Cellular Inhibitor of Apoptosis 1 and 2 Are Ubiquitin Ligases for the Apoptosis Inducer Smac/DIABLO

Abstract

Inhibitors of apoptosis (IAPs) are crucial regulators of programmed cell death. The mechanism by which IAPs prevent apoptosis has previously been attributed to the direct inhibition of caspases. The function of mammalian IAPs is counteracted by cell death inducer second mitochondria-derived activator of caspases (Smac)/DIABLO during apoptosis. Here we show that cIAP1 and cIAP2 are E3 ubiquitin-protein isopeptide ligases (ubiquitin ligases) for Smac. cIAPs stimulate Smac ubiquitination both in vivo andin vitro, leading to Smac degradation. cIAP1 and cIAP2 associate with overlapping but distinct subsets of E2 (ubiquitin carrier protein) ubiquitin-conjugating enzymes. The substrate-dependent E3 activity of cIAPs is mediated by their RING domains and is dependent on the specific interactions between cIAPs and Smac. Similarly, Drosophila IAP1 also possesses ubiquitin ligase activity that mediates the degradation of the Drosophila apoptosis inducers Grim and HID. These results suggest a novel and conserved mechanism by which IAPs block apoptosis through the degradation of death inducers. Inhibitors of apoptosis (IAPs) are crucial regulators of programmed cell death. The mechanism by which IAPs prevent apoptosis has previously been attributed to the direct inhibition of caspases. The function of mammalian IAPs is counteracted by cell death inducer second mitochondria-derived activator of caspases (Smac)/DIABLO during apoptosis. Here we show that cIAP1 and cIAP2 are E3 ubiquitin-protein isopeptide ligases (ubiquitin ligases) for Smac. cIAPs stimulate Smac ubiquitination both in vivo andin vitro, leading to Smac degradation. cIAP1 and cIAP2 associate with overlapping but distinct subsets of E2 (ubiquitin carrier protein) ubiquitin-conjugating enzymes. The substrate-dependent E3 activity of cIAPs is mediated by their RING domains and is dependent on the specific interactions between cIAPs and Smac. Similarly, Drosophila IAP1 also possesses ubiquitin ligase activity that mediates the degradation of the Drosophila apoptosis inducers Grim and HID. These results suggest a novel and conserved mechanism by which IAPs block apoptosis through the degradation of death inducers. second mitochondria-derived activator of caspases inhibitor of apoptosis X-linked inhibitor of apoptosis cellular inhibitor of apoptosis baculoviral IAP repeat hemagglutinin ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase Apoptosis is an evolutionarily conserved process of cell autodestruction that plays a critical role in development, the maintenance of tissue homeostasis, and the protection against diseases (1Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar, 2Green D.R. Evan G.I. Cancer Cell. 2002; 1: 19-30Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases (3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar, 4Chang H.Y. Yang X. Microbiol. Mol. Biol. Rev. 2000; 64: 821-846Crossref PubMed Scopus (579) Google Scholar). A major mammalian apoptosis pathway leading to caspase activation is the intrinsic/mitochondrial pathway, which is induced by diverse intracellular death stimuli such as developmental cues, cellular stresses, severe DNA damages, and oncogenic transformation (5Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar, 6Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). These stimuli lead to the release of several cell death inducers from the mitochondria, including cytochrome c and Smac (also known as DIABLO). Cytochrome c promotes the formation of a cytosolic protein complex that activates an initiator caspase, caspase-9 (7Li 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 (6239) Google Scholar). Smac,1 on the other hand, promotes caspase-9 activation by neutralizing the inhibitory effect on caspases of the X-linkedinhibitor of apoptosis (XIAP) (8Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, 9Verhagen 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 (1972) Google Scholar). The IAPs comprise a group of conserved proteins crucial for the regulation of apoptosis (reviewed in Ref. 10Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2281) Google Scholar). Initially identified