and blood vessel development, maturation, or maintenance.

Knockout studies show that DIAP1 is required for the survival of many cell types in the fly. During larval development, knockout of DIAP1 in the Drosophila S2 cell line or a DIAP1 null mutation results in widespread caspasedependent cell death in the absence of any extrinsic cell death signals. Interestingly, DIAP2 knockout flies exhibit no gross cell death–related phenotypes.

In C. elegans, inhibition of BIR-1 expression using RNA interference (RNAi) does not affect apoptosis in adult somatic or germ cells. However, in the embryo, the lack of BIR-1 expression results in early lethality and a failure to complete cytokinesis.

4. SUBCELLULAR LOCATIONS OF IAPS

Clues to the functions of some IAPs can be found in their subcellular locations. Among the most striking is Survivin, which localizes to mitotic structures in dividing cells. Survivin is associated with the kinetochores of metaphase chromosomes and is recognized as a chromosomal passenger protein. As cells divide, Survivin leaves the chromosomes, moves to the microtubules during anaphase, and localizes to the midbody microtubules during telophase, where it concentrates until cytokinesis is completed. When Survivin production is perturbed, other components of the chromosomal passenger complex (CPC), such as inner centromere protein (INCENP), Aurora kinases, and Borealin, fail to localize properly to the centrosomes (kinetochore), resulting in chromosome segregation defects and sometimes, depending on the cellular background, in cell death. Cells reaching telophase with deficient Survivin fail to complete cytokinesis and become either binucleated (or multinucleated with repeated attempts at division) or tetraploid (and eventually aneuploid with successive attempts at cell division). Interestingly, c-IAP1 also localizes to midbody microtubules during telophase and reportedly associates with Survivin. Cells stably overexpressing c-IAP1 accumulate in G2-M phase, exhibit cytokinesis defects, and display a mitotic checkpoint abnormality, leading to polyploid cells when exposed to microtubule-targeting drugs. The fly Apollon/BRUCE ortholog, dBruce, also localizes to the midbody microtubule ring during cytokinesis, binding mitotic regulators and components of the vesicle-targeting machinery. Thus several IAPs appear to regulate events associated with cytokinesis, although specific details of mechanisms are lacking.

Several IAPs traffic between the cytosol and nucleus, including XIAP, c-IAP1, and c-IAP2. Translocation of XIAP from the cytosol to the nucleus has been associated

with induction of cell death. For example, during neuronal cell death as a result of hypoxia-ischemia, XIAP is reported to move into the nucleus in complex with an endogenous inhibitory factor, XAF1, which binds XIAP, stimulating its nuclear translocation. In contrast, a pool of c-IAP1 redistributes into the cytosolic compartment in a caspase-dependent manner after apoptotic stimuli activate extrinsic (TNF and TNF-related apoptosisinducing ligand [TRAIL]) and intrinsic (ultraviolet irradiation and staurosporine) pathways.

Association of IAPs with organelles has also been reported. In cancer cells (but not normal cells), a pool of Survivin is localized to the mitochondria. Evidence from cell imaging, subcellular fractionation, and electron microscopy suggests that the pool of mitochondrial Survivin translocates into the cytosol in response to apoptotic stimuli, where it binds XIAP and other proteins to aid in suppression of apoptosis.

