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Proteasome-mediated Degradation of Smac during Apoptosis: XIAP Promotes Smac Ubiquitination in Vitro

2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês

10.1074/jbc.m200317200

1083-351X

Marion MacFarlane, Wendy Merrison, Shawn B. Bratton, Gerald M. Cohen,

Autophagy in Disease and Therapy

During apoptosis, Smac (secondmitochondria-derived activator ofcaspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitroubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation. During apoptosis, Smac (secondmitochondria-derived activator ofcaspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitroubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation. tumor necrosis factor-related apoptosis-inducing ligand inhibitor of apoptosis protein secondmitochondria-derived activator ofcaspases ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin protein ligase death-inducing signaling complex glutathioneS-transferase benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone phosphate-buffered saline Apoptosis is a form of cell death that is essential for the correct development and homeostasis of multicellular organisms. Two major apoptotic pathways have been identified: one activated via death receptor activation and the other by stress-inducing stimuli (1Bratton S.B. MacFarlane M. Cain K. Cohen G.M. Exp. Cell Res. 2000; 256: 27-33Crossref PubMed Scopus (288) Google Scholar). Triggering of cell surface death receptors of the tumor necrosis factor receptor superfamily, including CD95 (Fas/Apo-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),1 result in a rapid activation of caspase-8 following its recruitment to a trimerized receptor/ligand complex via adaptor molecules (2Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar). Second, stress-induced apoptosis caused by chemicals and growth factor deprivation results in perturbation of mitochondria and the ensuing release of proteins, such as cytochrome c, from the intermitochondrial membrane space (3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Once released cytochrome c binds to Apaf-1, which in the presence of dATP results in the recruitment and activation of caspase-9 (4Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6182) Google Scholar, 5Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 6Zou H., Li, Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar). The release of cytochrome c from mitochondria is also regulated, in part, by Bcl-2 family members with anti-apoptotic and pro-apoptotic members inhibiting or promoting the release, respectively (7Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4254) Google Scholar, 8Li H.,. Zhu H., Xu, C-J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3765) Google Scholar, 9Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3061) Google Scholar, 10Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4382) Google Scholar). The activated initiator caspases-8 and -9 then activate the effector caspases-3, -6, and -7, which are responsible for the cleavage of important cellular substrates resulting in the classical biochemical and morphological changes associated with the apoptotic phenotype (11Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar, 12Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2428) Google Scholar, 13Slee E.A. Harte M.T. Kluck R.M. Wolf B.B. Casiano C.A. Newmeyer D.D. Wang H.G. Reed J.C. Nicholson D.W. Alnemri E.S. Green D.R. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1664) Google Scholar).Caspase activity is also regulated by the inhibitor of apoptosis proteins (IAPs), which are characterized by one or more baculovirus IAP repeats, called BIR domains that are responsible for their anti-apoptotic activity (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar, 15Holcik M. Korneluk R.G. Nat. Rev. 2001; 2: 550-556Crossref Scopus (233) Google Scholar), and in some IAPs a RING finger domain near their COOH terminus. One major function of IAPs, particularly XIAP, c-IAP1, and c-IAP2, is their propensity to bind to and inhibit the processed forms of key initiator and effector caspases, including caspase-9, -3, and -7 (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar, 16Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1707) Google Scholar). XIAP has also recently been shown to bind to and inhibit active caspase-9 and -3 within the apoptosome complex (17Srinivasula S.M. Hedge R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.-A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (850) Google Scholar, 18Bratton S.B. Walker G. Srinivasula S.M. Sun X-M. Butterworth M. Alnemri E.S. Cohen G.M. EMBO J. 2001; 20: 998-1009Crossref PubMed Scopus (336) Google Scholar).Protein ubiquitination is a post-translational protein modification that involves the sequential action of ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (19Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1175) Google Scholar). Recently, the RING finger domain has been implicated in the ubiquitination of proteins with a functional relationship being established between the RING finger and ubiquitin ligase activity (20Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (936) Google Scholar,21Joazeiro C.A.P. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar). Several E3s, including Mdm2, are highly homologous to IAP with respect to their RING finger domains and promote degradation of both themselves and specific substrates, such as p53 (22Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). Recent reports indicate that XIAP and c-IAP1 promote self-ubiquitination in response to apoptotic stimuli (23Yang Y. Fang Y. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar) and that both XIAP and c-IAP2 promotein vitro ubiquitination of caspase-3 and -7 (24Huang H. Joazeiro C.A.P. Bonfoco E. Kamada S. Leverson J.D. Hunter T. J. Biol. Chem. 2000; 275: 26661-26664Abstract Full Text Full Text PDF PubMed Google Scholar, 25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar)Recently, a novel protein Smac (secondmitochondria-derived activator ofcaspases), and its murine homologue DIABLO, were described which promote caspase activation by eliminating IAP inhibition of caspases (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar). Smac is synthesized as a 239-amino acid precursor protein, with the NH2-terminal 55 amino acids serving as a mitochondrial matrix-targeting signal. In response to apoptotic stimuli, mature Smac is released into the cytoplasm and binds to XIAP, thereby relieving XIAP inhibition of caspases. Recently this interaction has been mapped to the NH2-terminal 20 amino acids of mature Smac, and removal of this region completely blocks its ability to bind XIAP (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 29Srinivasula S.M. Datta P. Fan X.-J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). As Smac acts to prevent IAP activity, it is proposed to be a human equivalent of the Drosophila proteins Reaper, Grim, and Hid (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar). However, we have shown recently that Smac β, a Smac variant lacking the NH2-terminal IAP binding domain, still potentiates apoptosis, suggesting that Smac may also possess proapoptotic activity independent of its IAP inhibition (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar).In this study, we investigate the kinetics and modulation of Smac release from mitochondria following death receptor-mediated apoptosis. Caspase-8 is the most apical caspase in TRAIL-induced apoptosis in MCF-7 cells (31MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar) and is recruited to the native TRAIL death-inducing signaling complex (DISC) in various cell types (32Sprick M.R. Weigand M.A. Reiser E. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 33Kischkel F. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 34Harper N. Farrow S.N. Kaptein A. Cohen G.M. MacFarlane M. J. Biol. Chem. 2001; 276: 34743-34752Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In MCF-7 cells, which are caspase-3 null (35Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar), TRAIL induces rapid Bid cleavage resulting in engagement of the mitochondrial amplification loop that is essential for apoptosis in certain cells (31MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar). MCF-7 cells therefore provide a good model in which to study death receptor-mediated, and hence apical caspase-induced, release of pro-apoptotic molecules from mitochondria. We now demonstrate that TRAIL induces a rapid release of mitochondrial Smac in MCF-7 cells, which is inhibited by Bcl-2. Once released, Smac is rapidly degraded by the proteasome, an effect promoted in vitro by the ubiquitin-protein ligase activity of XIAP. This study identifies Smac as a target for ubiquitination and further demonstrates the versatility of XIAP as a potent inhibitor of apoptosis.DISCUSSIONRecent studies have shown that Smac can act as a potent pro-apoptotic molecule, an effect primarily attributed to its propensity to bind to IAP family proteins (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar), although other mechanisms cannot be excluded (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar). However, to exert its pro-apoptotic effect Smac must be released from mitochondria, and until now, few studies have investigated the kinetics or modulation of Smac release in intact cells. We now demonstrate that death receptor-mediated apoptosis induces rapid and complete release of mitochondrial Smac, with kinetics similar to that observed for cytochrome c. As studies with Bid-deficient mice have confirmed that Bid is a critical substrate forin vivo signaling by death receptor agonists (42Yin X.-M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar), TRAIL-induced release of Smac and cytochrome c in MCF-7 cells is likely to be tBid-dependent. Our observation that z-VAD.FMK prevented the release of both Smac and cytochromec would support this, however, we cannot exclude the possibility that other apical caspase substrates may also play a role. Importantly, our results demonstrate that mitochondrial release of pro-apoptotic molecules is an apical caspase-dependent event in receptor-mediated apoptosis. This is in contrast to that recently reported for cell stress-associated mitochondrial Smac release, which is proposed to be a caspase-catalyzed event occurring downstream of cytochrome c release (43Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (354) Google Scholar).We also show that Smac release in MCF-7 cells is sensitive to inhibition by Bcl-2/Bcl-xL, with Bcl-2 exhibiting greater potency than Bcl-xL. The ability of Bcl-2 family members to inhibit the release of mitochondrial intermemebrane pro-apoptotic molecules, such as cytochrome c, is now well documented (3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Intriguingly, in some experiments, Bcl-2 appeared more potent in preventing Smac than cytochrome c release. Structural analysis revealed that Smac exists as a homodimer (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar), with an apparent molecular mass of ∼100 kDa (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar), whereas cytochrome c is known to have a monomeric molecular weight of only ∼12 kDa. If Bcl-2 were to act simply by blocking a tBid-induced pore, then one might predict that Smac release would be more readily inhibited than release of cytochromec. Thus, mitochondrial release of Smac and cytochromec may occur by different mechanisms, perhaps involving tBid and/or another as yet unidentified pore-forming molecule.Following its release from mitochondria, Smac was rapidly degraded by the proteasome, an effect we observed in both death receptor-mediated and chemical-induced apoptosis. Recent studies have revealed that XIAP can function as an E3 ligase and promotes the degradation of both itself and its substrates. Until now, the only XIAP substrates identified in vitro have been caspase-3, -7, and -9 (25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar). Thus, XIAP exhibits its anti-apoptotic effect by directly binding to and inhibiting active caspase-3, -7, and -9 (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar), but in some circumstances may additionally exert its effect through ubiquitination and degradation of active caspases (25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar). We now demonstrate that XIAP can also act as an E3 ligase for Smac and thus may promote the proteasome-mediated degradation of Smac in intact cells. Importantly, the BIR3-RING domain of XIAP was both necessary and sufficient to ubiquitinate Smac in vitro. Structural analysis has revealed that the primary binding of Smac to XIAP occurs via the NH2-terminal four residues of Smac and the BIR3 domain of XIAP, with significantly weaker binding observed between Smac and the BIR2 domain of XIAP (41Wu G. Chai J. Suber T.L., Wu, J.-W., Du, C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar). Smac is proposed to compete with, and thereby antagonize the “caspase inhibitory” effect of XIAP (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 29Srinivasula S.M. Datta P. Fan X.-J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 40Liu Z. Sun C. Olejnicak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J., Wu, J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar,41Wu G. Chai J. Suber T.L., Wu, J.-W., Du, C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar). Importantly, our data suggest that ubiquitination and degradation of Smac may represent another mechanism for modulating apoptosis. Degradation of small amounts of Smac released from mitochondria would provide a safeguard against inadvertent perturbation of mitochondria and subsequent caspase activation. It may be the case that cells exhibiting high levels of XIAP are protected from apoptosis in several ways: through the ability of XIAP to directly inhibit and degrade caspases and additionally through its ability to bind and target Smac for degradation. Recently, the serine protease Omi/HtrA2, which contains an NH2-terminal AVPS motif similar to that found in mature Smac, was shown to be released from mitochondria and inhibit XIAP by direct binding (44Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 45Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopolos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 46Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufman H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 47Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Thus Omi, in addition to being a Smac-like inhibitor of IAP activity, may be another pro-apoptotic molecule with the potential to be regulated by proteasomal degradation. Other E3 ligases, in addition to XIAP, may also target Smac or Omi for proteasomal-mediated degradation. Others have reported that c-IAP2 also possesses E3 ligase activity and can monoubiquitinate caspase-3 and -7in vitro. However, as mature Smac binds preferentially to XIAP, but not c-IAP1 or -2 in intact cells (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar), it is unlikely that c-IAP2 contributes greatly to the proteasomal-mediated degradation of Smac in vivo.Taken together, our results demonstrate that receptor-mediated Smac release from mitochondria occurs prior to or in parallel with the release of cytochrome c. Once released, Smac is rapidly degraded by the proteasome, an effect we propose from our in vitro data may be mediated in part via the E3 ligase activity of the BIR3-RING domain of XIAP. Based on our results and those of others, apoptosis can now be viewed as a process that has features in common with the control of other cellular processes by ubiquitination. Apoptosis is a form of cell death that is essential for the correct development and homeostasis of multicellular organisms. Two major apoptotic pathways have been identified: one activated via death receptor activation and the other by stress-inducing stimuli (1Bratton S.B. MacFarlane M. Cain K. Cohen G.M. Exp. Cell Res. 2000; 256: 27-33Crossref PubMed Scopus (288) Google Scholar). Triggering of cell surface death receptors of the tumor necrosis factor receptor superfamily, including CD95 (Fas/Apo-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),1 