SMAC negatively regulates the anti-apoptotic activity of ML-IAP

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1 negatively regulates the anti-apoptotic activity of Domagoj Vucic, Kurt Deshayesξ, Heidi Ackerlyξ, Maria Teresa Pisabarroξ, Saloumeh Kadkhodayan#, Wayne J. Fairbrotherξ, and Vishva M. Dixit Departments of Molecular Oncology, Protein Engineeringξ, and Bioanalytical R&D# Genentech, Inc. South San Francisco, CA Running title: negatively regulates To whom correspondence should be addressed: Vishva M. Dixit, Department of Molecular Oncology, Genentech, Inc., South San Francisco, CA 94080, Tel: , Fax: , 1

2 SUMMARY Inhibitors of apoptosis (IAPs) physically interact with a variety of proapoptotic proteins and inhibit apoptosis induced by diverse stimuli. X-linked IAP (X-IAP) is a prototype IAP family member that inhibits several caspases, the effector proteases of apoptosis. The inhibitory activity of X-IAP is regulated by, a protein that is processed to its active form upon receipt of a death stimulus. Cleaved binds X-IAP and antagonizes its anti-apoptotic activity. Here we show that melanoma IAP (), a potent anti-cell death protein and caspase inhibitor, physically interacts with through its BIR domain. In addition to binding full-length, BIR associates with peptides that are derived from the amino-terminus of active, processed. This high affinity interaction is very specific and can be completely abolished by single amino acid mutations either in the amino terminus of active or in the BIR domain of. In cells expressing and X-IAP, coexpression or addition of peptides abrogates the ability of the IAPs to inhibit cell death. These results demonstrate the feasibility of using peptides as a way to sensitize IAP-expressing cells to pro-apoptotic stimuli such as chemotherapeutic agents. 2

3 INTRODUCTION Programmed cell death, or apoptosis, is a genetically regulated mechanism that plays an important role in development and homeostasis in metazoans (1). Abnormalities in programmed cell death that lead to early cell death or the absence of normal cell death have been linked to a variety of human diseases including neurodegenerative disorders and cancer (2). Currently there are two well-characterized apoptotic pathways, one initiated through the engagement of cell surface death receptors by their specific ligands (3), and the other triggered by changes in internal cellular integrity (4). Both pathways eventually converge, resulting in activation of caspases, cysteine-dependent aspartate-specific proteases that comprise the effector arm of the apoptotic process (5). The major regulators of caspases are the IAPs or inhibitors of apoptosis (6). Originally identified in baculoviruses by their ability to substitute functionally for P35, a potent anti-apoptotic gene product (7-9), IAPs have been discovered in both invertebrates and vertebrates (10-18). Members of the IAP family are characterized by one to three tandem baculovirus IAP repeat (BIR) motifs and most of them also possess a carboxy-terminal RING finger motif (6). IAPs inhibit apoptosis induced by a variety of stimuli and interact with multiple cellular partners (19). The anti-apoptotic activity of several IAPs has been attributed to their ability to inhibit caspases (15,20,21). Human X- chromosome-linked IAP (X-IAP), for example, inhibits active caspases-3 and -7 and 3

4 Apaf-1-cytochrome-c-mediated activation of caspase-9 (22,23). This inhibitory activity is mediated through distinct BIR domains of X-IAP; the BIR2 domain and preceding linker region inhibit caspases-3 and 7, while BIR3 blocks caspase-9 (24-26). Similarly, the anti-apoptotic activity of melanoma IAP, or, is attributed to its lone BIR domain (15). can bind and inhibit caspase-9 through its BIR domain and mutations in the BIR reduce both inhibition of caspase-9and general anti-apoptotic activity (15). IAPs are themselves regulated, by proteins that block their anti-apoptotic activity (27). In Drosophila, Reaper (RPR), HID and GRIM physically interact with and inhibit the anti-cell death activity of D-IAP1 and D-IAP2, fly members of the IAP family (28-30). /DIABLO performs a similar function to RPR, HID and GRIM in mammals (31,32). An amino-terminal signal sequence targets to mitochondria (31), but during apoptosis, is processed into the active form and released into the cytosol where it binds IAPs and prevents them from inhibiting caspases (31,33). Thus,, by binding to the BIR2 and BIR3 domains of X-IAP, abrogates inhibition of the caspases-3 and -9 (34). Interestingly, the only sequence homology between insect RPR, HID and GRIM and human is in the four amino-terminal residues of the active proteins (35). The same region is present in the linker peptide of processed caspase-9 (35) and in HtrA2, a recently identified -like IAP antagonist (36-39). This short peptide fits into a small hydrophobic pocket on the surface of the X-IAP BIR3 domain 4