5. IAPS AS CASPASE INHIBITORS

IAPs are among the few types of cellular proteins that are capable of binding active caspases. In humans, XIAP is recognized as a potent inhibitor of effector caspases-3 and -7, as well as initiator caspase-9. Thus XIAP operates both within the intrinsic pathway, downstream of Apaf-1/cytochrome c to suppress apoptosis, and at the point of convergence of several apoptosis pathways, where caspases-3 and -7 operate as executioners of the cell death program. Dissection of XIAP has revealed that its second BIR domain (BIR2) and a short upstream sequence (“linker”) N-terminal to BIR2 are necessary and sufficient for potent (low nanomolar and even sub-nanomolar) inhibition of active caspases- 3 and -7. In contrast, the third BIR domain (BIR3) of XIAP is necessary and sufficient for potent inhibition of active caspase-9. BIR domains from c-IAP1, c-IAP2, Livin, Apollon, and ML-IAP have also been shown to bind specific caspases, although with lower affinity (micromolar). Structural studies have demonstrated that the higher affinity interaction of XIAP is caused by having two points of contact as compared with only one in the other IAPs. In this regard, all caspase-binding BIR domains contain a surface crevice that accommodates a tetrapeptide sequence corresponding to the N- terminus of the cleaved caspase’s small subunit of the catalytic domain (Figure 2-4). The tetrapeptide sequence has been dubbed the IAP-binding motif (IBM). The IBM mode of binding is shared by all caspases that bind BIRs. XIAP, however, has two additional modes of binding. The linker associated with BIR2 binds across the active site of caspases-3 and -7, whereas an α-helix of BIR3 makes an additional contact with caspase-9. The

Figure 2-4. Structure of the XIAP BIR3 domain complexed with SMAC tetrapeptide. SMAC peptide bound to BIR3 of XIAP. The BIR3 domain of XIAP (shown as a space-filling model) complexed with the SMAC tetrapeptide, AVPI. See Color Plate 3.

reported inhibitory constants (Ki values) for the eight mammalian members of the IAP family are provided in Table 2-2. The interaction of Survivin with caspases may require additional accessory proteins and posttranslational modifications.

6. IAPS AS E3 LIGASES

Several of the IAP family members (XIAP, c-IAP1, c-IAP2, ML-IAP, and ILP-2 in mammals) contain a C-terminal RING domain that binds ubiquitin-conjugating enzymes (E2), endowing them with E3 ubiquitin ligase activity. The types of ubiquitin modifications that IAPs induce on their substrates may vary, with K48-linked polyubiquitin chains representing the best documented and the modification typically associated with targeting for proteasomal degradation. However, some IAPs may also mediate non-degradative ubiquitinylation of substrates (involving K63-linked polyubiquitin chains), such as the receptor interacting kinase (RIP1) protein by c-IAP1 and c-IAP2. The UBA domain, found in XIAP, c-IAP1, c-IAP2, and ILP-2, binds ubiquitin chains and plays a crucial role in several facets of IAP function related to their E3 ligase activities.

Factors regulating the E3 ligase activity of IAPs are not fully understood. IAPs auto-ubiquitinylate themselves, constituting a mechanism for self-induced destruction. Binding of endogenous antagonists such as “second mitochondria-derived activator of caspases” (SMAC) in mammals and analogous proteins in insects can stim-

ulate the self-directed E3 ligase activity of IAPs, leading to more rapid proteasome-dependent degradation, thereby promoting apoptosis. The interaction of survivin with XIAP has been reported to reduce self-directed ubiquitinylation and thereby result in higher levels of XIAP and protection from apoptosis.

Several substrates of IAP-mediated ubiquitinylation have been identified thus far, and more are being discovered as research advances. Generally, all IAP-interacting proteins are candidates for IAP-promoted ubiquitinylation. Among the unanswered questions is how IAPs choose among various E2s to induce K48-linked versus alternatively (e.g., K63) linked polyubiquitin, with very different consequences for substrate degradation versus activation.