result in a rapid activation of caspase-8 following its recruitment to a trimerized receptor/ligand complex via adaptor molecules (2Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar). Second, stress-induced apoptosis caused by chemicals and growth factor deprivation results in perturbation of mitochondria and the ensuing release of proteins, such as cytochrome c, from the intermitochondrial membrane space (3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Once released cytochrome c binds to Apaf-1, which in the presence of dATP results in the recruitment and activation of caspase-9 (4Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6182) Google Scholar, 5Cain K. Brown D.G. Langlais C. Cohen G.M. J. Biol. Chem. 1999; 274: 22686-22692Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 6Zou H., Li, Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar). The release of cytochrome c from mitochondria is also regulated, in part, by Bcl-2 family members with anti-apoptotic and pro-apoptotic members inhibiting or promoting the release, respectively (7Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4254) Google Scholar, 8Li H.,. Zhu H., Xu, C-J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3765) Google Scholar, 9Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3061) Google Scholar, 10Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4382) Google Scholar). The activated initiator caspases-8 and -9 then activate the effector caspases-3, -6, and -7, which are responsible for the cleavage of important cellular substrates resulting in the classical biochemical and morphological changes associated with the apoptotic phenotype (11Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar, 12Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2428) Google Scholar, 13Slee E.A. Harte M.T. Kluck R.M. Wolf B.B. Casiano C.A. Newmeyer D.D. Wang H.G. Reed J.C. Nicholson D.W. Alnemri E.S. Green D.R. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1664) Google Scholar). Caspase activity is also regulated by the inhibitor of apoptosis proteins (IAPs), which are characterized by one or more baculovirus IAP repeats, called BIR domains that are responsible for their anti-apoptotic activity (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar, 15Holcik M. Korneluk R.G. Nat. Rev. 2001; 2: 550-556Crossref Scopus (233) Google Scholar), and in some IAPs a RING finger domain near their COOH terminus. One major function of IAPs, particularly XIAP, c-IAP1, and c-IAP2, is their propensity to bind to and inhibit the processed forms of key initiator and effector caspases, including caspase-9, -3, and -7 (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar, 16Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1707) Google Scholar). XIAP has also recently been shown to bind to and inhibit active caspase-9 and -3 within the apoptosome complex (17Srinivasula S.M. Hedge R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.-A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (850) Google Scholar, 18Bratton S.B. Walker G. Srinivasula S.M. Sun X-M. Butterworth M. Alnemri E.S. Cohen G.M. EMBO J. 2001; 20: 998-1009Crossref PubMed Scopus (336) Google Scholar). Protein ubiquitination is a post-translational protein modification that involves the sequential action of ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (19Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1175) Google Scholar). Recently, the RING finger domain has been implicated in the ubiquitination of proteins with a functional relationship being established between the RING finger and ubiquitin ligase activity (20Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (936) Google Scholar,21Joazeiro C.A.P. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar). Several E3s, including Mdm2, are highly homologous to IAP with respect to their RING finger domains and promote degradation of both themselves and specific substrates, such as p53 (22Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). Recent reports indicate that XIAP and c-IAP1 promote self-ubiquitination in response to apoptotic stimuli (23Yang Y. Fang Y. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar) and that both XIAP and c-IAP2 promotein vitro ubiquitination of caspase-3 and -7 (24Huang H. Joazeiro C.A.P. Bonfoco E. Kamada S. Leverson J.D. Hunter T. J. Biol. Chem. 2000; 275: 26661-26664Abstract Full Text Full Text PDF PubMed Google Scholar, 25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar) Recently, a novel protein Smac (secondmitochondria-derived activator ofcaspases), and its murine homologue DIABLO, were described which promote caspase activation by eliminating IAP inhibition of caspases (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar). Smac is synthesized as a 239-amino acid precursor protein, with the NH2-terminal 55 amino acids serving as a mitochondrial matrix-targeting signal. In response to apoptotic stimuli, mature Smac is released into the cytoplasm and binds to XIAP, thereby relieving XIAP inhibition of caspases. Recently this interaction has been mapped to the NH2-terminal 20 amino acids of mature Smac, and removal of this region completely blocks its ability to bind XIAP (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 29Srinivasula S.M. Datta P. Fan X.