Smac/DIABLO Selectively Reduces the Levels of c-IAP1 and c-IAP2 but Not That of XIAP and Livin in HeLa Cells
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m401253200
ISSN1083-351X
Autores Tópico(s)Ubiquitin and proteasome pathways
ResumoThe inhibitor of apoptosis (IAP) proteins bind and inhibit caspases via their baculovirus IAP repeat domains. Some of these IAPs are capable of ubiquitinating themselves and their interacting proteins through the ubiquitin-protein isopeptide ligase activity of their RING domain. The Drosophila IAP antagonists Reaper, Hid, and Grim can accelerate the degradation of Drosophila IAP1 and some mammalian IAPs by promoting their ubiquitin-protein isopeptide ligase activity. Here we show that Smac/DIABLO, a mammalian functional homolog of Reaper/Hid/Grim, selectively causes the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although it efficiently promotes the auto-ubiquitination of them all. Smac binding to c-IAP via its N-terminal IAP-binding motif is the prerequisite for this effect, which is further supported by the findings that Smac N-terminal peptide is sufficient to enhance c-IAP1 ubiquitination, and Smac no longer promotes the ubiquitination of mutant c-IAP1 lacking all three baculovirus IAP repeat domains. In addition, different IAPs require the same ubiquitin-conjugating enzymes UbcH5a and UbcH6 for their ubiquitination. Taken together, Smac may serve as a key molecule in vivo to selectively reduce the protein level of c-IAPs through the ubiquitin/proteasome pathway. The inhibitor of apoptosis (IAP) proteins bind and inhibit caspases via their baculovirus IAP repeat domains. Some of these IAPs are capable of ubiquitinating themselves and their interacting proteins through the ubiquitin-protein isopeptide ligase activity of their RING domain. The Drosophila IAP antagonists Reaper, Hid, and Grim can accelerate the degradation of Drosophila IAP1 and some mammalian IAPs by promoting their ubiquitin-protein isopeptide ligase activity. Here we show that Smac/DIABLO, a mammalian functional homolog of Reaper/Hid/Grim, selectively causes the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although it efficiently promotes the auto-ubiquitination of them all. Smac binding to c-IAP via its N-terminal IAP-binding motif is the prerequisite for this effect, which is further supported by the findings that Smac N-terminal peptide is sufficient to enhance c-IAP1 ubiquitination, and Smac no longer promotes the ubiquitination of mutant c-IAP1 lacking all three baculovirus IAP repeat domains. In addition, different IAPs require the same ubiquitin-conjugating enzymes UbcH5a and UbcH6 for their ubiquitination. Taken together, Smac may serve as a key molecule in vivo to selectively reduce the protein level of c-IAPs through the ubiquitin/proteasome pathway. Apoptosis is genetically controlled cell death that is essential for development and homeostasis of multicellular organisms. Both excessive and insufficient cell death can lead to anomalies or diseases. The molecular machinery involved in apoptosis is highly conserved across divergent species. The apoptotic death of cells requires proteolytic activation of caspases that are synthesized as latent proenzymes. Once activated, caspases cleave a wide range of molecules that eventually result in the dismantlement of cells. Caspases are therefore tightly controlled within the cells. Active caspases can be specifically inhibited by the inhibitor of apoptosis (IAP), 1The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; WT, wild type; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzymes; E3, ubiquitin-protein isopeptide ligase; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid; TNFR, tumor necrosis factor receptor; IBM, IAP-binding motif. a family of proteins containing one to three copies of characteristic baculovirus IAP repeat (BIR) (1Liston P. Fong W.G. Korneluk R.G. Oncogene. 2003; 22: 8568-8580Crossref PubMed Scopus (390) Google Scholar). The BIR domains, in some cases together with the intervening linker regions, directly bind and inhibit caspases. IAP proteins are counteracted by Reaper, Hid, Grim, Sickle, and Jafrac2 in Drosophila, and by Smac/DIABLO, Omi/HtrA2, and GSPT1/eRF3 in mammals (2Wang 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 (438) Google Scholar, 3Srinivasula S.M. Datta P. Kobayashi M. Wu J.W. Fujioka M. Hegde R. Zhang Z. Mukattash R. Fernandes-Alnemri T. Shi Y. Jaynes J.B. Alnemri E.S. Curr. Biol. 2002; 12: 125-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 4Wing J.P. Karres J.S. Ogdahl J.L. Zhou L. Schwartz L.M. Nambu J.R. Curr. Biol. 2002; 12: 131-135Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 5Christich A. Kauppila S. Chen P. Sogame N. Ho S.I. Abrams J.M. Curr. Biol. 2002; 12: 137-140Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Tenev T. Zachariou A. Wilson R. Paul A. Meier P. EMBO J. 2002; 21: 5118-5129Crossref PubMed Scopus (78) Google Scholar, 7Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 8Verhagen 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 (1985) Google Scholar, 9Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol. Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 10Hegde 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 (633) Google Scholar, 11Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopoulos 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 (469) Google Scholar, 12Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufmann 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 (480) Google Scholar, 13van Loo G. van Gurp M. Depuydt B. Srinivasula S.M. Rodriguez I. Alnemri E.S. Gevaert K. Vandekerckhove J. Declercq W. Vandenabeele P. Cell Death Differ. 2002; 9: 20-26Crossref PubMed Scopus (277) Google Scholar). Despite the overall sequence differences, these IAP antagonists share a conserved N-terminal IAP-binding motif (IBM) or Reaper/Hid/Grim motif. This motif is indispensable and sufficient for counteracting IAP inhibition. The small subunit of active caspase-9 also binds to XIAP through the same IBM produced by auto-processing (14Srinivasula 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 (863) Google Scholar). This mechanism allows these IAP antagonists to compete with caspases for IAP binding and consequently relieves caspases and promotes cell death. Some IAPs also regulate apoptosis through the ubiquitin-protein isopeptide ligase (E3) activity of their RING domain (1Liston P. Fong W.G. Korneluk R.G. Oncogene. 2003; 22: 8568-8580Crossref PubMed Scopus (390) Google Scholar, 15Yang Y. Yu X. FASEB J. 2003; 17: 790-799Crossref PubMed Scopus (176) Google Scholar). These IAPs are capable of targeting the poly-ubiquitination of IAP-binding proteins such as caspases and IAP antagonists (16Huang 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, 17Wilson R. Goyal L. Ditzel M. Zachariou A. Baker D.A. Agapite J. Steller H. Meier P. Nat. Cell Biol. 2002; 4: 445-450Crossref PubMed Scopus (253) Google Scholar, 18Muro I. Hay B.A. Clem R.J. J. Biol. Chem. 2002; 277: 49644-49650Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 19MacFarlane M. Merrison W. Bratton S.B. Cohen G.M. J. Biol. Chem. 2002; 277: 36611-36616Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 20Hu S. Yang X. J. Biol. Chem. 2003; 278: 10055-10060Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 21Olson M.R. Holley C.L. Yoo S.J. Huh J.R. Hay B.A. Kornbluth S. J. Biol. Chem. 2003; 278: 4028-4034Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). On the other hand, the E3 activity of IAP also leads to the ubiquitination of IAPs themselves, and such autoubiquitination can be enhanced by Reaper, Hid, and Grim (22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar, 23Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 24Hays R. Wickline L. Cagan R. Nat. Cell Biol. 2002; 4: 425-431Crossref PubMed Scopus (109) Google Scholar, 25Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar, 26Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (182) Google Scholar, 27Silke J. Kratina T. Ekert P.G. Pakusch M. Vaux D.L. J. Biol. Chem. 2003; 279: 4313-4321Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). An alternatively spliced form of Smac, Smac3, was recently reported to promote XIAP ubiquitination and degradation (28Fu J. Jin Y. Arend L.J. J. Biol. Chem. 2003; 278: 52660-52672Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These observations demonstrate that these IAP antagonists accelerate the disposal of IAPs in addition to releasing captive caspases from IAPs. Moreover, the mammalian IAP-binding protein Omi/HtrA2 can directly degrade IAP molecules through its serine protease activity (29Yang Q.H. Church-Hajduk R. Ren J. Newton M.L. Du C. Genes Dev. 2003; 17: 1487-1496Crossref PubMed Scopus (272) Google Scholar, 30Srinivasula S.M. Gupta S. Datta P. Zhang Z. Hegde R. Cheong N. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2003; 278: 31469-31472Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 31Suzuki Y. Takahashi-Niki K. Akagi T. Hashikawa T. Takahashi R. Cell Death Differ. 