7. IAPS AND SIGNAL TRANSDUCTION

The IAP family plays an important role in the regulation of several signaling pathways, including activation of protein kinases. For example, c-IAP1 and c-IAP2 mediate TNF-α–induced NF-κB activation through nondegradative, K63-linked polyubiquitinylation of RIP1 via interaction with TNF receptor (TNFR)–associated factors 1 (TRAF1) and 2 (TRAF2). In this regard, it is presumed that c-IAP1 and c-IAP2 partner with nonclassical ubiquitin-conjugating enzymes (E2s) responsible for non–K48-linked polyubiquitinylation of RIP1 (such as UBC13, which mediates K63-linked ubiquitinylation), but firm details are lacking. In contrast to stimulating

Table 2-2. Reported inhibitory constants (Ki values) for the eight mammalian members of the IAP family

Note: Some values may require further verification and should be treated only as an indication of what has been reported in the literature. ND, not determined; NI, not inhibited.

the TNFR1→ TNF receptor–associated death domain (TRADD)→RIP pathway for NF-κB activation, the c-IAP1 and c-IAP2 proteins also ubiquitinylate the serine/threonine kinase, “NF-κB–inducing kinase” (NIK). NIK is a protein that controls the non-canonical NF-κB signaling cascade involving p100/p105 NF-κB family proteins, which undergo limited degradation to produce p50/p52 transcription factor subunits that partner principally with RelB. The c-IAPs promote the destabilization of NIK (presumably involving K48-linked polyubiquitinylation) via proteasomal degradation, thus blunting signaling via the non-canonical NF-κB signaling pathway. Thus effects of c-IAP1 and c-IAP2 on NF-κB signal transduction pathways are complex. The first BIR domain of c-IAP1 and c-IAP2 binds TRAF1 and TRAF2, the latter of which is also a RING domain-containing E3 ligase. TRAFs are critical intermediaries in signaling for essentially all TNF family receptors and toll-like receptors (TLRs). TRAF1 and TRAF2 collaborate with TNFRs, but not with TLRs.

XIAP, c-IAP1, and c-IAP2 are involved in controlling the stability of c-RAF kinase, a serine/threonine protein kinase that activates Erk1/2-dependent signaling pathways mediating cell proliferation, differentiation, migration, and survival. Knockdown of these IAPs stabilizes c- RAF protein. In this context, XIAP is indirectly involved in the ubiquitinylation of c-RAF by promoting the association of the ubiquitin ligase carboxy terminal Hsc70interacting protein (CHIP) to a protein complex that contains c-RAF.

XIAP is involved in NF-κB and MAPK activation, which is mediated by transforming growth factor β (TGF-β) and the BMPs by direct interaction of the BIR1 domain with TGF-β–activated protein kinase 1

(TAK1) binding protein (TAB1), which in turn activates TAK1 to induce NF-κB and downstream MAPKs. XIAP, as well as cIAP-1 and cIAP-2 also participate in NLRC1 (NOD1) and NLRC2 (NOD2) signalling to stimulate NF-κb activation and stress kinase activity. Though mechanistic details are lacking, those IAP-family members bind Rip2, a protein kinase that associates via CARD-CARD interactions with NOD1 and NOD2. It is speculated that the IAPs enable NOD1/NOD2 signaling, either by recruiting the TAB/TAK complex directly (XIAP binds TAB/TAK) or indirectly by catalyzing K63linked polyubiquitination a post-translational modification that binds TAB.

In the context of Survivin’s role in mitosis, it is essential in providing proper localization of the chromosomal passenger proteins INCENP, Borealin, and Aurora B, thus ensuring that the kinase Aurora B finds its mitotic substrates. Activation of Aurora B requires its autophosphorylation and binding to INCENP, which then allows for association with Borealin and Survivin. Disruptions in this chromosomal passenger complex result in mitotic catastrophe and cell death.

Table 2-3 lists the human IAP family members and their associated binding partners involved in various signaling pathways.

