Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP ؊/؊ cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.Among the regulators of programmed cell death, or apoptosis (1), Bcl-2 proteins (2) control the release of apoptogenic proteins from mitochondria, notably cytochrome c (3), whereas members of the inhibitor of apoptosis (IAP) 1 gene family act as endogenous inhibitors of caspases (4), the enzymatic effectors of apoptosis (1). The structural requirements of IAP-caspase(s) complexes have been defined in considerable detail (5).Survivin is a structurally unique IAP protein that has been implicated in protection from apoptosis and regulation of mitosis (6). A role of survivin in cell division has been linked to assembly/stability of metaphase and anaphase microtubules (7) and spindle checkpoint function (8). In contrast, despite its ability to counteract apoptosis in vitro, and in transgenic animals (6), the mechanism(s) by which survivin inhibits apoptosis has remained elusive. This is important because IAPs, especially XIAP (9) and survivin (6), have emerged as critical regulators of cell survival in tumors and promising targets for rational anti-cancer therapy (10,11).In this study, we investigated the mechanism(s) of survivin cytoprotection. We found that in response to cell death stimulation, survivin physically associates with XIAP, and this complex promotes enhanced XIAP stability and synergistic inhibition of caspase-9 activation. MATERIALS AND METHODSCell Culture-Breast carcinoma MCF-7, lymphoblastoid Raji, and kidney embryonic HEK293T cells were from the American Type Culture Collection (ATCC, Manassas, VA). Wild type (WT) or XIAP Ϫ/Ϫ mouse embryonic fibroblasts (MEF) (12) were the gift of Dr. C. Duckett (University of Michigan).Protein-Protein Interactions-Affinity fractionation and immunoprecipitation experiments were carried out as described (10, 13). Fulllength survivin, truncated survivin BIR-(1-87), full-length XIAP, cIAP1, cIAP2, or the three isolated XIAP, BIR1-(1-123), BIR2-(123-259) and BIR3-(260 -336) were expressed as GST fusion proteins (14). Replication-deficient adenoviruses encoding GFP (pAd-GFP) or survivin (pAd-survivin) were described (15). Pull-down experiments with recombinant survivin (0.1-0.4 g) and GST, GST-XIAP, GST-cIAP1, GST-cIAP2 (8 g), or the individual GST-BIR1, -BIR2, or -BIR3 of XIAP (10 g) bound to glutathione beads (100 l) were as described (13). Alternatively, XIAP was translated in vitro in the presence of [ 35 S]methionine (Amersham Sciences), mixed with 5 g of GST or GST-survivin, and u...

Inhibitor of apoptosis proteins (IAPs) 3 are a family of anti-apoptotic proteins characterized by the presence of baculoviral IAP repeat (BIR) domains (for view, see Ref. 1). IAPs modulate apoptosis by binding and inactivating caspases (2). Several IAPs contain E2 binding RING domains and also operate as E3 ligases that catalyze attachment of ubiquitin to protein substrates. This E3 ligase activity can be directed either toward caspases and caspase regulators or toward signal-transducing proteins primarily involved in activation of NF-B and Jun N-terminal kinase.The human genome includes eight genes that encode BIR-containing proteins. Among these are cIAP1 and cIAP2, which are highly homologous in amino acid sequence and domain structure, containing (from the N to C terminus) three tandem BIR domains followed by a CARD and RING. The cIAP1 and cIAP2 proteins are unique among the BIRfamily proteins in their ability to form complexes with TRAF-family adapter proteins involved in TNF receptor signaling (3-5). These members of the IAP family are known to induce ubiquitination and proteasome-dependent degradation of the TRAFs to which they bind (e.g. TRAF1, TRAF2), but the structural basis for this phenomenon is largely unknown.In addition to binding certain caspases (6), cIAP1 and cIAP2 are reported to bind SMAC, a mitochondrial protein that competes with caspases for binding to IAPs when released into the cytosol (7). In the IAP-family member, XIAP, the binding site for SMAC has been mapped to the third BIR domain (BIR3), where it competes for binding to caspase-9 (8). However, the site on cIAP1 and cIAP2, where SMAC binds has not been previously elucidated, and its relation to the sites required for binding to other targets such as TRAF1 and TRAF2 have not been heretofore defined.Here, we undertook a structure-function analysis of cIAP1 to compare the binding sites for TRAF2 and SMAC and to reveal the functional consequences of disruptions in these binding sites. Our findings indicate that TRAF2 binds the BIR1 domain, whereas SMAC binds the BIR3 domain of cIAP1. The integrity of these binding sites on BIR1 and BIR3 is required for cIAP1-mediated ubiquitination of TRAF2 and SMAC, respectively, revealing the basis for differential targeting of protein substrates by this E3 ligase. We also show evidence of BIR1-dependent modulation of TRAF2-mediated regulation of NF-B. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single IAP-family protein can direct its E3 ligase activity toward different substrates and can alter the cellular functions of different protein targets in accordance with differences in the specificity of individual BIR domains. MATERIALS AND METHODSPlasmids, Mutagenesis, Recombinant Proteins-A cDNA clone encoding cIAP1 (ID 627116) was obtained from IMAGE consortium through ResGen (Invitrogen) and PCR-subcloned into the pcDNA3-FLAG expression vector. Deletion or point mutations were introduced by PCR or using the QuikChange site-directed mutagen...