5 and is essential for binding IAPs and blocking their caspase-inhibitory activity (34,40,41). In this study we demonstrate that physically interacts with and abrogates the ability of to inhibit apoptosis. Peptides derived from the amino terminus of active are also shown to bind and attenuate its antiapoptotic activity. The specificity of the : interaction is supported by the finding that single amino acid changes in the amino terminus of active or the BIR domain of completely abolish their association. EXPERIMENTAL PROCEDURES Expression constructs - Plasmids expressing β-galactosidase, P35, Myc-XIAP and Myc-, as well as deletions and site-specific mutants, have been previously described (15,42,43). cdna encoding human was PCR-amplified from a HeLa cdna library with -specific primers. cdna was then subcloned into the mammalian expression vector pcdna3.1 with a carboxy-terminal Flag tag (Invitrogen). The 55M construct (amino acids ) was also PCRamplified and similarly subcloned into the pcdna3.1 vector. Sequences encoding X- IAP BIR3 (amino acid residues ) and BIR (amino acid residues ) were subcloned into pet15b vector (Novagen) for bacterial expression. 5

6 Cell culture, antibodies and immunoprecipitations - Human 293T embryonic kidney cells and MCF7 human breast carcinoma cells were cultured as described previously (43).The primary antibodies used were anti-flag M2 (Sigma-Aldrich), anti- Myc (Covance), anti-caspase-9 (Pharmingen), and anti- (Imgenex). Immunoprecipitations were performed as previously described (15,43). Protein purification, peptide generation and peptide binding assays E. coli strain BL21 (DE3) transformed with pet-15b-x-iap-bir3 or pet-15b--bir was induced with 1mM isopropyl β-d-thiogalactoside (IPTG) for 4 hours at 30 C, pelleted and resuspended in 100 ml Buffer A (50 mm Tris (ph 8.0), 300 mm NaCl, 5 mm β-mercaptoethanol, 0.5 mm PMSF, 2 mm benzamidine) containing 5 mm imidazole. Lysate produced by homogenization and centrifugation was passed through Ni-agarose and Superdex 75 sizing columns, washed, eluted and dialyzed against buffer containing 50 mm Tris (ph 8.0), 120 mm NaCl, 5 mm DTT, 0.5 mm PMSF, 2 mm benzamidine, 50 µm zinc acetate and 1 mm sodium azide. Antennapedia (RQIKIWFQNRRMKWKK-NH2) fusions were constructed using standard solid phase methods utilizing Wang resin with a rink amide linker and fluorenylmethyl (Fmoc) chemistry. The peptides were cleaved using a mixture of 1:0.05:0.02 trifluoroacetic acid (TFA):triisopropyl silane:ethyl methyl sulfide. The 6

7 resulting peptides were purified by preparative HPLC and analyzed by LCMS. The Fluorescein labeled peptides AVPIAQKSEK-5-FAM (), ATPFQEGLRK-5-FAM (Caspase-9) and AVPSPPPASK-5-FAM (HtrA2) and biotin derivatives AVPIAQKSEKbiotin (), ATPFQEGLRK-biotin (Caspase-9), and AVPSPPPASK-biotin (HtrA2) were constructed as C-terminal amides using modified Fmoc chemistry with the aminoterminal amino acid N-α-protected with tert-butoxycarbonyl (Boc). The labels were attached through N-ε of a lysine attached to the C-terminus of the nine residue peptides by selectively removing the N-ε [4,4 dimethyl-2,6-dioxoxocyclohex-1-ylidene]-3- methylbutyl (ivdde) protecting group with 2% hydrazine in dimethylacetamide (DMA). Coupling with either 5-carboxyfluorescein (5-FAM) or biotin was achieved using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexaphosphate (PyBOP) as the condensation reagent. The control peptide biotin-tgwetwvcooh was made by biotinylating the amino-terminus of the peptide. TFA cleavage and HPLC purification obtained the labeled peptides. Fluorescence polarization experiments were performed in 96-well plates on the Analyst HT (Molecular Devices Corporation). Binding experiments were performed using 1:3 serial dilutions of BIR and X-IAP BIR3 domains starting from 300 µm in 50mM Tris buffer ph=7.2, 120mM NaCl, 5mM DTT. Approximately 1nM of each 5-carboxyfluorescein-tagged probe was added to a set of wells containing 7

