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J. Biol. Chem., Vol. 275, Issue 35, 26661-26664, September 1, 2000

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ACCELERATED PUBLICATION
The Inhibitor of Apoptosis, cIAP2, Functions as a Ubiquitin-Protein Ligase and Promotes in Vitro Monoubiquitination of Caspases 3 and 7^*

Han-kuei Huang, Claudio A. P. Joazeiro, Emanuela Bonfoco^§, Shinji Kamada, Joel D. Leverson, and Tony Hunter^¶

From the  Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies and the ^§ Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, March 26, 2000, and in revised form, June 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The inhibitor of apoptosis, cIAP2, contains a putative Ring finger motif at the C terminus. Using in vitro ubiquitination assays, we found that the Ring finger of cIAP2 alone possesses intrinsic ubiquitin ligase activity and promotes substrate-independent ubiquitination. It also promotes ubiquitination of caspases 3 and 7 but not caspase-1. The Ring fingers of c-Cbl and Apc11 failed to promote caspase-7 ubiquitination, suggesting that the Ring finger of cIAP2 itself is involved in substrate recognition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Programmed cell death, often referred to as apoptosis, is required for normal embryonic development, growth, and homeostasis (1-4). Caspases, proteases that cleave at the carboxyl side of Asp residues, are required for intracellular protein degradation and execution of cell death (5-7). Full-length caspases are inactive and contain pro-domains at the N terminus followed by sequences that are cleaved by other caspases to yield active large and small subunits (5-7). Recent studies have shown that inhibitors of apoptosis (IAP;¹ for review see Ref. 8) can inhibit the proteolytic activities of caspases 3 and 7 (9, 10), block activation of pro-caspase-9 (11), and prevent apoptosis induced by Fas signaling (10) and etoposide (9). All IAPs contain 1 to 3 baculoviral IAP repeat (BIR) domains, which resemble zinc finger motifs (12). It has been reported that individual BIR domains, together with the intervening linker regions, are required for the inhibition of caspase activity (10, 12-14). Some IAPs such as cIAP1 and cIAP2 also contain a CARD domain (15) and interact with tumor necrosis factor receptor-associated factor 2 (9, 16). cIAP1, cIAP2, and XIAP also contain a Ring finger motif (see Fig. 1A). However, the function of the Ring finger motif of IAPs remains unclear.

Recent studies have established a functional relationship between Ring finger and ubiquitin ligase activity (17-20). Ubiquitination is a post-translational protein modification that requires ATP and three different enzymes, a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3) (21, 22). In short, free ubiquitin is recruited to E1 by thioester bond formation and subsequently transferred to E2 through a second thioester bond between E2 and ubiquitin. E2, in conjunction with E3, transfers ubiquitin to target proteins. There are two major families of E3, which possess either a homologous to E6-AP C terminus (HECT) domain or a Ring finger motif. Ubiquitination is reportedly involved in cell cycle control (17, 23), endocytosis (24, 25), signal transduction (18, 19, 26), DNA repair (27), and apoptosis (28). However, the function of ubiquitination in apoptosis is not clear. Given that cIAP2 contains a Ring finger motif, we tested whether cIAP2 possesses ubiquitin-protein ligase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Constructs-- The human cIAP2 and caspase-7 cDNA clones were purchased from Research Genetics (IMAGE Consortium Clones 1992226 and 563750, respectively). cDNA clones of human caspases 1 and 3 were described previously (29, 30). All the His-tagged constructs (see Fig. 1B) were generated by standard polymerase chain reaction procedures (31) using the plasmid pHis8, a derivative of pET28 (Novagen). The truncated cIAP2 constructs are indicated in Fig. 1B. Point mutations were generated using Pfu polymerase (QuikChange^TM, Stratagene) according to the manufacturer's protocol.