2004; 11: 208-216Crossref PubMed Scopus (166) Google Scholar). Five of the eight human IAPs have a C-terminal RING domain (32Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar). However, little is known about the characteristics of the E3 activity of various IAPs. Even though the ubiquitination and proteasomal degradation of XIAP have been reported to be enhanced by Smac3 rather than Smac, it is not known if c-IAP1 and c-IAP2 are also modulated by their cognate antagonists via ubiquitin/proteasomal degradation. In this paper we show that Smac stimulates the rapid degradation of c-IAP1 and c-IAP2 but not XIAP and Livin in HeLa cells, although Smac does promote auto-ubiquitination of all four of these human IAPs. These human IAPs require the same ubiquitin-conjugating enzymes (E2) UbcH5a and UbcH6 for their E3 activity in vitro. We also demonstrate that c-IAP degradation promoted by Smac is dependent on RING E3 activity and direct binding to Smac, and the Smac N-terminal peptide is sufficient to accelerate c-IAP1 auto-ubiquitination. These observations, together with the a recent report that Smac is recruited by the tumor necrosis factor receptor (TNFR) signaling pathway to disrupt TRAF2-c-IAP1 complex (33Deng Y. Ren X. Yang L. Lin Y. Wu X. Cell. 2003; 115: 61-70Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), indicate that Smac may selectively down-regulate the levels of c-IAP1 and c-IAP2, thereby facilitating caspase-8 activation initiated by TNFR signaling in vivo. Antibodies and Reagents—The polyclonal antibodies that recognize, respectively, the residues 527–546 of human c-IAP1, residues 507–524 of human c-IAP2, and residues 244–263 of human XIAP, and the monoclonal antibody against human ubiquitin were purchased from R & D Systems. Horseradish peroxidase-conjugated anti-GST antibody, anti-FLAG M2 antibody, and anti-c-Myc monoclonal antibody were from Sigma. The monoclonal anti-Livin antibody was from IMGENEX, and the monoclonal anti-actin antibody was from Santa Cruz Biotechnology. The polyclonal antiserum against Smac was generated by immunizing rabbits with recombinant Smac protein as described previously (7Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar). The mammalian ubiquitin, rabbit ubiquitin-activating enzyme (E1), and various recombinant human E2 were from Boston Biochem, Inc. Plasmids—The plasmids for the GST fusion proteins of human c-IAP1, c-IAP2, and XIAP (29Yang Q.H. Church-Hajduk R. Ren J. Newton M.L. Du C. Genes Dev. 2003; 17: 1487-1496Crossref PubMed Scopus (272) Google Scholar) and the C-terminal His9-tagged mature form of Smac (7Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar) have been described elsewhere. The cDNAs for Livin α and Livin β were individually inserted into the pGEX-4T-2 (Amersham Biosciences) to generate GST fusion proteins. The p3×FLAG-CMV-7 vector (Sigma) was used for the expression of IAP proteins in mammalian cells. The N-terminal ubiquitin-fused and C-terminal c-Myc-tagged Smac wild type (WT) and the Ala1 deletion mutant (ΔA) were generated by PCR and subcloned into the pcDNA3.1(–) mammalian expression vector (Invitrogen) according to the reported technique (34Varshavsky A. Methods Enzymol. 2000; 327: 578-593Crossref PubMed Scopus (47) Google Scholar, 35Baker R.T. Smith S.A. Marano R. McKee J. Board P.G. J. Biol. Chem. 1994; 269: 25381-25386Abstract Full Text PDF PubMed Google Scholar). The mutation of the conserved His residue to Ala in the RING domain of respective IAPs (c-IAP1 H588A, c-IAP2 H574A, XIAP H467A, Livin α H269A, and Livin β H251A) was made using the QuickChange Multisite-directed Mutagenesis kit (Stratagene). Full-length c-IAP1 and XIAP and different c-IAP1 truncation mutants were generated by PCR and subcloned into the pTYB11 (New England Biolabs). All constructs were confirmed by sequencing. Protein Expression and Purification—All of the recombinant proteins were expressed in Escherichia coli strain BL21 (DE3). The GST-fused IAP proteins were purified with glutathione-Sepharose affinity chromatography, followed by Superdex 200 gel filtration chromatography if necessary. The C-terminal His9-tagged wild type Smac and Smac ΔA were purified with nickel-nitrilotriacetic acid-Sepharose affinity chromatography. The non-tagged full-length c-IAP1 and XIAP and the truncated c-IAP1 mutants were purified from the Chitin affinity column after dithiothreitol induction according to the manufacturer's protocols (New England Biolabs). The protein concentrations were determined by the modified Bradford method (36Zor T. Selinger Z. Anal. Biochem. 