-J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). As Smac acts to prevent IAP activity, it is proposed to be a human equivalent of the Drosophila proteins Reaper, Grim, and Hid (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar). However, we have shown recently that Smac β, a Smac variant lacking the NH2-terminal IAP binding domain, still potentiates apoptosis, suggesting that Smac may also possess proapoptotic activity independent of its IAP inhibition (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar). In this study, we investigate the kinetics and modulation of Smac release from mitochondria following death receptor-mediated apoptosis. Caspase-8 is the most apical caspase in TRAIL-induced apoptosis in MCF-7 cells (31MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar) and is recruited to the native TRAIL death-inducing signaling complex (DISC) in various cell types (32Sprick M.R. Weigand M.A. Reiser E. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 33Kischkel F. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 34Harper N. Farrow S.N. Kaptein A. Cohen G.M. MacFarlane M. J. Biol. Chem. 2001; 276: 34743-34752Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In MCF-7 cells, which are caspase-3 null (35Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1701) Google Scholar), TRAIL induces rapid Bid cleavage resulting in engagement of the mitochondrial amplification loop that is essential for apoptosis in certain cells (31MacFarlane M. Merrison W. Dinsdale D. Cohen G.M. J. Cell Biol. 2000; 148: 1239-1254Crossref PubMed Scopus (147) Google Scholar). MCF-7 cells therefore provide a good model in which to study death receptor-mediated, and hence apical caspase-induced, release of pro-apoptotic molecules from mitochondria. We now demonstrate that TRAIL induces a rapid release of mitochondrial Smac in MCF-7 cells, which is inhibited by Bcl-2. Once released, Smac is rapidly degraded by the proteasome, an effect promoted in vitro by the ubiquitin-protein ligase activity of XIAP. This study identifies Smac as a target for ubiquitination and further demonstrates the versatility of XIAP as a potent inhibitor of apoptosis. DISCUSSIONRecent studies have shown that Smac can act as a potent pro-apoptotic molecule, an effect primarily attributed to its propensity to bind to IAP family proteins (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar), although other mechanisms cannot be excluded (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar). However, to exert its pro-apoptotic effect Smac must be released from mitochondria, and until now, few studies have investigated the kinetics or modulation of Smac release in intact cells. We now demonstrate that death receptor-mediated apoptosis induces rapid and complete release of mitochondrial Smac, with kinetics similar to that observed for cytochrome c. As studies with Bid-deficient mice have confirmed that Bid is a critical substrate forin vivo signaling by death receptor agonists (42Yin X.-M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar), TRAIL-induced release of Smac and cytochrome c in MCF-7 cells is likely to be tBid-dependent. Our observation that z-VAD.FMK prevented the release of both Smac and cytochromec would support this, however, we cannot exclude the possibility that other apical caspase substrates may also play a role. Importantly, our results demonstrate that mitochondrial release of pro-apoptotic molecules is an apical caspase-dependent event in receptor-mediated apoptosis. This is in contrast to that recently reported for cell stress-associated mitochondrial Smac release, which is proposed to be a caspase-catalyzed event occurring downstream of cytochrome c release (43Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (354) Google Scholar).We also show that Smac release in MCF-7 cells is sensitive to inhibition by Bcl-2/Bcl-xL, with Bcl-2 exhibiting greater potency than Bcl-xL. The ability of Bcl-2 family members to inhibit the release of mitochondrial intermemebrane pro-apoptotic molecules, such as cytochrome c, is now well documented (3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Intriguingly, in some experiments, Bcl-2 appeared more potent in preventing Smac than cytochrome c release. Structural analysis revealed that Smac exists as a homodimer (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar), with an apparent molecular mass of ∼100 kDa (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar), whereas cytochrome c is known to have a monomeric molecular weight of only ∼12 kDa. If Bcl-2 were to act simply by blocking a tBid-induced pore, then one might predict that Smac release would be more readily inhibited than release of cytochromec. Thus, mitochondrial release of Smac and cytochromec may occur by different mechanisms, perhaps involving tBid and/or another as yet unidentified pore-forming molecule.Following its release from mitochondria, Smac was rapidly degraded by the proteasome, an effect we observed in both death receptor-mediated and chemical-induced apoptosis. Recent studies have revealed that XIAP can function as an E3 ligase and promotes the degradation of both itself and its substrates. Until now, the only XIAP substrates identified in vitro have been caspase-3, -7, and -9 (25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar). Thus, XIAP exhibits its anti-apoptotic effect by directly binding to and inhibiting active caspase-3, -7, and -9 (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar), but in some circumstances may additionally exert its effect through ubiquitination and degradation of active caspases (25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar). We now demonstrate that XIAP can also act as an E3 ligase for Smac and thus may promote the proteasome-mediated degradation of Smac in intact cells. Importantly, the BIR3-RING domain of XIAP was both necessary and sufficient to ubiquitinate Smac in vitro. Structural analysis has revealed that the primary binding of Smac to XIAP occurs via the NH2-terminal four residues of Smac and the BIR3 domain of XIAP, with significantly weaker binding observed between Smac and the BIR2 domain of XIAP (41Wu G. Chai J. Suber T.L., Wu, J.