Protein Purification-- All His-tagged recombinant proteins were purified using TALON resin (CLONTECH) according to the manufacturer's protocol with minor modifications. Induction of protein expression was carried out at 20-23 °C by adding 8 ml of 100 mM isopropyl-1-thio--D-galactopyranoside to an 800-ml bacterial culture when cell density (A₆₀₀) reached 0.6-0.7. Bacterial cells were resuspended in binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM imidazole, pH 7.9) followed by sonication. After centrifugation (39,000 × g), cell extract was incubated with 300 µl (bed volume) of TALON resin at 4 °C for 1-2 h. Beads were washed three times with 10 ml of washing buffer (same as binding buffer except with 10 mM imidazole). Proteins were eluted with 300-500 µl of elution buffer (same as binding buffer except with 100 mM imidazole). Eluted proteins were concentrated to 1-2 mg per ml using a microconcentrator (Filtron). During the concentration step, the buffer was changed to 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.05% Nonidet P-40, and 10% glycerol. For full-length cIAP2, truncated cIAP2s, and caspases 1 and 7, a fraction of the protein was insoluble, and the soluble fractions were purified by using TALON resin.

Ubiquitination Assays-- Ubiquitination assay was performed as described previously (18) with minor modifications. Reactions (10-12 µl) contained His-tagged E1 (50-500 nM), His-tagged human Ubc4 (0.5- 5 µM), GST-ubiquitin (5 µM), ATP (2 mM), 1 µg of caspase protein, and 3 µg of cIAP2 protein in reaction buffer (50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl₂, 0.05% Nonidet P-40, and 0.5 mM dithiothreitol). Reactions were incubated at 25 °C for 90 min and then stopped by adding 2× SDS sampling buffer (4% SDS and 5.8 M -mercaptoethanol). Preparation of GST-ubiquitin was performed by GST affinity purification according to the manufacturer's protocol (Amersham Pharmacia Biotech). Preparations of c-Cbl Ring finger and GST-Apc11 proteins were described previously (18, 32).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To test whether cIAP2, like other Ring finger proteins, can promote autoubiquitination (17-19, 26, 33), full-length cIAP2 was tagged with 8 histidine residues, and recombinant protein was purified from Escherichia coli (see "Experimental Procedures"). We also tested whether cIAP2 can promote ubiquitination of caspase-7 (Casp7), as it has been reported that the BIR domains of cIAP1 and cIAP2 physically interact with caspases 3 and 7 (9) and that XIAP interacts with caspases 3 and 7 through its second BIR domain (BIR2) (10). Full-length His-tagged Casp7 was purified from E. coli as described previously (34, 35). The in vitro ubiquitination assay was performed with purified recombinant His-tagged E1 and E2 (human Ubc4) and GST-ubiquitin fusion proteins followed by immunoblot analysis using anti-GST antibody to detect substrate-independent ubiquitination and anti-Casp7 antibody to detect ubiquitinated Casp7.

Slower migrating proteins consistent with ubiquitinated species were detected by the anti-GST antibody (see Fig. 2A, cf. lanes 2 and 3), suggesting that cIAP2 functions as a ubiquitin-protein ligase and promotes substrate-independent ubiquitination. Presumably, this results from autoubiquitination of cIAP2, but these ubiquitinated proteins could result from ubiquitination of Ubc4, E1, or from formation of poly-GST-ubiquitin not conjugated to other proteins. This substrate-independent ubiquitination required both E1 and E2 (data not shown). To determine whether the BIR or CARD domains are required for substrate-independent ubiquitination, we generated different truncated cIAP2 constructs (Fig. 1B) and purified recombinant truncated cIAP2 proteins followed by ubiquitination assay. Compared with the negative control (no incubation, data not shown), full-length cIAP2 (Fig. 2B, lane 1) truncation of one (B2R, Fig. 2B, lane 2), two (B3R, Fig. 2B, lane 3), or three (CR, Fig. 2B, lane 4) BIR domains did not significantly affect the ubiquitination activity. The Ring finger motif alone (R, Fig. 2C, lane 1) can catalyze ubiquitination. In contrast, mutant cIAP2 either lacking the Ring finger motif (B1C, Fig. 2B, lane 5 and Fig. 2C, lane 2) or bearing a point mutation in the Ring finger motif, in which the first zinc-binding Cys residue is mutated to Ala (C557A, Fig. 2C, lane 6), failed to promote ubiquitination. Point mutation at the corresponding position in the c-Cbl and Apc11 Ring fingers also abolishes ubiquitination activity (18, 32). These observations indicate that the cIAP2 Ring finger motif alone is sufficient to promote substrate-independent ubiquitination.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Sequence comparison of the Ring finger motifs of cIAP2 and c-Cbl. A, amino acid sequence alignment of the Ring fingers of cIAP2 (top) and c-Cbl (bottom). The amino acid sequences are compared for maximal alignment. The shaded regions indicate identical amino acid residues among these two proteins. The asterisks indicate the Cys or His residues required for zinc binding. B, schematic diagram of cIAP2 truncation mutants generated for this study.