Integrity of the blood vessel wall is essential for vascular homeostasis and organ function. A dynamic balance between endothelial cell survival and apoptosis contributes to this integrity during vascular development and pathological angiogenesis. The genetic and molecular mechanisms regulating these processes in vivo are still largely unknown. Here, we show that Birc2 (also known as cIap1) is essential for maintaining endothelial cell survival and blood vessel homeostasis during vascular development. Using a forward-genetic approach, we identified a zebrafish null mutant for birc2, which shows severe hemorrhage and vascular regression due to endothelial cell integrity defects and apoptosis. Using genetic and molecular approaches, we show that Birc2 positively regulates the formation of the TNF receptor complex I in endothelial cells, thereby promoting NF-kappaB activation and maintaining vessel integrity and stabilization. In the absence of Birc2, a caspase-8-dependent apoptotic program takes place that leads to vessel regression. Our findings identify Birc2 and TNF signaling components as critical regulators of vascular integrity and endothelial cell survival, thereby providing an additional target pathway for the control of angiogenesis and blood vessel homeostasis during embryogenesis, regeneration and tumorigenesis.

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Defects in apoptosis play important roles in cancer pathogenesis and progression and have been linked to chemoresistance, radioresistance, and hormonal therapy failures (1, 2). A need exists therefore to reduce the roadblocks to apoptosis in advanced cancers, placing tumor cells into a more vulnerable state and making them easier to eradicate.Caspases are the principal mediators of apoptosis (3, 4). These intracellular proteases become activated in response to various cell death stimuli, triggering a characteristic set of biochemical reactions responsible for cellular demise in the context of normal cell turnover and immune-based elimination of virus-infected and malignant cells. Caspases cleave and activate each other in proteolytic networks, characterized by upstream initiator proteases (e.g. caspases 2, 8, 9, and 10) and downstream effector proteases (e.g. caspases 3, 6, and 7) (5). At least four major pathways for apoptosis have been delineated (6 -11), including pathways activated by: (a) TNF 1 /Fas family cytokines receptors, which recruit certain caspases to ligandactivated receptor complexes; (b) granzyme B, a protease injected into target cells by cytolytic T-cells and NK cells, which directly cleaves and activates caspases; (c) mitochondria, which release caspase-activating proteins into the cytosol in response to various stimuli; and (d) endoplasmic reticulum, where organellar stress links to caspase activation through various mechanisms (12, 13). These pathways for apoptosis are kept in check by endogenous antagonists that operate at specific points in caspase signaling cascades to interrupt the cell death program. Unfortunately, tumor cells commonly over-express apoptosis-suppressing proteins, thus avoiding elimination by either natural immune defenses or iatrogenic means (hormonal therapy, chemotherapy).Certain caspases are inhibited by IAP family proteins (14). Several members of the IAP family have been shown to bind and directly suppress the activity of selected caspases, thereby suppressing apoptosis (1, 15-28). Over-expression of IAPs has been documented in several types of human cancers (1, 20, 27, 29 -38), and proof-of-concept data using antisense and peptide * This work was supported by National Institutes of Health Grant CA-78040, the Prostate Cancer Foundation, and Accelerate Brain Care Cure, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ Present address: Princess Margaret Hospital, Toronto M5G 2M9, Canada.ʈ To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. E-mail: reedoffice@ burnham.org. 1 The abbreviations used are: TNF, tumor necrosis factor; IAP, inhibitor of apoptosis protein; XIAP, X-chromosome linked inhibitor of apoptosis; E2, ubiquitin carrier protein; GST, glutathione S-transferase; AFC, 7-amino-4-trifluoromethylcoumarin; MEF, mou...

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© 2 0 0 2 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n . ABSTRACTCell cycle checkpoints constitute a network of signal transduction mechanisms to monitor DNA damage and replication and thereby regulate progression through the cell cycle. A series of events is triggered in cells upon DNA damage. Here we describe a framework for the understanding of the functions of the core components involved in the cell cycle response to DNA damage and the relevance to the origin of cancer.

The tumor suppressor ARF is transcribed from the INK4a/ ARF locus in partly overlapping reading frames with the CDK inhibitor p16 Ink4a . ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19 ARF . Little is known about the dependency of human p14 ARF function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14 ARF -induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53 +/+ p21 CIP1+/+ 14-3-3s +/+ ), but not p53 7/7 counterparts, underwent G 1 and G 2 cell cycle arrest following infection with a p14 ARF -adenovirus. In p21 CIP17/7 cells, p14 ARF did not induce G 1 or G 2 arrest, while 14-3-3s 7/ 7 counterparts were mainly arrested in G 1 , pointing to essential roles of p21 CIP1 in G 1 and G 2 arrest and cooperative roles of p21 and 14-3-3s in ARF-mediated G 2 arrest. Our data demonstrate a strict p53 and p21 CIP1 dependency of p14 ARF -induced cell cycle arrest in human cells.