-W., Du, C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar). Smac is proposed to compete with, and thereby antagonize the “caspase inhibitory” effect of XIAP (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 29Srinivasula S.M. Datta P. Fan X.-J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 40Liu Z. Sun C. Olejnicak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J., Wu, J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar,41Wu G. Chai J. Suber T.L., Wu, J.-W., Du, C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar). Importantly, our data suggest that ubiquitination and degradation of Smac may represent another mechanism for modulating apoptosis. Degradation of small amounts of Smac released from mitochondria would provide a safeguard against inadvertent perturbation of mitochondria and subsequent caspase activation. It may be the case that cells exhibiting high levels of XIAP are protected from apoptosis in several ways: through the ability of XIAP to directly inhibit and degrade caspases and additionally through its ability to bind and target Smac for degradation. Recently, the serine protease Omi/HtrA2, which contains an NH2-terminal AVPS motif similar to that found in mature Smac, was shown to be released from mitochondria and inhibit XIAP by direct binding (44Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 45Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopolos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 46Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufman H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 47Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Thus Omi, in addition to being a Smac-like inhibitor of IAP activity, may be another pro-apoptotic molecule with the potential to be regulated by proteasomal degradation. Other E3 ligases, in addition to XIAP, may also target Smac or Omi for proteasomal-mediated degradation. Others have reported that c-IAP2 also possesses E3 ligase activity and can monoubiquitinate caspase-3 and -7in vitro. However, as mature Smac binds preferentially to XIAP, but not c-IAP1 or -2 in intact cells (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar), it is unlikely that c-IAP2 contributes greatly to the proteasomal-mediated degradation of Smac in vivo.Taken together, our results demonstrate that receptor-mediated Smac release from mitochondria occurs prior to or in parallel with the release of cytochrome c. Once released, Smac is rapidly degraded by the proteasome, an effect we propose from our in vitro data may be mediated in part via the E3 ligase activity of the BIR3-RING domain of XIAP. Based on our results and those of others, apoptosis can now be viewed as a process that has features in common with the control of other cellular processes by ubiquitination. Recent studies have shown that Smac can act as a potent pro-apoptotic molecule, an effect primarily attributed to its propensity to bind to IAP family proteins (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar, 27Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar), although other mechanisms cannot be excluded (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar). However, to exert its pro-apoptotic effect Smac must be released from mitochondria, and until now, few studies have investigated the kinetics or modulation of Smac release in intact cells. We now demonstrate that death receptor-mediated apoptosis induces rapid and complete release of mitochondrial Smac, with kinetics similar to that observed for cytochrome c. As studies with Bid-deficient mice have confirmed that Bid is a critical substrate forin vivo signaling by death receptor agonists (42Yin X.-M. Wang K. Gross A. Zhao Y. Zinkel S. Klocke B. Roth K.A. Korsmeyer S.J. Nature. 1999; 400: 886-891Crossref PubMed Scopus (860) Google Scholar), TRAIL-induced release of Smac and cytochrome c in MCF-7 cells is likely to be tBid-dependent. Our observation that z-VAD.FMK prevented the release of both Smac and cytochromec would support this, however, we cannot exclude the possibility that other apical caspase substrates may also play a role. Importantly, our results demonstrate that mitochondrial release of pro-apoptotic molecules is an apical caspase-dependent event in receptor-mediated apoptosis. This is in contrast to that recently reported for cell stress-associated mitochondrial Smac release, which is proposed to be a caspase-catalyzed event occurring downstream of cytochrome c release (43Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (354) Google Scholar). We also show that Smac release in MCF-7 cells is sensitive to inhibition by Bcl-2/Bcl-xL, with Bcl-2 exhibiting greater potency than Bcl-xL. The ability of Bcl-2 family members to inhibit the release of mitochondrial intermemebrane