View larger version (63K): [in this window] [in a new window]   Fig. 2.   cIAP2 promotes substrate-independent ubiquitination. Purified recombinant cIAP2 (wild-type or truncated mutants as indicated in Fig. 1B) was incubated with purified E1, E2, and Casp7 for either 0 or 90 min, as indicated, at 25 °C. Reactions were stopped by adding 2× sample buffer and were subjected to SDS polyacrylamide gel electrophoresis followed by immunoblot analysis. The transfer membrane was probed with anti-Casp7 antibody (Transduction Laboratories) (see Fig. 3), stripped, and then re-probed with anti-GST antibody (Santa Cruz Biotechnology) to detect ubiquitinated proteins. Panels A, B, and C represent three different experiments that were performed independently with both positive and negative controls and the indicated recombinant proteins.

When recombinant Casp7 was included in the assay, monoclonal anti-Casp7 antibody detected slower migrating proteins corresponding to monoubiquitinated full-length Casp7 (Fig. 3A, cf. lanes 2 and 3). The additional bands may be cleavage products, such as Casp7 lacking the small subunit, or they might represent Casp7 ubiquitinated at different Lys residues. In contrast, in the absence of cIAP2 Casp7 did not show any slower migrating protein bands (Fig. 3A, lane 1). These results suggest that cIAP2 promotes monoubiquitination of Casp7. Next we tested whether the BIR and CARD domains are required for Casp7 monoubiquitination using the various truncation mutants. We found that truncation of one (B2R, Fig. 3B, lane 2), two (B3R, Fig. 3B, lane 3), or three (CR, Fig. 3B, lane 4) BIR domains did not affect Casp7 ubiquitination significantly, suggesting that BIR domains are not required for Casp7 monoubiquitination in vitro. Furthermore, a point mutation (I235P) in the BIR2 domain of the truncated cIAP2 lacking the first BIR domain (B2R-I235P), which at the corresponding position in the p35 viral IAP abolishes interaction between p35 and Casp3 (13), had no effect on either substrate-independent ubiquitination or Casp7 monoubiquitination (Fig. 2C, lane 5 and Fig. 3C, lane 5, respectively). This suggests that the BIR2 domain is not needed to target Casp7 for ubiquitination in vitro even though this domain can interact with Casp7.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   cIAP2 promotes monoubiquitination of caspase-7. In vitro ubiquitination assays were performed as described in the legend to Fig. 2. The transfer membrane was probed with anti-Casp7 antibody to detect ubiquitination of Casp7. Samples are the same as in Fig. 2. The truncated mutants of cIAP2 are described in Fig. 1B. Panels A, B, and C represent three different experiments that were performed independently with both positive and negative controls and the indicated recombinant proteins.

Interestingly, the cIAP2 Ring finger motif alone was able to promote Casp7 monoubiquitination (Fig. 3C, lane 1). In contrast, cIAP2 mutants either lacking the Ring finger motif (B1C, Fig. 3B, lane 5 and Fig. 3C, lane 2) or bearing a point mutation in the Ring finger motif (C557A, Fig. 3C, lane 6) failed to promote Casp7 ubiquitination. These observations suggest that Casp7 is a substrate for cIAP2 ubiquitin-ligase activity and that the Ring finger motif alone is sufficient to promote Casp7 monoubiquitination. As a control for specificity of Casp7 ubiquitination by cIAP2, we tested whether the Ring fingers of c-Cbl and Apc11 can promote Casp7 ubiquitination. Both GST-c-Cbl Ring finger and GST-Apc11 promoted substrate-independent ubiquitination (Fig. 2C, lanes 3 and 4), but neither catalyzed Casp7 ubiquitination (Fig. 3C, lanes 3 and 4, respectively). Furthermore, the same negative result was obtained with two independent His-tagged Apc11 preparations (data not shown). Taken together our results suggest that the Ring finger motif of cIAP2 not only catalyzes ubiquitination but also is sufficient for recognition of Casp7 as a substrate.

