An IAP-IAP complex inhibits apoptosis.

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 apo-ptosis 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 METHODS
Cell 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).

Identification of a Survivin-XIAP Complex during
Apoptosis-We fractionated Raji cell extracts by affinity chromatography over an antibody to survivin (14) and analyzed bound proteins by immunoblotting. Survivin co-eluted with the mo-* This work was supported by National Institutes of Health Grants HL54131, CA78810, and CA90917 (to D. C. A.) and AG15393 (to G. S. S. and J. C. R.) and by the Department of Defense Prostate Cancer Research Program, postdoctoral fellowship program. 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.
We next determined whether survivin and XIAP interacted directly. Recombinant survivin bound GST-XIAP in a concentration-dependent manner, whereas no interaction with GST was observed (Fig. 1D). Reciprocally, 35 S-labeled in vitro translated XIAP associated with GST-survivin but not GST (Fig.  1D). We next expressed IAP family proteins cIAP1 and cIAP2 (4) and tested their association with survivin. Both cIAP1 and cIAP2 bound survivin directly, whereas no interaction with GST was detected (Fig. 1E). A role of homologous BIR se-quences in IAP-IAP complex formation was investigated. Recombinant survivin associated with XIAP-BIR1 and XIAP-BIR3 and more weakly with XIAP-BIR2 (Fig. 1F). Reciprocally, a truncated survivin 1-87 mutant containing the single survivin BIR bound full-length XIAP (not shown).
Regulation of XIAP Stability by Survivin-MCF-7 cells transduced with pAd-GFP and exposed to staurosporine exhibited a time-dependent loss of XIAP expression ( Fig. 2A). In contrast, transduction of MCF-7 cells with pAd-survivin preserved XIAP expression over time in the presence of staurosporine ( Fig. 2A). We then performed [ 35 S]L-methionine pulse-chase experiments in HEK293T cells expressing XIAP together with survivin or a control vector. Co-transfection of HEK293T cells with survivin resulted in persistence of XIAP levels over a 6-h interval after chase (Fig. 2B) and prolongation of XIAP half-life from 2 h in vector-transfected cells to 5.5 h (Fig.  2C). Addition of the proteasome inhibitor lactacystin prevented loss of XIAP expression in staurosporine-treated MCF-7 cells, whereas a caspase inhibitor, Z-VAD-fmk, was ineffective (Fig. 2D).
Regulation of XIAP Ubiquitination by Survivin-When mixed with ubiquitin, in vitro translated, 35 S-labeled XIAP exhibited a ladder of bands typical of polyubiquitinated conjugates (Fig. 3A). In contrast, a XIAP (Lys 328 3 Arg) mutant lacking a critical lysine residue involved in ubiquitination of this protein exhibited minimal ubiquitin conjugation in vitro (Fig. 3A). Addition of recombinant survivin suppressed XIAP ubiquitin conjugate formation, whereas a control Traf3 protein was ineffective (Fig. 3B). Increasing concentrations of recombinant survivin inhibited the binding of the ubiquitin-conjugat-

FIG. 1. Survivin-XIAP interaction.
A, affinity chromatography. Raji cell extracts were fractionated on an anti-survivin column, and eluted fractions were analyzed by immunoblotting. B, co-immunoprecipitation. MCF-7 cells were immunoprecipitated (IP) with IgG or an antibody to survivin with or without staurosporine (STS), and immune complexes were analyzed by immunoblotting. P, pellet; S, supernatant; E, cell extract. C, co-immunoprecipitation from overexpressing cells. MCF-7 cells were transduced with pAd-survivin without or with staurosporine (STS) and immunoprecipitated with IgG or an antibody to survivin, followed by immunoblotting. D, survivin-XIAP interaction. Upper panel, GST-XIAP or GST was incubated with survivin, and bound proteins were analyzed by immunoblotting. Input, survivin (0.4 g). Lower panel, 35 S-labeled in vitro translated XIAP (Input) was incubated with GST-survivin or GST, followed by autoradiography. E, survivin-IAP interaction. The indicated GST-IAPs were incubated with survivin, and bound proteins were detected by immunoblotting. Input, survivin (0.4 g). F, BIR interaction. The indicated GST-BIRs were mixed with survivin, followed by immunoblotting. The membrane was stained with Coomassie Blue for equal loading.