as baculoviral proteins that can functionally substitute the baculovirus-encoded caspase inhibitor p35, the IAP family now includes several mammalian cellular proteins (e.g. XIAP/MIHA/h-ILP, cIAP1/HIAP2/hMIHB, cIAP2/HIAP-1/hMIHC, NAIP, ML-IAP, and survivin) and two Drosophila proteins (DIAP1 and DIAP2/dILP). Structurally, all IAPs contain one to three baculovirus IAP repeats (BIRs). IAPs can directly inhibit caspases through their BIR domains and intervening linker regions. For example, XIAP has been shown to inhibit caspases-3, -7, and -9 through the BIR region (11Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1719) Google Scholar, 12Deveraux Q.L. Roy N. Stennicke H.R. Van Arsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1242) Google Scholar), whereas the corresponding region of DIAP1 inhibits Drosophilacaspases, including DRICE and DCP-1 (13Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 14Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (142) Google Scholar). Smac counteracts the inhibitory effect of XIAP by masking the caspase-9-binding site on XIAP (8Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, 9Verhagen 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 (1972) Google Scholar, 15Srinivasula 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 (315) Google Scholar, 16Srinivasula S.M. Hegde 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 (860) Google Scholar). The IAP binding motif of Smac was subsequently found to be conserved in three Drosophila death inducers, HID, Grim, and Reaper (17Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (710) Google Scholar), that are required for Drosophila cell death (reviewed in Ref. 18Bergmann A. Agapite J. Steller H. Oncogene. 1998; 17: 3215-3223Crossref PubMed Scopus (109) Google Scholar). These three proteins induce apoptosis through the inhibition of DIAP1 (13Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 19Goyal L. McCall K. Agapite J. Hartwieg E. Steller H. EMBO J. 2000; 19: 589-597Crossref PubMed Scopus (380) Google Scholar), likely via a mechanism similar to that of Smac (17Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (710) Google Scholar). In addition to XIAP, Smac also interacts with cIAP1 and cIAP2 (8Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, 9Verhagen 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 (1972) Google Scholar). Unlike XIAP, the inhibitory effect of the cIAPs on caspases is weak (100–1,000-fold less than that of XIAP) (20Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar). The mechanism by which cIAP1 and cIAP2 regulate apoptosis remains less understood. Many IAPs, including XIAP, cIAP1, cIAP2, and DIAP1, also contain a COOH-terminal RING domain. The RING domains on XIAP and cIAP1, like those found in many other proteins (21Lorick 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 (944) Google Scholar), have been shown to possess E3 ubiquitin ligase activity (22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (869) Google Scholar, 23Huang H. Joazeiro C.A. Bonfoco E. Kamada S. Leverson J.D. Hunter T. J. Biol. Chem. 2000; 275: 26661-26664Abstract Full Text Full Text PDF PubMed Google Scholar). Protein polyubiquitination, which typically targets proteins for degradation in the 26 S proteasome, involves the sequential action of three enzymes: the ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (Ubc or E2), and a ubiquitin ligase (E3) (24Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 25Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar). The substrate specificity is mainly determined by E3, which binds to both E2 and the substrate and facilitates the assembly of a multiubiquitin chain on the substrate. The E3 activity of XIAP and cIAP1 can mediate their self-degradation during the apoptosis of thymocytes in response to certain apoptosis stimuli. cIAP1 also regulates the level of TRAF2, an intracellular signaling molecule for the tumor necrosis factor receptor 2. The degradation of TRAF2 by cIAP1 appears to sensitize cells to TNF-mediated apoptosis (26Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-349Crossref PubMed Scopus (394) Google Scholar). On the other hand, the RING domain of IAPs has been shown to inhibit apoptosis in a cell type- and/or death stimulus-dependent manner (27Harvey A.J. Soliman H. Kaiser W.J. Miller L.K. Cell Death Differ. 