c-IAP1 Is Cleaved by Caspases to Produce a Proapoptotic C-terminal Fragment

Abstract

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor α treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment. Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor α treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment. caspase recruitment domain baby hamster kidney Chinese hamster ovary N-acetyl-Asp-Glu-Val-aspartinal hemagglutinin polyacrylamide gel electrophoresis tumor necrosis factor α N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone glutathione S-tranferase The IAP (inhibitor of apoptosis) proteins Op-IAP and Cp-IAP were originally identified in baculoviruses because they could functionally replace the baculovirus-caspase inhibitor P53 (1Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar, 2Clem R.J. Duckett C.S. Trends Cell Biol. 1997; 7: 337-339Abstract Full Text PDF PubMed Scopus (89) Google Scholar). The closest homologues to the baculovirus IAPs are theDrosophila IAPs, D-IAP1 and D-IAP2, and the human IAPs, c-IAP1 (MIHB), c-IAP2 (MIHC), and XIAP (hILP, MIHA) (3Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2255) Google Scholar, 4Miller L.K. Trends Cell Biol. 1999; 9: 323-328Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). These molecules all share two or three copies of the BIR (baculovirus IAP repeat) motif at their N termini and a RING finger at their C termini and are direct inhibitors of caspases, a family of death-inducing proteases (5Huang Q. Deveraux Q.L. Maeda S. Salvsen G.S. Stennicke H.R. Hammock B.D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1427-1432Crossref PubMed Scopus (112) Google Scholar, 6Kaiser W.J. Vucic D. Miller L.K. FEBS Lett. 1998; 440: 243-248Crossref PubMed Scopus (100) Google Scholar, 7Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1129) Google Scholar, 8Deveraux Q.L. Roy N. Stennicke H.R. Van, A.rsdale T. Zhou Q. Srinivasula S.M. Alnemri E.S. Salvesen G.S. Reed J.C. EMBO J. 1998; 17: 2215-2223Crossref PubMed Scopus (1232) Google Scholar, 9Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1707) Google Scholar, 10Takahashi R. Deveraux Q. Tamm I. Welsh K. Assamunt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). All of these IAP proteins have been shown to inhibit apoptosis in one or more paradigms. D-IAP1 was consistently retrieved in a genetic screen for caspase inhibitors (11Hawkins C.J. Wang S.L. Hay B.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2885-2890Crossref PubMed Scopus (140) Google Scholar), and both D-IAP1 and D-IAP2 overexpression inhibits apoptosis in the Drosophila retina (12Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (639) Google Scholar). Human c-IAP1 and c-IAP2 are less potent inhibitors of apoptosis compared with XIAP, correlating with the observation that XIAP is a more potent caspase inhibitor (∼100-fold) than c-IAP1 or c-IAP2in vitro (3Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2255) Google Scholar). Both biochemical and structural data support a model where the individual BIR motifs of XIAP are specific inhibitors of different caspases. That is, the second BIR motif (BIR-2) and adjacent sequences of XIAP interact directly with activated caspase-3 and are sufficient to inhibit caspase-3 (13Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 812-818Crossref Scopus (294) Google Scholar, 14Deveraux, Q., Leo, E., Stennicke, H., Welsh, K., Salvesen, G., and Reed, J. (1999) EMBO J. 5242–5251.Google Scholar). In addition, BIR-3 of XIAP binds to and inhibits caspase-9. However, NMR structure analysis predicts that the mechanism of caspase inhibition by BIR-3 will be distinct from that of BIR-2 (15Sun C. Cai M. Meadows R.P. Xu N. Gunasekera A.H. Herrmann J. Wu J.C. Fesik S.W. J. Biol. Chem. 2000; 275: 33777-33781Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar), supporting a model where the individual BIR repeats have independent and nonredundant functions (14Deveraux, Q., Leo, E., Stennicke, H., Welsh, K., Salvesen, G., and Reed, J. (1999) EMBO J. 5242–5251.Google Scholar). The role of the C-terminal RING finger seems to be cell type- and/or death stimulus-dependent. In some situations the RING is required for antiapoptotic activity, but in others the RING inhibits the antiapoptotic function of IAP proteins. Although the RING finger of Op-IAP is required for protection of insect cells from apoptosis induced by actinomycin D treatment or baculovirus infection, this domain is not required for inhibition of Hid-induced apoptosis in the same insect cell line (16Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (492) Google Scholar, 17Harvey A.J. Soliman H. Kaiser W.J. Miller L.K. Cell Death Differ. 