To determine whether other caspases could be substrates for cIAP2-mediated ubiquitination, we tested Casp1, whose proteolytic activity is not inhibited by cIAP2 (9). His-tagged full-length recombinant Casp1 was purified from E. coli followed by ubiquitination assay. As shown in Fig. 4B (cf. lanes 1 and 2), cIAP2 promoted substrate-independent ubiquitination in the presence of Casp1. However, no slower migrating Casp1 protein bands were detected by anti-Casp1 antibody even after long exposure (Fig. 4A, lanes 1 and 2) suggesting that cIAP2 does not promote ubiquitination of Casp1. Alternatively, a small fraction of Casp1 might be ubiquitinated, but this was below the sensitivity of the anti-Casp1 antibody. As a control, we mixed Casp1 protein with Casp7 protein and repeated the ubiquitination assay. cIAP2 promoted substrate-independent ubiquitination and Casp7 monoubiquitination (data not shown), indicating that our Casp1 preparation was not contaminated with inhibitory impurities.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Caspase-1 is not ubiquitinated by cIAP2. A, purified recombinant Casp-1 was subjected to ubiquitination assay as described in the legend to Fig. 2, and the transfer membrane was probed with anti-Casp1 antibody (Santa Cruz Biotechnology) to detect ubiquitination of Casp1. Lane 1, non-reacted; lane 2, reacted for 90 min. B, the transfer membrane was stripped and re-probed with anti-GST antibody to detect substrate-independent ubiquitination. Lane 1, non-reacted; lane 2, reacted for 90 min.

In addition to Casp1, we also tested whether Casp3 can be ubiquitinated by cIAP2, as it has been reported that Casp3 interacts with cIAP2 (10), and the amino acid sequences of Casp3 and Casp7 are more closely related to each other than to other caspases (5-7). The pro-domain of Casp3 was substituted with a His tag, and recombinant Casp3, a mixture of inactive Casp3 and active large and small subunits, was purified (29, 30) followed by ubiquitination assay. cIAP2 and the Ring finger promoted substrate-independent ubiquitination in the presence of Casp3 (Fig. 5B, lanes 2 and 3). Furthermore, polyclonal anti-Casp3 antiserum detected slower migrating proteins corresponding to monoubiquitinated forms of pro-Casp3 and active Casp3 subunits (Fig. 5A, lanes 2 and 3). In addition to monoubiquitinated Casp3, slower migrating protein bands corresponding to polyubiquitinated Casp3 were also detected after long exposure (data not shown). In contrast, mutant cIAP2 lacking the Ring finger failed to promote substrate-independent ubiquitination (Fig. 5B, lane 4) or ubiquitination of Casp3 (Fig. 5A, lane 4). These results suggest that Casp3 is another substrate for cIAP2 ubiquitin ligase and that the Ring finger of cIAP2 alone is sufficient to mediate ubiquitination of Casp3.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   cIAP2 promotes ubiquitination of caspase-3. In vitro ubiquitination assays were performed as described in the legend to Fig. 2. Purification of active Casp3 has been described (29, 30). A, the transfer membrane was probed with anti-Casp3 antiserum (Pharmingen) to detect ubiquitination of Casp3. Lane 1, non-reacted; lane 2, reacted for 90 min; lane 3, the Ring finger alone; lane 4, mutant cIAP2 lacking the Ring finger. B, the transfer membrane was stripped and re-probed with anti-GST antibody to detect substrate-independent ubiquitination. Samples loaded are the same as for panel A.

Using in vitro ubiquitination assays, we have identified ubiquitin-protein ligase activity as a novel function of cIAP2. Similarly, Yang et al. (36) have recently reported that other IAPs, cIAP1 and XIAP, promote autoubiquitination during apoptosis induced by glucocorticoid and etoposide. cIAP2 contains three BIR domains, a CARD domain, and a Ring finger motif. BIR domains are reportedly required for inhibition of proteolytic activities of Casp3 and Casp7 in vitro (9, 10). Our biochemical analyses of cIAP2 indicate that the Ring finger motif can function independently of the BIR and CARD domains and promote both substrate-independent and -dependent ubiquitination. More importantly, we found that Casp7 and Casp3, but not Casp1, can be ubiquitinated by cIAP2, suggesting that Casp7 and Casp3 are specific caspase targets for cIAP2-mediated ubiquitination.