1996; 236: 302-308Crossref PubMed Scopus (888) Google Scholar). In Vitro Ubiquitination Assays—In vitro ubiquitination assays were carried out as described previously (29Yang Q.H. Church-Hajduk R. Ren J. Newton M.L. Du C. Genes Dev. 2003; 17: 1487-1496Crossref PubMed Scopus (272) Google Scholar). IAP proteins (200 nm) were incubated with or without Smac for 2 h at 30 °C in a reaction system containing 50 mm Tris-HCl (pH 7.5), 50 mm NaCl, 2 mm Mg-ATP, 20 μm mammalian ubiquitin, 100 nm rabbit E1, and 400 nm recombinant human E2. The reactions were stopped by adding equal volumes of 2× SDS sample loading buffer followed by Western blot analysis. Transfection of Cultured Cells—HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were seeded onto 6-well plates the day before transfection and transfected at ∼70% confluence by using Lipofectin combined with Plus reagent (Invitrogen) according to the manufacturer's protocols. The IAP expression plasmids (3 μg each for c-IAP1, c-IAP2, and XIAP, and 250 ng each for Livin α and Livin β) together with 2.5 μg of Smac expression plasmids or pcDNA3.1(–) blank plasmid were used for single-well transfection in the absence of antibiotics. The cells were harvested 12 h after the transfection and lysed with 0.5% CHAPS in 20 mm HEPES (pH 7.4), 10 mm KCl, 1 mm MgCl2, and 1mm dithiothreitol. The lysates were centrifuged, and the protein concentrations of the supernatants were quantified by the modified Bradford method (36Zor T. Selinger Z. Anal. Biochem. 1996; 236: 302-308Crossref PubMed Scopus (888) Google Scholar). Western Blot Analysis—Proteins were resolved by SDS-PAGE and transferred to nitrocellulose filters. The filters were blocked with 5% milk and probed with antibodies as indicated in the text. The signals were visualized with the enhanced chemiluminescence method. Smac Prevents the Accumulation of Both c-IAP1 and c-IAP2 in HeLa Cells—To investigate the effect of Smac on IAP protein levels, we transfected HeLa cells with the expression vectors for Smac and various IAPs. The ubiquitin fusion technique was used here to secure the production of mature Smac protein with the expected N-terminal AVPI motif (6Tenev T. Zachariou A. Wilson R. Paul A. Meier P. EMBO J. 2002; 21: 5118-5129Crossref PubMed Scopus (78) Google Scholar, 37Hunter A.M. Kottachchi D. Lewis J. Duckett C.S. Korneluk R.G. Liston P. J. Biol. Chem. 2003; 278: 7494-7499Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The HeLa cells transiently transfected with c-IAP1 or c-IAP2 expression vector produced not only the expected full-length c-IAP1 or c-IAP2 but also the multiple higher molecular mass forms that are characteristic of ubiquitinated products (lanes 2 and 4 of Fig. 1). This was consistent with the previous reports that both c-IAP1 and c-IAP2 are E3s and are capable of directing auto-ubiquitination (16Huang 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, 22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar). In contrast, the transiently expressed XIAP, under the same conditions, showed only one or two bands above the major band of full-length XIAP (lane 6 of Fig. 1). Surprisingly, the protein levels of both c-IAP1 and c-IAP2, but not XIAP, were significantly decreased when the mature form of Smac was co-expressed (lanes 2, 4, and 7 of Fig. 1). Previous works have revealed that the Drosophila functional homologs of Smac, Reaper, Hid, and Grim can promote the degradation of mammalian IAP proteins bearing normal E3 activity (23Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 24Hays R. Wickline L. Cagan R. Nat. Cell Biol. 2002; 4: 425-431Crossref PubMed Scopus (109) Google Scholar, 25Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar, 26Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (182) Google Scholar). Two of these Smac homologs, Grim and Reaper, also reduce the levels of IAPs by suppressing global protein synthesis (23Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 26Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (182) Google Scholar). It was unlikely that the decrease of c-IAP1 and c-IAP2 was caused by the Smac-mediated inhibition of protein synthesis because XIAP under similar conditions was unaffected. To test whether Smac stimulated the ubiquitination-mediated degradation of both c-IAP1 and c-IAP2, we mutated His588 in c-IAP1 and His574 in c-IAP2 to Ala. This conserved His residue in the RING finger domain has proven important for the IAP E3 activity (22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar). The effects of Smac on these IAP E3 activities were examined