1997; 4: 733-744Crossref PubMed Scopus (43) Google Scholar, 18Hawkins C.J. Uren A.G. Hacker G. Medcalf R.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13786-13790Crossref PubMed Scopus (96) Google Scholar). Partial inhibition of cell death induced by HID as well as Reaper and Grim is conferred by BIR-2 of Op-IAP (most equivalent to BIR-3 of c-IAP1) plus a critical carboxyl-proximal flanking sequence. However, BIR-2 plus the RING finger is a more potent protector than BIR-2 (with proximal sequences) alone, implying that the RING finger contributes to the antiapoptotic function of Op-IAP (19Vucic D. Kaiser W.J. Miller L.K. J. Biol. Chem. 1998; 273: 33915-33921Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The RING fingers of a number of proteins including c-IAP1 and XIAP were recently shown to function as E3 ubiquitin ligases in proteosome-dependent protein degradation (20Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (859) Google Scholar). This activity may predominantly facilitate self-destruction as shown for c-IAP1 and XIAP, resulting in cell death. Alternatively, the antiapoptotic function of IAP proteins may be explained by ubiquitination and degradation of caspases as suggested for the RING finger of c-IAP2 (21Huang 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). The RING finger domains of XIAP, D-IAP1, and D-IAP2 also were shown to interact with signaling factors for the bone morphogenetic protein kinase receptors and may participate in these signal transduction pathways as well (22Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (323) Google Scholar). Earlier work in mammalian cells had suggested that the RING finger of full-length c-IAP1 does not interfere with its antiapoptotic activity in mammalian cells. That is, in a variety of stably or transiently transfected cells, c-IAP1 and c-IAP2 were reported to inhibit apoptosis induced by different death stimuli including serum withdrawal, menadione, staurosporine, caspase-1, Bak, or K+depolarization (23Liston P. Roy N. Tamai K. Lefebvre C. Baird S. Cherton-Horvat G. Farahani R. McLean M. Ikeda J.-E. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (867) Google Scholar, 24Uren A.G. Pakusch M. Hawkins C.J. Puls K.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4974-4978Crossref PubMed Scopus (445) Google Scholar, 25Orth K. Dixit V.M. J. Biol. Chem. 1997; 272: 8841-8844Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26Simons M. Beinroth S. Gleichmann M. Liston P. Korneluk R.G. MacKenzie A.E. Bahr M. Klockgether T. Robertson G.S. Weller M. Schulz J.B. J. Neurochem. 1999; 72: 292-301Crossref PubMed Scopus (103) Google Scholar). In contrast to these reports using mammalian cells, full-length human c-IAP1 failed to inhibit Reaper-induced cell death in a Drosophila eye model unless the RING finger was deleted (12Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (639) Google Scholar). Thus, in insect cells, the RING finger appears to negatively regulate the antiapoptotic function of human c-IAP1. Consistent with this finding, of the spacer-RING region of D-IAP1 in in a eye by cell death in the eye Furthermore, the RING finger of baculovirus Cp-IAP and Drosophila D-IAP1 apoptosis in the cell line A.J. Soliman H. Kaiser W.J. Miller L.K. Cell Death Differ. 1997; 4: 733-744Crossref PubMed Scopus (43) Google Scholar). found that the Drosophila c-IAP1 and c-IAP2 failed to protect mammalian cells unless the RING finger domain was Furthermore, c-IAP1 was cleaved during apoptosis to the C-terminal spacer-RING domain that was of cells in The human IAPs were identified in an data by with a for to baculovirus were as to full-length from the that the of These were to c-IAP1 and C-terminal were by of in all three at the in The deletion an fragment. The RING was by recombinant and to at a at IAPs and were the of the Sindbis virus and recombinant were as reported Nature. 