cIAPs would therefore potentially have a dual means of blocking caspase function, through direct inhibition and through ubiquitination leading to degradation or other consequences. Our data suggest that ubiquitination may be a novel mechanism for controlling the induction of apoptosis through negative regulation of caspases. Degradation of spontaneously activated caspase molecules would be a safeguard against their escape from direct inhibition by cIAPs in nonapoptotic cells, which might otherwise lead to catastrophic caspase activation. In addition, monoubiquitination of full-length caspase-3 might inhibit formation of active Casp3 complexes or their proteolytic activity. Given that monoubiquitination is involved in receptor trafficking and endocytosis, it is conceivable that monoubiquitination might be an intracellular targeting signal for caspases (24, 25). Alternatively, caspases might be modified by ubiquitin-related proteins such as SUMO. It will be important to determine whether active caspases are targets for cIAP-mediated ubiquitination in vivo. Based on our results, apoptosis can be viewed as a process that has parallels to the control of other biological processes by ubiquitination (17-20, 24-26, 37, 38).

An unexpected outcome of our study is that the cIAP2 Ring finger motif itself is directly involved in substrate recognition. The spacing and the amino acid residues between the zinc-binding Cys/His residues of Ring fingers are variable (20), and conceivably, different Ring finger motifs might preferentially recognize different substrates. In keeping with this idea, we found that the Ring finger of cIAP2 but not c-Cbl or Apc11 promotes Casp7 ubiquitination, suggesting that the cIAP2 Ring finger alone is sufficient to confer substrate specificity. Nonetheless, the inhibitory interaction of caspases with the BIR domains of cIAPs may be used as an additional mechanism for targeting caspases for Ring finger-mediated ubiquitination and degradation.

	FOOTNOTES

^* This work was supported by Fellowship DRG-1531 from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (to H.-k. H.), by Fellowship 2-41-98 from the American Cancer Society, California Division (to C. A. P. J.), by Grant PF9922801CCG from the American Cancer Society (to J. D. L.), and by Public Health Service Grants CA39780 and CA82683 from the National Cancer Institute (to T. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Frank and Else Schilling American Cancer Society Professor. To whom correspondence should be addressed: Molecular Biology and Virology Laboratory, The Salk Inst. for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-453-4100; Fax: 858-457-4765; E-mail: hunter@salk.edu.

Published, JBC Papers in Press, June 20, 2000, DOI 10.1074/jbc.C000199200

	ABBREVIATIONS

The abbreviations used are: IAP, inhibitor(s) of apoptosis; BIR, baculoviral IAP repeat; CARD, caspase-recruitment domain; HECT, homologous to E6-AP C terminus; GST, glutathione S-transferase; Casp, caspase.

	REFERENCES

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				E. M. Creagh, B. M. Murphy, P. J. Duriez, C. S. Duckett, and S. J. Martin Smac/Diablo Antagonizes Ubiquitin Ligase Activity of Inhibitor of Apoptosis Proteins J. Biol. Chem., June 25, 2004; 279(26): 26906 - 26914. [Abstract] [Full Text] [PDF]

				Q.-H. Yang and C. Du Smac/DIABLO Selectively Reduces the Levels of c-IAP1 and c-IAP2 but Not That of XIAP and Livin in HeLa Cells J. Biol. Chem., April 23, 2004; 279(17): 16963 - 16970. [Abstract] [Full Text] [PDF]

				E. R. McDonald III and W. S. El-Deiry Suppression of caspase-8- and -10-associated RING proteins results in sensitization to death ligands and inhibition of tumor cell growth PNAS, April 20, 2004; 101(16): 6170 - 6175. [Abstract] [Full Text] [PDF]

J. Silke, T. Kratina, P. G. Ekert, M. Pakusch, and D. L. Vaux Unlike Diablo/smac, Grim Promotes Global Ubiquitination and Specific Degradation of X Chromosome-linked Inhibitor of Apoptosis (XIAP) and Neither Cause Apoptosis J. Biol. Chem., February 6, 2004; 279(6): 4313 - 4321. [Abstract] [Full Text]