growth factor(s) is not known, we propose a feedback mechanism whereby Myc interferes with growth factor signalling.     

Functional analysis of the Autographa californica nucleopolyhedrovirus IAP1 and IAP2

The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in de-tail. AcMNPV with p35-deletion inhibited the apoptosis of BTI-Tn-5B1-4 (Tn-Hi5) cells induced by a Helicoverpa armigera single nucleocapsid NPV (HearNPV) infection and rescued the replication of HearNPV and BV production in these cells. Transient-expression experiments indicated that both IAP1 and IAP2 suppress apoptosis of Tn-Hi5 cells during HearNPV infection. Recombinant HearNPVs ex-pressing AcMNPV iap1, iap2 and p35, respectively, not only prevented apoptosis but also allowed HearNPV to replicate in Tn-Hi5 cells. However, the iap1, iap2 and p35 genes when expressed in HearNPV were unable to rescue BV production. These results indicate that both AcMNPV iap1 and iap2 function independently as apoptosis inhibitors of and are potential host range factors.     

Myc Potentiates Apoptosis by Stimulating Bax Activity at the Mitochondria

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.     

The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein

Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation.     

The role of IAP antagonist proteins in the core apoptosis pathway of the mosquito disease vector <Emphasis Type="Italic">Aedes aegypti</Emphasis>

While apoptosis regulation has been studied extensively in Drosophila melanogaster, similar studies in other insects, including disease vectors, lag far behind. In D. melanogaster, the inhibitor of apoptosis (IAP) protein DIAP1 is the major negative regulator of caspases, while IAP antagonists induce apoptosis, in part, by binding to DIAP1 and inhibiting its ability to regulate caspases. In this study, we characterized the roles of two IAP antagonists, Michelob_x (Mx) and IMP, in apoptosis in the yellow fever mosquito Aedes aegypti. Overexpression of Mx or IMP caused apoptosis in A. aegypti Aag2 cells, while silencing expression of mx or imp attenuated apoptosis. Addition of recombinant Mx or IMP, but not cytochrome c, to Aag2 cytosolic extract caused caspase activation. Consistent with this finding, AeIAP1 bound and inhibited both initiator and effector caspases from A. aegypti, and Mx and IMP competed with caspases for binding to AeIAP1. However, a difference was observed in the BIR domains responsible for Dronc binding by AeIAP1 versus DIAP1. These findings demonstrate that the mechanisms by which IAP antagonists regulate apoptosis are largely conserved between A. aegypti and D. melanogaster, although subtle differences exist.     

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.     

Unlike Diablo/smac, Grim promotes global ubiquitination and specific degradation of X chromosome-linked inhibitor of apoptosis (XIAP) and neither cause apoptosis

Grim is a Drosophila inhibitor of apoptosis (IAP) antagonist that directly interferes with inhibition of caspases by IAPs. Expression of Grim, or removal of DIAP1, is sufficient to activate apoptosis in fly cells. Transient expression of Grim in mammalian cells induces apoptosis, arguing for the conservation of apoptotic pathways, but cytoplasmic expression of the mammalian IAP antagonist Diablo/smac does not. To understand why, we compared Grim and Diablo. Although they have the same IAP binding specificity, only Grim promoted XIAP ubiquitination and degradation. Grim also synergized with XIAP to promote an increase in total cellular ubiquitination, whereas Diablo antagonized this activity. Surprisingly, Grim-induced ubiquitination of XIAP did not require the IAP RING finger. Analysis of a Grim mutant that promoted XIAP degradation, but was not cytotoxic, suggests that Grim killing in transient assays is due to a combination of IAP depletion, blocking of IAP-mediated caspase inhibition, and at least one other unidentified function. Unlike transiently transfected cells, inducible mammalian cell lines can sustain continuous expression of Grim and selective degradation of XIAP without undergoing apoptosis, demonstrating that down-regulation and antagonism of IAPs is not sufficient to cause apoptosis of mammalian cells.     

RIP1 Protein-dependent Assembly of a Cytosolic Cell Death Complex Is Required for Inhibitor of Apoptosis (IAP) Inhibitor-mediated Sensitization to Lexatumumab-induced Apoptosis

Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death.     

IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition

The antiapoptotic properties of the inhibitor of apoptosis (IAP) family of proteins have been linked to caspase inhibition. We have previously described an alternative mechanism of XIAP inhibition of apoptosis that depends on the selective activation of JNK1. Here we report that two other members of the IAP family, NAIP and ML-IAP, both activate JNK1. Expression of catalytically inactive JNK1 blocks NAIP and ML-IAP protection against ICE- and TNF-alpha-induced apoptosis, indicating that JNK1 activation is necessary for the antiapoptotic effect of these proteins. The MAP3 kinase, TAK1, appears to be an essential component of this antiapoptotic pathway since IAP-mediated activation of JNK1, as well as protection against TNF-alpha- and ICE-induced apoptosis, is inhibited when catalytically inactive TAK1 is expressed. In addition, XIAP, NAIP, and JNK1 bind to TAK1. Importantly, expression of catalytically inactive TAK1 did not affect XIAP inhibition of caspase activity. These data suggest that XIAP's antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.