8. IAP–IAP INTERACTIONS

Several IAP family proteins are capable of forming homoor hetero complexes that contribute to their functional properties. Survivin, for example, forms homodimers, assisted by a coiled-coil domain located downstream of its BIR. The three-dimensional (3D) structures of the Survivin homodimer have been solved, providing

a firm understanding of their structural basis. In addition, Survivin interacts with XIAP, c-IAP1, and c-IAP2, apparently via BIR–BIR interactions. The association of survivin with XIAP has been reported to reduce autoubiquitinylation of XIAP, thus causing accumulation of XIAP and enhancing apoptosis resistance. In another example of a BIR–BIR interaction, XIAP homodimerization via its BIR1 domain has been sited as a crucial event for NF-κB activation mediated by XIAP. To date, the structures of BIR–BIR complexes have not yet been solved; therefore, the molecular basis for this type of interaction and firm insights are lacking in terms of which BIRs are capable of associating. RING–RING domain interactions among IAP family members have also been reported in the case of XIAP and c-IAP1, where they have been suggested to stimulate ubiquitinylation of XIAP and its subsequent degradation via the proteasome. Further details are needed about the structural basis and functional consequences of IAP–IAP interactions.

9. POST-TRANSLATIONAL MODIFICATIONS

OF BIR PROTEINS

At least three types of post-translational modifications of IAPs contribute to their biological roles: ubiquitinylation, proteolysis, and phosphorylation. With respect to phosphorylation, examples of regulatory phosphorylation events have been identified thus far for XIAP and Survivin. Akt/protein kinase B (PKB) is a member of a family of phosphatidylinositol 3-OH-kinase (PI- 3K)–regulated serine/threonine kinases that promote cell survival and suppress apoptosis. XIAP is phosphorylated by activated Akt at Ser87, preventing both auto-ubiquitinylation and cisplatin-induced ubiquitinylation of XIAP. Because Akt is hyperactive in many cancers, this post-translational modification may be a common mechanism contributing to tumor cell survival. The cellular functions of Survivin are regulated by multiple kinases, including Cdk1, p34 Cdc2/cyclin B1, Aurora B, and protein kinase A (PKA). Phosphorylation of Survivin on Thr34 by Cdk1 stabilizes Survivin from proteasomal degradation. The mitotic kinase p34 Cdc2/cyclin B is among the kinases capable of Thr34 phosphorylation of Survivin, an event required for at least some of the functions of Survivin as a regulator of cell division. The molecular events reportedly regulated by Thr34 phosphorylation include (1) association with caspase-9 and hepatitis B virus X-interacting protein (HBXIP) for apoptosis suppression; and (2) association with Cdk1 to stabilize Survivin during prometaphase and metaphase. Cyclic AMP (cAMP)–dependent PKA

phosphorylates Survivin at Ser20, resulting in loss of binding to XIAP and other potential cofactors. This phosphorylation appears to occur exclusively on cytosolic pools of Survivin. The Aurora B protein kinase is capable of phosphorylating Survivin at Thr117 during mitosis. However, this phosphorylation event has a negative effect on Survivin and its function as a regulator of cell division. Dephosphorylation of Thr117 on Survivin is required for chromosome orientation and centromere stabilization. It seems likely that additional examples of regulation of IAP family proteins by phosphorylation will eventually be revealed.

With respect to ubiquitinylation, the polyubiquitinylation of IAPs has been alluded to earlier in this chapter as a means of controlling cellular levels of IAPs. Monoubiquitinylation of XIAP has also been reported to regulate the subcellular distribution of XIAP in neurons. Interestingly, the yeast Bir1p protein undergoes modification with small ubiquitin-like modifier (SUMO), a ubiquitin-like protein. SUMO modification is lost in Bir1p variants lacking the BIR repeats.

Regarding proteolysis, XIAP is cleaved by caspases, separating the BIR1-2 region from the BIR3-RING segment of the protein. The functional consequence of this caspase-mediated cleavage event is probably to eliminate XIAP as a barrier to apoptosis.