as described below. Smac Promotes the Degradation of c-IAP1 and c-IAP2 by Enhancing Their Auto-ubiquitination in HeLa Cells—The H588A mutant c-IAP1 exhibited a much higher expression level than the wild type in HeLa cells (compare lanes 3 and 6 of Fig. 2A). The amount of wild type c-IAP1 was also dramatically increased by the addition of proteasome inhibitor MG132 to the culture medium (lane 10, Fig. 2A). MG132, however, did not cause a significant increase in the H588A c-IAP1 protein level (lane 13). These observations were in agreement with the previous report that c-IAP1 is continuously down-regulated by ubiquitination-dependent degradation, and such ubiquitination is mediated by the E3 activity of c-IAP1 itself (22Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar). In the presence of MG132, co-expression of wild type Smac led to a reduction in the level of non-ubiquitinated and mono-/di-ubiquitinated wild type c-IAP1 and a concomitant increase in the level of highly ubiquitinated c-IAP1 forms (lane 11 of Fig. 2A). In the absence of MG132, co-expression of Smac resulted in nearly complete disappearance of c-IAP1 (lane 4 of Fig. 2A). These data indicated that Smac promoted the poly-ubiquitination and degradation of c-IAP1. It is known that the processed Smac and other IAP antagonists have a conserved N-terminal IBM with an initial Ala residue. Mutation of this Ala or deletion of the IBM abolishes the specific binding of these proteins to IAP (38Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (716) Google Scholar, 39Srinivasula 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). To assess whether Smac enhancement of c-IAP1 ubiquitination and degradation requires the same binding, we co-transfected c-IAP1 with a Smac mutant lacking the initial Ala (Smac ΔA) that was also produced by using the ubiquitin fusion expression vector. As expected, Smac ΔA did not enhance the auto-ubiquitination of c-IAP1 (Compare lanes 5 and 3 and lanes 13 and 10 of Fig. 2A), suggesting that the specific binding of Smac to c-IAP1 via its IBM is required for Smac to enhance the auto-ubiquitination of c-IAP1. In agreement with the above observations, Smac did not cause a drastic change in the level of E3-negative H588A c-IAP1 either in the absence or in the presence of MG132 (lanes 6 and 7, and lanes 13 and 14 of Fig. 2A, respectively). Smac likewise strongly promoted the ubiquitination and degradation of c-IAP2 (Fig. 2B). Mutation of His574 to Ala in c-IAP2 also significantly increased the expression level of c-IAP2 (Fig. 2B). As mentioned above, the level of c-IAP1 was significantly elevated by the addition of MG132. In contrast, c-IAP2 did not show such a significant change when MG132 was added (compare lanes 3 and 10 in Fig. 2B). Such a difference made it reasonable to speculate that in the absence of Smac, the E3 of c-IAP2 was much less active than c-IAP1 in directing its auto-ubiquitination. It should be noted that although His588 in c-IAP1 and His574 in c-IAP2 are critical for their E3 activities, mutation of this residue to Ala greatly reduced but did not completely abolish their E3 activity, as revealed by the background ubiquitinated product ladders (lanes 6, 7, 13, and 14 of Fig. 2). This residual activity was also observed in an in vitro ubiquitination assay using purified mutant c-IAP proteins (data not shown). Smac Does Not Promote the Degradation of XIAP and Livin in HeLa Cells—XIAP has been reported to be ubiquitinated by its own E3 activity when expressed in HEK 293T cells, and this auto-ubiquitination and subsequent degradation is stimulated by Reaper (26Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (182) Google Scholar). In this current study, XIAP transiently expressed in HeLa cells was ubiquitinated as well; however, mono-ubiquitination rather than poly-ubiquitination of XIAP seemed to be the major product (Fig. 3A). Similar to c-IAP1 and c-IAP2, XIAP ubiquitination also depended on its E3 activity, for the H467A E3-negative mutant gave either no band or a much weaker mono-ubiquitination band (lanes 3, 5, 8, and 10 of Fig. 3A). Although more poly-ubiquitinated XIAP products could be detected in the presence of MG132, they were much less significant compared with that of c-IAP1 and c-IAP2. Unlike that of c-IAP1 and c-IAP2, the overall amount of XIAP was not quite affected by the co-transfected Smac, although Smac still promoted XIAP ubiquitination. In the absence of MG132, co-expression of Smac only had a negligible effect on the level of XIAP (lane 4 of Fig. 3A), whereas under similar