1996; 379: PubMed Scopus Google Scholar, 2000; PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). C-terminal c-IAP1 fragments and fragments of c-IAP1 were by and from a for in transfected cells. All were by and cells were at or in a or and the with recombinant Sindbis at a of Cell was at by was shown to the apoptotic death induced by Sindbis virus Nature. 1996; 379: PubMed Scopus Google Scholar, Q. Reed J.C. Nature. 1993; PubMed Scopus Google Scholar). of of of and of cells were and with unless were within Cell was by and as in the cells by were with of protein and of the of using of was and cell was by cells apoptotic to the number of cells. to of c-IAP1 was purified from cleaved from with and to to Cell were with and a of inhibitors at with recombinant or and by analysis was with or c-IAP1 protein and were produced by in of was with of cell as J. Zhang K. S.W. Genes Dev. 1997; PubMed Scopus Google and at for in the or of caspase inhibitor or with recombinant caspase-3 was as R.J. Ueno K. Takahashi A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). proteins were purified using and against to of by protein were to and the at for was compared with a of to the of to protein in To the antiapoptotic of IAP family human c-IAP1 and c-IAP2 were the Sindbis virus and for their to inhibit Sindbis cell death. Sindbis virus apoptotic death in cell types, a analysis of the function of a variety of cell death Q. Reed J.C. Nature. 1993; PubMed Scopus Google Scholar). and baculovirus were reported to protect cells in this C.S. Clem R.J. Van, H. EMBO J. 1996; PubMed Scopus Google Scholar), and both the and antiapoptotic functions of several family have been in this Nature. 1996; 379: PubMed Scopus Google Scholar). and cells were with recombinant human or XIAP, and cell was by Although and XIAP cells from cell both c-IAP1 and c-IAP2 failed to protect cells compared with Sindbis virus the same in and is that Sindbis virus a cell death that is to c-IAP1. In an c-IAP1 was for the to inhibit apoptosis induced by or in transiently transfected cells. Although cells from apoptotic death induced by Bax or Bak, c-IAP1 failed to protect in contrast to work in the same To the RING finger could interfere with antiapoptotic activity, were or XIAP sequences within the Sindbis virus to proteins the C-terminal RING finger or both the spacer region and RING cells were with recombinant or IAPs, and cell was by the spacer-RING or RING domains of XIAP had antiapoptotic This with apoptosis is consistent with the observation by others that the RING the spacer region of XIAP is required for inhibition of cell death in cells C.S. Wang Y. Mol. Cell Biol. 1998; 18: PubMed Scopus Google and that the BIR domain of XIAP is sufficient to inhibit caspases R. Deveraux Q. Tamm I. Welsh K. Assamunt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). However, the C-terminal of c-IAP1 conferred a of antiapoptotic activity, indicating that the BIR domain was sufficient to inhibit cell death in this and that the antiapoptotic function of the BIR domain was suppressed by the RING domain were observed in cells not The RING deletion was also at to its activity but of the BIR domain alone was and to Thus, in protein may not for the by C-terminal in cells that deletion of the C-terminal RING finger of c-IAP1 the antiapoptotic function of the BIR domain against Sindbis virus In contrast to deletion of the C-terminal spacer-RING of c-IAP2 failed to antiapoptotic activity c-IAP2 in the Sindbis virus Both full-length and C-terminal c-IAP2 proteins were consistently to by analysis in Sindbis cells, and of c-IAP2 in this model were However, c-IAP2 the spacer-RING region was of apoptosis induced by treatment of cells, full-length c-IAP2 was in this of the C-terminal spacer-RING domain of c-IAP2 c-IAP2 in transfected cells, of c-IAP1 failed to protect the RING domains of c-IAP1 and c-IAP2 but not XIAP were of the antiapoptotic functions of their BIR domains in the may in be to protein To the of c-IAP1 protein during from cells with recombinant or c-IAP1 were with a against recombinant c-IAP1. The c-IAP1 from recombinant Sindbis virus with c-IAP1 c-IAP1 with the in BIR and in increased c-IAP1 protein In to full-length an of and a less 35-kDa in were with virus full-length that c-IAP1 may be cleaved during apoptosis. were in cells not The BIR domain alone was also the of c-IAP1 in cells, but the less 35-kDa was and of c-IAP1 were to of c-IAP1 is in these cleavage fragments. of of the motif within the spacer region produced