1997; 4: 733-744Crossref PubMed Scopus (44) Google Scholar, 28Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (495) Google Scholar). The cIAP1 RING domain can mediate the monoubiquitination of caspases-3 and -7 (23Huang H. Joazeiro C.A. Bonfoco E. Kamada S. Leverson J.D. Hunter T. J. Biol. Chem. 2000; 275: 26661-26664Abstract Full Text Full Text PDF PubMed Google Scholar), and the XIAP RING domain promotes caspase-3 degradation through polyubiquitination (29Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (546) Google Scholar). However, it remains unclear whether the E3 activity of IAPs targets proapoptotic proteins other than caspases. Here we present evidence that cIAP1 and cIAP2 are E3 ubiquitin ligases for Smac and promote the degradation of this critical death inducer. The substrate-dependent E3 activity of cIAPs requires an intact RING finger domain. We also identify putative ubiquitin-conjugating enzymes (E2) for cIAP1 and -2. In addition, DIAP1 is able to ubiquitinate Grim and HID, two Drosophilafunctional homologues of Smac. These results show that cIAPs and DIAP1 can also inhibit apoptosis through the degradation of death inducers. Human embryonic kidney 293T and cervix carcinoma HeLa cells were cultured in complete Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Rabbit polyclonal antibodies against FLAG and hemagglutinin (HA) tags (Santa Cruz Biotechnology), anti-FLAG monoclonal antibody M2 and M2 conjugated on agarose beads (Sigma), and yeast ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) GST-UbcH5c, and His6-ubiquitin (Boston Biochem) were purchased from the indicated sources. All mammalian expression constructs were made in pRK5. The cDNAs for cIAP1, cIAP2, XIAP, caspase-9, and all E2s were of human origin. The cDNA fragments for the full-length E2s were obtained from a lymph node or a leukocyte cDNA library (Clontech) by PCR. All E2s were tagged with an HA at the NH2 terminus, and the position of the tags for other constructs is indicated in the figures and/or text. The amino acid residues present in the following deletion mutant constructs are indicated in parentheses: cIAP1mut(1–587), cIAP2mut(1–573), cIAP1C(452–618), cIAP2C(441–604), cIAP2ΔBIR1(101–587), and DIAP1mut(1–381). Transient transfection was performed using the calcium phosphate precipitation method. Unless indicated otherwise, cells were transfected with 0.1–10 μg of the indicated plasmids to minimize the difference in the expression levels of various proteins. Sixteen to 22 h after transfection, cell lysates were prepared in 1% Nonidet P-40 lysis buffer (50 mm HEPES, 150 mm NaCl, 10% glycerol, 2 mm dithiothreitol, 1% Nonidet P-40, 1 mm EDTA) and immunoprecipitated with anti-FLAG monoclonal antibody M2 beads. The immunoprecipitates and cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Mature Smac protein was obtained by the tnt-coupled transcription/translation system (Promega) according to the manufacturer's instructions. FLAG-tagged cIAP1, XIAP, and cIAP2mut (which lacked the first BIR domain) were expressed in 293T cells and immunoprecipitated from the cell lysates with the anti-FLAG M2 monoclonal antibody conjugated on agarose beads. For the experiments shown in A the immunoprecipitates were with buffer mm mm NaCl, Nonidet P-40, 10% glycerol, 1 mm EDTA) and to a ubiquitination of yeast μg of μg of and of Smac in at for with the was by in the of the was resolved by SDS-PAGE by For the in in Smac was first with the FLAG beads at for to Smac to to the the ubiquitination the beads were three with 1% Nonidet P-40 lysis resolved on and analyzed by HeLa cells were transfected with μg of with μg of a and 1 μg of DIAP1, or h after transfection, cells were in and with The of cells was determined by the of cells by the of as previously S. Yang X. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The cell death inducer Smac is as a which the with the of the (8Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar). Smac is the it with whether IAPs can Smac for we in 293T cells the Smac protein with cIAP1, cIAP2, or the of Smac protein with cIAP1 or cIAP2 but with XIAP The of the Smac protein was to the inhibition of apoptosis XIAP apoptosis In addition, the expression levels of XIAP were than those of cIAP1 and cIAP2 1 the in Smac expression was by the effect of the of proteins. the that of cIAPs to the inhibition of protein we the effect of cIAPs on Smac and found that expression level was by 1 The of Smac and the expression levels of cIAPs suggest that cIAP1 and cIAP2 Smac as as for degradation via the of the cIAPs with XIAP, we expressed FLAG-tagged cIAP1, cIAP2, and XIAP in 293T cells with cIAP1 and cIAP2 were modified with as indicated by the of In the of XIAP was with expression levels whether cIAPs and XIAP can ubiquitinate we expressed cIAPs and XIAP with Smac in 293T The Smac protein that to cIAP1 and cIAP2 was a of and polyubiquitination 2 The Smac ubiquitination was that the of Smac proteins were modified the addition of The and Smac ubiquitination were by immunoblotting using an antibody against ubiquitin 2 XIAP, induce ubiquitination of Smac The RING domains of cIAPs are required for their (22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (869) Google Scholar). whether domains are also for the ubiquitination of we the RING domains of cIAP1 and shown in 2 RING deletion to ubiquitinate Smac. ubiquitination of Smac by IAPs is RING whether other cellular are in the Smac we expressed cIAPs and in HeLa a we also expressed in 293T We found that the binding of Smac to cIAPs in two cells was However, ubiquitination of Smac was in HeLa cells whereas the Smac ubiquitination was in 293T cells two the that the ubiquitination of Smac is by other cellular The interactions between IAPs and Smac are mediated by the IAP BIR domains and the Smac a acid the specificity of ubiquitination of we a mutant cIAP1 all the BIR domains and a mutant Smac with the motif The BIR deletion mutant of cIAP1 to to and Smac this region E3 activity (22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (869) Google Scholar). In addition, the deletion mutant of Smac with mutant was by cIAP1 and in the of cIAP1 with the Smac Smac ubiquitination and degradation requires specific interactions between cIAPs and Smac. A or at a In it a or which E2s can associate with cIAP1, cIAP2, and XIAP, IAP was expressed in 293T cells with the E2s that have been previously found to associate with various RING domain The COOH-terminal region of cIAP2 was in experiments it that cIAP1 with and as as with and whereas cIAP2 with all family E2s and with but with and XIAP, on the other hand, with all E2s but with a weak binding to and is for an E3 to to For example, a E3 that is with interacts with several E2s Y. M. H. N. Y. Takahashi R. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). E2 in vivo can also be determined by the of other proteins and the of XIAP binds to several the weak ligase activity of XIAP Smac was by a E2 In addition, the cIAP2 COOH-terminal region that the RING domain was for the binding the BIR domains are for E2 identified putative E2s for cIAP1, cIAP2, and XIAP, we the ubiquitination of Smac by IAPs using ubiquitination FLAG-tagged full-length cIAP1 and XIAP and a deletion mutant of cIAP2 the first BIR domain were expressed in mammalian cells and with an anti-FLAG The mutant cIAP2 was it was expressed at a level than the proteins the to to Smac the cIAP1 or protein was to a ubiquitination and E2 and in ubiquitination of Smac was as shown by the of A The ubiquitination was ubiquitin was also in the In XIAP Smac the of XIAP was than that with cIAP1 These results that both cIAP1 and cIAP2 are ubiquitin ligases but suggest that XIAP is a weak ubiquitin ligase for Smac. Drosophila IAP1 plays a critical role in apoptosis with DIAP1 in the embryonic of cell death (13Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). activation of the Drosophila death inducers Grim, HID, and Reaper to and the inhibitory effect of DIAP1 on via an motif that is with other and with the mammalian Smac (13Wang S.L. Hawkins C.J. Yoo S.J. Muller H.A. Hay B.A. Cell. 1999; 98: 453-463Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (710) Google L. McCall K. Agapite J. Hartwieg E. Steller H. EMBO J. 2000; 19: 589-597Crossref PubMed Scopus (380) Google Scholar). that Grim, and Reaper promote the and degradation of DIAP1 R. L. M. A. Agapite J. Steller H. P. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, S. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, S.J. I. H. Wang L. Wang S.L. R.J. Muller H.A. Hay B.A. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, A. H. A. Steller H. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). whether DIAP1, like can mediate the ubiquitination of the death inducers, we the effect of DIAP1 on Grim and expression of DIAP1 in 293T cells in with the of a of of DIAP1 on an intact RING domain A and 2 DIAP1 also the Grim and A and and The expression levels of Grim and were than the expression of DIAP1 and with a that the BIR domains of DIAP1 the of likely through a direct D. Kaiser W.J. Miller L.K. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). However, the levels of Grim and were in the of the RING deletion mutant of DIAP1, which the to ubiquitinate and A that the ubiquitination to degradation of Grim, we the in the Grim The Grim mutant be by DIAP1, and the level of Grim protein was with the protein results show that the E3 activity of DIAP1 can degradation of both Grim and HID. the of the IAP E3 activity in apoptosis an was made to the apoptosis inhibition of the and the of cIAPs apoptosis in 293T an function of cIAPs has been previously A.G. Pakusch M. Hawkins C.J. K. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. Roy N. K. C. S. R. M. A. Nature. PubMed Scopus Google Scholar), of cIAPs in cells has also been to the RING domain was (26Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-349Crossref PubMed Scopus (394) Google Scholar, R.J. N.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). proapoptotic activity of cIAPs be of the of cIAP1 such as (26Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-349Crossref PubMed Scopus (394) Google Scholar). The E3 activity of plays a complex role in the regulation of apoptosis. of DIAP1, on the other hand, was in 293T or HeLa DIAP1 apoptosis in cells in a transfection apoptosis and In the E3 RING mutant of DIAP1 was less in apoptosis the DIAP1 E3 activity to apoptosis inhibitory The IAPs family is a major group of apoptosis IAPs have been shown to directly block caspase In this we found that cIAP1 and cIAP2 can also the cell death inducer Smac for degradation via the COOH-terminal RING Similarly, Drosophila DIAP1 possesses E3 activity that can mediate the ubiquitination and degradation of death inducers Grim and HID. These results suggest a novel and evolutionarily conserved mechanism for the function of the three mammalian IAPs that with cIAP1 and cIAP2 have E3 ligase activity Smac. In the substrate-dependent ligase activity of XIAP is it can to Smac. A that Smac is release from the during death apoptosis and that the Smac degradation is dependent on the pathway M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The also ubiquitination of Smac in However, by directly cIAPs and XIAP both in vivo and in vitro, we are able to show the difference in their E2 Smac. XIAP has been shown to be a inhibitor of caspases than cIAPs (20Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1137) Google Scholar). it is likely that cIAPs and XIAP inhibit apoptosis through XIAP directly to and inhibit cIAP1 and cIAP2 death inducers such as Smac to prevent it from the inhibitory effect of XIAP on caspases. has been shown that IAPs to Smac through their BIR We have found in this that cIAPs can also with several cIAPs function as molecule that with both the substrate and E2 and interactions be by other cellular as by the of ubiquitination in HeLa In in the ubiquitin ligase which plays an role in cell the E3 with the substrate through several proteins and the substrate specificity is determined mainly by the protein (24Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 25Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar). that both Smac and with the BIR region of IAPs, this region the substrate specificity of the IAPs, and it be to identify IAP that associate with this The inhibition of IAPs by the death inducer and the ubiquitination of death inducers by IAPs that are interactions between two of and proapoptotic proteins. These like those between the and proapoptotic of the family the and of We Wang for for and for