conditions Smac reduced both c-IAP1 and c-IAP2 to a nearly non-detectable level (Fig. 2). This result was consistent with the report by Silke et al. (27Silke J. Kratina T. Ekert P.G. Pakusch M. Vaux D.L. J. Biol. Chem. 2003; 279: 4313-4321Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) that Smac does not promote XIAP degradation. Unexpectedly, the Smac ΔA mutant, despite its failure to bind to various IAPs (Fig. 4), also stimulated the auto-ubiquitination of XIAP and Livin (lanes 4 and 5 and 11 and 12 in Fig. 3). The reason for this IBM-independent acceleration on XIAP and Livin ubiquitination remains unknown. Smac effectively stimulates auto-ubiquitination of Livin α, which slightly promoted the degradation of Livin α (lane 4 of Fig. 3B). Livin β, an alternatively spliced form of Livin, lacks 18 residues between the BIR and the RING domains compared with Livin α (40Vucic D. Stennicke H.R. Pisabarro M.T. Salvesen G.S. Dixit V.M. Curr. Biol. 2000; 10: 1359-1366Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 41Ashhab Y. Alian A. Polliack A. Panet A. Ben Yehuda D. FEBS Lett. 2001; 495: 56-60Crossref PubMed Scopus (189) Google Scholar). The transfection results of Livin β were basically the same as that of Livin α (data not shown). In contrast to c-IAP1 and c-IAP2, the mutation of the corresponding His residue in the RING finger domain of XIAP and Livin did not considerably enhance their expression, although the mutation did greatly reduce their E3 activity. Taken together, these observations suggested that the levels of XIAP and Livin were not mainly regulated by auto-ubiquitination/proteasomal degradation in HeLa cells. Different IAP Proteins Require the Same Ubiquitin-conjugating Enzymes in Vitro for Their E3 Activity—To compare the E3 activities of these human IAPs, we used an in vitro ubiquitination assay system containing purified ubiquitin, E1, and E2 to screen a panel of ubiquitin-conjugating enzymes available from Boston Biochem, Inc. All of these IAP proteins exhibited strong E3 activity when UbcH5a and UbcH6 were used as the E2 as manifested by the characteristic poly-ubiquitination ladders (lanes 4 and 7 of Fig. 5). Smac Does Not Promote Auto-ubiquitination of XIAP in Vitro—The purified XIAP was active in directing poly-ubiquitination. However, the auto-ubiquitination of XIAP was not enhanced by Smac. The amount of ubiquitinated forms of XIAP with a high molecular mass of ∼150 kDa was even slightly reduced by Smac (Fig. 6). Smac Promotes Auto-ubiquitination of c-IAP1 in Vitro through Specific Association with the BIR Domains—The observation that Smac promotes c-IAP1 auto-ubiquitination in HeLa cells was further confirmed in vitro by using UbcH6 as the E2. Smac at equimolar to c-IAP1 enhanced c-IAP1 autoubiquitination to the maximum (lane 5, Fig. 7A). Consistent with the transfection result, SmacΔA failed to promote such auto-ubiquitination (lane 7 of Fig. 7A). Similar results were also obtained with Smac and c-IAP2 (data not shown). The Smac mutant lacking the initial Ala residue did not bind IAP proteins and consequently did not promote the E3 activity of either c-IAP1 or c-IAP2 in HeLa cells or in vitro. These results strongly indicated that the interaction between the IBM of Smac and the BIR domains of IAP was essential and that the BIR domains may regulate the E3 activity of the RING finger. To verify this we made stepwise deletion of the first BIR (ΔBIR1, residues 1–161), the first two BIRs (ΔBIR1–2, residues 1–265), or all the three BIR domains (ΔBIR1–2-3, residues 1–339) from c-IAP1. All these mutants and the full-length wild type c-IAP1 were purified as a non-tagged form by using the pTYB11 intein system (New England Biolabs). The c-IAP1 mutants with one, two, or all three BIR domains deleted were still active in directing their auto-ubiquitination (Fig. 7A). The basal activity of ΔBIR1 and ΔBIR1–2 was very low but was significantly enhanced by Smac, whereas the basal activity of ΔBIR1–2-3 was comparably high and could no longer be enhanced by Smac. This suggested that the second and the third BIR domains of c-IAP1 strongly inhibit the E3 activity of the C-terminal RING. Smac binding to the BIR domains of c-IAP1 may help expose the RING domain to generate a higher E3 activity. Smac N-terminal Hexapeptide Is Sufficient to Stimulate the Ubiquitin-Protein Isopeptide Ligase Activity of c-IAP1—Previous work has shown that the N-terminal peptides of Smac can mimic Smac protein in removing the inhibition of XIAP on caspase-3 activation (38Chai J. Du C. Wu J.W. Kyin
Referência(s)