10. ENDOGENOUS ANTAGONISTS OF IAPS

In mammals, several endogenous antagonists of IAPs have been identified, including SMAC (Diablo), HtrA2 (Omi), apoptosis-related protein in the TGF-β signaling pathway (ARTS), and XAF-1. The SMAC and “hightemperature requirement serine protease” (HtrA2) are both targeted to the mitochondria by an N-terminal targeting sequence. Once inside these organelles, the targeting sequence is cleaved off, revealing a new N- terminus containing an IBM. In SMAC, this sequence is the tetramer Ala-Val-Pro-Ile, whereas in HtrA2, it is Ala- Val-Pro-Ser. SMAC and HtrA2 are released from the intermembrane space of mitochondria in response to apoptotic signals. SMAC is an elongated dimer for which the N-terminal Ala is essential for simultaneous binding to the IBM-binding grooves of BIR2 and BIR3 of XIAP and c-IAP1. (A 3D structure of the XIAP BIR3 domain binding the SMAC tetrapeptide AVPI is shown in Figure 2-4.) On binding, SMAC releases active caspase-3, -7, and -9 from XIAP, thus enabling apoptosis. HtrA2, a hexamer, acts in a similar manner as SMAC binding to displace caspases, but also possesses a serine protease activity that can induce cell death via a non–caspase-dependent mechanism. Recently, other mitochondrial proteins with

N-terminal IBMs have been identified that seem to primarily target BIR2 of XIAP, including Nipsnap (Nsp) 3 and 4, glutamate dehydrogenase (GdH), leucine-rich pentatricopeptide (LRPPR), and 3-hydroxyisobutyrate dehydrogenase (3HB) and other proteins. Several of these proteins (GdH, Nsp4, and LRPPR) have been shown to antagonize XIAP inhibition of caspases-3 in vitro, although not as potently as SMAC. The importance of these other IBM-containing proteins requires additional experimentation. An additional IAP antagonist (GstPT) is processed in the endoplasmic reticulum to remove an N-terminal leader and reveal an IBM, but the conditions that would permit its release from this organelle are unclear.

ARTS is a septin-like protein that resides in the mitochondria; it is released from these organelles and targets XIAP (Figure 2-5). ARTS lacks an IBM sequence and seems to interact with XIAP via a short C-terminal sequence, although other regions of the ARTS protein may also make contact in as much as the GTP-binding domain of ARTS is also required. When released from mitochondria, ARTS is reported to initially colocalize with XIAP initially in the cytoplasm, subsequently accumulating in the nucleus. The half-life of ARTS is regulated by ubiquitin-dependent mechanisms that appear to vary with apoptotic stimuli. Moreover, binding of ARTS to XIAP results in a decrease in XIAP in a proteosome-dependent manner, suggesting that ARTS may participate in controlling XIAP ubiquitinylation. Recently, ARTS was reported to bind and E3 ligase (SIAH,

“seven in absentia”), thus assisting with targeting of XIAP for K48-linked ubiquitination and proteasomal dyradiation.

XIAP antagonist factor-1 (XAF-1) is another endogenous inhibitor of XIAP. Its mechanism of antagonism seems to involve binding XIAP to induce its shuttling from the cytosol (where caspases reside) into the nucleus, thus effectively separating XIAP from the cellular compartment required for apoptosis suppression. Expression of XAF-1 is significantly reduced in cancer cell lines and primary tumors, apparently as a result of promoter hypermethylation. Additionally, XAF-1 promotes the degradation of Survivin, suggesting a role in both apoptosis and cell division.

In Drosophila, multiple IBM-containing proteins have been identified, including Reaper (Rpr), head involution defective (Hid), Grim, Sickle (Skl), and Jafrac2. These proteins function to release DIAPs from the Drosophila caspases, Drosophila Nedd-2 like caspase (DRONC; initiator caspase) and DCP-1 (effector caspase). In doing so, they contribute to programmed cell death during fly development. Several of the Drosophila IAP antagonists have also been reported to stimulate ubiquitin-mediated destruction of IAPs. The N-terminal methionine of Rpr, Hid, Grim, and Skl is removed by an endogenous exoprotease, thus revealing the conserved alanine that initiates the tetrapeptide IBM sequence. The activity of these IAP antagonists appears to be controlled predominantly at the level of gene transcription or mRNA stability via diverse mechanisms that