pro-apoptotic molecules, such as cytochrome c, is now well documented (3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Intriguingly, in some experiments, Bcl-2 appeared more potent in preventing Smac than cytochrome c release. Structural analysis revealed that Smac exists as a homodimer (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar), with an apparent molecular mass of ∼100 kDa (26Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2886) Google Scholar), whereas cytochrome c is known to have a monomeric molecular weight of only ∼12 kDa. If Bcl-2 were to act simply by blocking a tBid-induced pore, then one might predict that Smac release would be more readily inhibited than release of cytochromec. Thus, mitochondrial release of Smac and cytochromec may occur by different mechanisms, perhaps involving tBid and/or another as yet unidentified pore-forming molecule. Following its release from mitochondria, Smac was rapidly degraded by the proteasome, an effect we observed in both death receptor-mediated and chemical-induced apoptosis. Recent studies have revealed that XIAP can function as an E3 ligase and promotes the degradation of both itself and its substrates. Until now, the only XIAP substrates identified in vitro have been caspase-3, -7, and -9 (25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar). Thus, XIAP exhibits its anti-apoptotic effect by directly binding to and inhibiting active caspase-3, -7, and -9 (14Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2262) Google Scholar), but in some circumstances may additionally exert its effect through ubiquitination and degradation of active caspases (25Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar). We now demonstrate that XIAP can also act as an E3 ligase for Smac and thus may promote the proteasome-mediated degradation of Smac in intact cells. Importantly, the BIR3-RING domain of XIAP was both necessary and sufficient to ubiquitinate Smac in vitro. Structural analysis has revealed that the primary binding of Smac to XIAP occurs via the NH2-terminal four residues of Smac and the BIR3 domain of XIAP, with significantly weaker binding observed between Smac and the BIR2 domain of XIAP (41Wu G. Chai J. Suber T.L., Wu, J.-W., Du, C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar). Smac is proposed to compete with, and thereby antagonize the “caspase inhibitory” effect of XIAP (28Chai J., Du, C., Wu, J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 29Srinivasula S.M. Datta P. Fan X.-J. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 40Liu Z. Sun C. Olejnicak E.T. Meadows R.P. Betz S.F. Oost T. Herrmann J., Wu, J.C. Fesik S.W. Nature. 2000; 408: 1004-1008Crossref PubMed Scopus (540) Google Scholar,41Wu G. Chai J. Suber T.L., Wu, J.-W., Du, C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (702) Google Scholar). Importantly, our data suggest that ubiquitination and degradation of Smac may represent another mechanism for modulating apoptosis. Degradation of small amounts of Smac released from mitochondria would provide a safeguard against inadvertent perturbation of mitochondria and subsequent caspase activation. It may be the case that cells exhibiting high levels of XIAP are protected from apoptosis in several ways: through the ability of XIAP to directly inhibit and degrade caspases and additionally through its ability to bind and target Smac for degradation. Recently, the serine protease Omi/HtrA2, which contains an NH2-terminal AVPS motif similar to that found in mature Smac, was shown to be released from mitochondria and inhibit XIAP by direct binding (44Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 45Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopolos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 46Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufman H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 47Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Thus Omi, in addition to being a Smac-like inhibitor of IAP activity, may be another pro-apoptotic molecule with the potential to be regulated by proteasomal degradation. Other E3 ligases, in addition to XIAP, may also target Smac or Omi for proteasomal-mediated degradation. Others have reported that c-IAP2 also possesses E3 ligase activity and can monoubiquitinate caspase-3 and -7in vitro. However, as mature Smac binds preferentially to XIAP, but not c-IAP1 or -2 in intact cells (30Roberts D.L. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-227Crossref PubMed Scopus (92) Google Scholar), it is unlikely that c-IAP2 contributes greatly to the proteasomal-mediated degradation of Smac in vivo. Taken together, our results demonstrate that receptor-mediated Smac release from mitochondria occurs prior to or in parallel with the release of cytochrome c. Once released, Smac is rapidly degraded by the proteasome, an effect we propose from our in vitro data may be mediated in part via the E3 ligase activity of the BIR3-RING domain of XIAP. Based on our results and those of others, apoptosis can now be viewed as a process that has features in common with the control of other cellular processes by ubiquitination. We thank Dr. E. S. Alnemri for pGEX4T-XIAP cDNA; Dr. M. Butterworth for generating GST-XIAP recombinant proteins; Dr. M. Jaattela for MCF-7-Fas, MCF-7-Bcl-2, and MCF-7-Bcl-xL cells; Dr. D. L. Roberts for generating recombinant Smac β protein; and Dr. X. Wang for SmacΔ55 pet15b cDNA.