Survivin-XIAP Complex in Cytoprotection 34088
ing enzyme, UbcH5, to GST-XIAP (Fig. 3C), suggesting that a survivin-XIAP complex may prevent substrate accessibility to the ubiquitination machinery. We then explored whether survivin interfered with XIAP ubiquitination in vivo. Polyubiquitinated XIAP conjugates were readily detectable in HEK293T cells transfected with control pcDNA3 in the presence of a proteasome inhibitor (Fig. 3D). In contrast, expression of survivin attenuated XIAP polyubiquitination in vivo (Fig. 3D), and produced a Ͼ4-fold reduction in the relative amounts of XIAP-ubiquitin conjugates, as compared with control cells (Fig. 3E).
Cytoprotective Mechanism of the Survivin-XIAP Complex-We next used purified components in vitro to test whether a survivin-XIAP complex synergistically suppressed caspase activity. Whereas survivin alone had essentially no effect on caspase-9 generation, the combination with a suboptimal concentration of XIAP (Fig. 4A, left panel) dose-dependently inhibited caspase-9 generation (Fig. 4A, right panel).
Similarly, when combined with a suboptimal concentration of XIAP (Fig. 4B, left panel), survivin inhibited caspase activity in the context of a functional apoptosome in vitro (Fig. 4B, right  panel). To determine whether XIAP and survivin synergistically inhibited apoptosis in vivo, we transfected HEK293T cells with survivin and XIAP alone or in combination, and stimulated apoptosis by expressing Bax or Fas, which activate mitochondrial or death receptor apoptosis, respectively. Expression of suboptimal amounts of survivin or XIAP in HEK293T cells individually did not significantly reduce apoptosis (Fig. 4C). In contrast, the combination of XIAP and survivin synergistically suppressed cell death induced by Bax (Fig. 4C, left panel), or Fas (Fig. 4C, right panel). Next, we used XIAP Ϫ/Ϫ MEF to determine whether a survivin-XIAP complex was required for cytoprotection in vivo. Transduction of WT MEF with pAdsurvivin inhibited apoptosis stimulated by staurosporine or UVB (Fig. 4D). In contrast, survivin cytoprotection was completely lost in XIAP Ϫ/Ϫ MEF (Fig. 4D).

DISCUSSION
In this study, we have shown that IAP family members XIAP and survivin form a heterocomplex in response to cell death stimulation in vivo. This interaction promotes cell survival by enhancing the stability of XIAP against proteasomal destruction and by synergistically antagonizing apoptosome-mediated cell death, in a pathway abolished in XIAP Ϫ/Ϫ cells.
Formation of a survivin-XIAP complex resulted in increased stability of XIAP against polyubiquitination and proteasomal degradation in vitro and in vivo, potentially by excluding the ubiquitin-conjugating enzyme, UbcH5. Sudden changes in IAP stability influence cell viability (19), and ubiquitin-dependent proteasomal destruction of IAPs enhances cell death (20). Here, a stabilized survivin-XIAP complex synergistically suppressed caspase-9 processing/activity, alone or in the context of the apoptosome, and blocked apoptosis in vivo. Although the mechanism(s) of survivin cytoprotection have long remained elusive, these data suggest a model of intermolecular cooperation in which survivin enhances the anti-apoptotic activity of XIAP to suppress the upstream initiation of mitochondrial cell death (15,18). Apoptotic stimulation induces the formation of a survivin-XIAP complex in vivo, and future studies will investigate whether this reflects post-translational modifications in survivin (10) and/or XIAP (21) enabling protein interaction or, alternatively, redistribution of survivin from a specialized subcellular pool during cell death (14).
Targeted antagonists of the IAP-IAP complex may be suitable to disable apoptosis resistance in tumors, where survivin (6), and XIAP (9) are commonly overexpressed.