a c-IAP1 protein that was as in This deletion also the to in and the 35-kDa to In the cleavage fragments increased in with both fragments to be cleavage of and both contain the Both fragments also contain the C-terminal RING finger. of the RING the of the cleavage deletion of had the of the cleavage fragment. of the RING of the 35-kDa by the This is consistent with in the spacer region alone these data that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments that are both from the C The 35-kDa is to of the spacer and RING domains because the of this region is and of an spacer-RING at The BIR-2 the C The N-terminal fragments of c-IAP1 could not be identified the cleavage fragments and may be To the 52- and 35-kDa fragments of c-IAP1 are by caspase in vitro c-IAP1 was with cell extracts that contain activated caspases J. Zhang K. S.W. Genes Dev. 1997; PubMed Scopus Google Scholar, A. R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D.A. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of c-IAP1 by the apoptotic cell produced a 35-kDa the same as that observed in apoptotic cells. This cleavage was by a caspase inhibitor and a caspase-3 inhibitor but was not by the inhibitor not c-IAP1 was also cleaved to a 35-kDa by purified recombinant caspase-3 that was by The cleavage observed in cells was not in this in vitro unless the domain was stabilizing the cleavage Consistent with in apoptotic cells, cell extracts cleaved the in to the and fragments and data not This that vitro cleavage fragments also contain the C-terminal region of c-IAP1. Consistent with in apoptotic cells the of c-IAP1 was more in vitro compared with full-length c-IAP1 of the of c-IAP1 was as as of and of the protein that was cleaved by To the cleavage for the several the of the spacer region were in the of c-IAP1. of to of the cleavage of and had of this D and data not of also of the fragment. of may cleavage at a second but we the that the is a of the fragment. the RING finger the antiapoptotic activity of full-length c-IAP1 and because a C-terminal of c-IAP1 the RING finger is by caspases in in apoptotic cells, we the that the C of c-IAP1 proapoptotic of cells with a the spacer-RING region of c-IAP1 induced cell death in a were with a not To of the spacer-RING was for the of several were in transfected cells. of the domain from the spacer-RING proapoptotic activity and with protein that the domain contributes to the of the spacer-RING region were with in and cells D and and that the of was a more potent in these cell types, correlating with increased protein compared with the The spacer or RING alone activity compared with and The RING domain alone was not by the but was to be with an The spacer region alone was consistently at an and Thus, these not the that the spacer region was sufficient for proapoptotic However, of three in the RING proapoptotic activity of that the RING was required for activity The activity of spacer-RING the in cells was by with the caspase inhibitor that caspases apoptosis induced by the of the and spacer-RING domains were that these proteins are in cells and To that the spacer-RING domain of c-IAP1 interfere with the antiapoptotic activity of the BIR we spacer-RING could protection by the BIR domain from cell death. of cells with Bax and the BIR domain that BIR suppressed cell death. Furthermore, with spacer-RING the activity of BIR in and cells To that the RING domain of c-IAP1 binds a protein was purified from E. and for its to during Consistent with the that RING fingers of proteins are to of the same of as two BIR domains that one BIR domains contain a in the recently structure of several BIR domains (13Sun C. Cai M. Gunasekera A.H. Meadows R.P. Wang H. Chen J. Zhang H. Wu W. Xu N. Ng S.C. Fesik S.W. Nature. 1999; 401: 812-818Crossref Scopus (294) Google Scholar, C. Cai M. Meadows R.P. Xu N. Gunasekera A.H. Herrmann J. Wu J.C. Fesik S.W. J. Biol. Chem. 2000; 275: 33777-33781Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, Vaux D.L. Biol. 1999; PubMed Scopus Google Scholar). This also that the of cell activity by the RING finger alone (with or an is not to in protein In contrast to XIAP, full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by one or more death stimuli including Bax overexpression, virus infection, and However, deletion of the C-terminal spacer-RING domains of c-IAP1 and c-IAP2 restored the antiapoptotic function of the N-terminal BIR domains This finding that the C of c-IAP1 and c-IAP2 negatively regulate the antiapoptotic activity of the BIR deletion of the C-terminal spacer-RING of c-IAP2 as the of a found in J. M. I. M. C. A. H. P. 1999; 93: PubMed Google Scholar). this a antiapoptotic c-IAP2 However, the to is in the was recently found to a that is also to to A.G. K. S. Dixit V.M. Mol. Cell. 2000; Full Text Full Text PDF PubMed Google Scholar). The the of the in of these M. A. K. P. C. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), to the BIR domain of in mammalian cells are consistent with in eye model where deletion of the RING finger domain of D-IAP1 its antiapoptotic function against Reaper overexpression and during Drosophila eye (12Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (639) Google Scholar). deletion of the c-IAP1 RING was required to protect the eye from Reaper-induced cell death. However, in some situations the RING finger may be required for c-IAP1 was reported to protect human cells from Reaper-induced apoptosis Dixit V.M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and deletion of the RING finger of D-IAP1, D-IAP2, and their to inhibit apoptosis in mammalian cells C. P. Uren A. S. Vaux D. Cell Death Differ. 1998; PubMed Scopus Google Scholar). Furthermore, the RING of baculovirus Cp-IAP is required for antiapoptotic activity and to inhibit (5Huang Q. Deveraux Q.L. Maeda S. Salvsen G.S. Stennicke H.R. Hammock B.D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1427-1432Crossref PubMed Scopus (112) Google Scholar). all of these the RING finger of IAP proteins may or negatively the function of IAP and this function is in cell two factors to to the of antiapoptotic activity by full-length c-IAP1 during protein and to caspase of the RING of c-IAP1 was reported to autoubiquitination and degradation of c-IAP1 (20Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (859) Google Scholar). of the RING to c-IAP1 in cells but the observed in protein may not the of antiapoptotic c-IAP1 was also cleaved to 52- and 35-kDa fragments by recombinant by apoptotic cell and in cells apoptosis. Furthermore, the 35-kDa spacer-RING was of apoptosis and was of the antiapoptotic function of the BIR domain function been to the of and is not required for direct to caspases N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1129) Google for the proapoptotic function of the spacer-RING fragment. To the contrary, the of c-IAP1 appears to both the full-length and cleaved fragments of the mechanism is not or recruitment to a protein the degradation of or one or more of several in the as ubiquitination However, both the spacer and RING domains of c-IAP1 to be to in This is consistent with of c-IAP1 in apoptotic cells appears to be by caspases as the same fragments were with and a caspase inhibitor cleavage of c-IAP1 by apoptotic cell that the 35-kDa caspase cleavage of c-IAP1 the spacer-RING domain. This domain of c-IAP1 apoptosis in transfected cells, consistent with the finding that the region of D-IAP1 cell death in the Drosophila eye (12Hay B.A. Wassarman D.A. Rubin G.M. Cell. 1995; 83: 1253-1262Abstract Full Text PDF PubMed Scopus (639) Google Scholar). The of proapoptotic cleavage of c-IAP1 during cell death is of the finding that and also from antiapoptotic to proapoptotic factors by caspase cleavage R.J. Ueno K. Takahashi A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, Clem R.J. R. A. Ueno K. Science. 1997; PubMed Scopus Google Scholar). However, the in function of the c-IAP1 spacer-RING that is during apoptosis is not This of c-IAP1 could cell an independent function ubiquitination of the BIR or antiapoptotic The RING domain of was found to be sufficient for in vitro E3 activity that ubiquitination of caspases and but not caspase (21Huang 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). This finding seems with the proapoptotic function of spacer-RING in cells but may a role for the spacer region in the proapoptotic function of spacer-RING cells. Alternatively, spacer-RING could function as a inhibitor of degradation of caspases. Consistent with this deletion of the N-terminal BIR domain of in a C-terminal that appears to interfere with by for G. J. S. Nature. 1998; PubMed Scopus Google J. S. A. Cell Biol. 1999; PubMed Scopus Google Scholar). for with the