8 the protein dilutions. The Kd values of the probe-protein interactions were calculated using Klotz plots and confirmed by Scatchard analysis. Three-dimensional modeling and sequence analysis The high resolution crystal structure of /DIABLO complexed with the BIR3 domain of X-IAP (40) was used to model the N-terminal four residues (Ala-Val-Pro-Ile) of /DIABLO into the binding groove of the previously modeled structure of (15). Both protein structures, X-IAP and, were superimposed by their alpha carbons (rmscα = 0.47 Å), and docking of the peptide was manually performed on the binding site. The / complex was energy minimized using DISCOVER (Molecular Simulations, Inc.). Amino acid sequence alignments were performed using ClustalW (44). Apoptosis assay Apoptosis assays were performed essentially as described previously (15,42). RESULTS AND DISCUSSION physically interacts with has been shown to physically associate with several IAP family members, most prominently with X-IAP (31). To determine if can bind, we co-expressed with X-IAP, or 8

9 vector control. Upon overexpression, was processed to its active form whereby the first amino-terminal 55 amino acids are cleaved (Fig. 1A). Association of and X-IAP with active was demonstrated by immunoprecipitation and the interactions occurred with similar efficiency (Fig. 1A). We investigated the portion of that is responsible for interaction with using truncation mutants of ML- IAP containing either the BIR or the RING finger domains (Fig. 1B). The BIR domain, like full-length, immunoprecipitated active but the RING finger domain did not (Fig. 1B). Thus, physically interacts with through its BIR domain. BIR binds RPR-like peptides Sequence comparison of the active forms of human, HtrA2 and caspases-9, and fly RPR, HID and GRIM revealed a strong similarity in their amino-termini (Fig. 2A) (35). Since the four amino-terminal residues (Ala, Val, Pro, Ile) of active fit into the binding groove on the surface of the X- IAP BIR3 (40), we investigated whether a similar complex might form between BIR and peptide. A three-dimensional model based on the reported structure of the /X-IAP BIR3 complex predicted that peptide should bind BIR much as it does X-IAP BIR3 (Fig. 2B). To test the validity of our model, we examined whether, HtrA2, caspase-9 or control peptides could bind the BIR3 domain of X-IAP or the BIR of., HtrA2 and caspase-9 peptides efficiently pulled down purified X-IAP BIR3 and 9

10 BIR, whereas a control peptide did not (Fig. 3A). The binding affinities of these peptides for X-IAP BIR3 and BIR were determined by a fluorescence polarization based assay (Fig. 3B and C)., HtrA2 and caspase-9 peptides exhibited binding affinities in the low micromolar range for X-IAP BIR3 (Fig. 3B) and in the sub-micromolar range for BIR (Fig. 3C). These results indicate that BIR binds and other mammalian RPR-like peptides with high affinity and in a manner similar to X-IAP BIR3. blocks the anti-apoptotic activity of Processing of exposes the four amino-terminal residues that mediate binding to X-IAP and are required for to block caspase inhibition by X-IAP. We investigated whether this region of is also required for to bind and abrogate its anti-cell death activity. Active was mimicked in coimmunoprecipitation experiments using an amino-terminally truncated form of (amino acids ); (55M) (Fig. 4A). Full-length that was processed to its active form was able to bind or X-IAP (Fig. 4B). In contrast, there was no interaction between 55M and or X-IAP (Fig. 4B). Consistent with these results, but not 55M was able to abrogate -mediated inhibition of adriamycin-induced apoptosis (Fig. 4C). Addition of a single methionine to the amino terminus of active was therefore sufficient to block its inhibitory effect on. 10

11 We also tested whether peptides corresponding to the nine residues at the amino terminus of active would reverse -mediated inhibition of apoptosis. and M- peptides, the latter having an additional amino-terminal methionine, were synthesized as fusions with antennapedia peptide. Antennapedia peptides permit chimeric fusions to gain entry into the cell where they can engage their targets (45). Expression of or X-IAP efficiently blocked adriamycin-induced apoptosis and addition of M- peptides did not have a significant inhibitory effect on the protective activity of IAPs (Fig. 4D). However, addition of peptides almost completely negated the ability of and X-IAP to inhibit apoptosis (Fig. 4D). Thus, co-expression of full-length or addition of -like peptides abrogates the anti-apoptotic activity of. disrupts binding of to processed caspase-9 To better understand the mechanism by which antagonizes the anti-apoptotic function of, we investigated the effect of on the ability of to bind caspase-9. When overexpressed, caspase-9 undergoes autocatalytic processing and it is the processed form that physically interacts with X-IAP (35,46). Similarly, co-immunoprecipitated processed caspase-9 but not its zymogen precursor (Fig. 5A). The interaction between and caspase-9 is highly specific because mutation of aspartate 138 to alanine in the BIR domain of completely abolished the ability of to bind processed caspase-9 (Fig. 5A). 11

12 Co-expression of caspase-9 and with prevented from interacting with caspase-9 (Fig. 5B). Instead, associated with (Fig. 5B), indicating that binding to disrupts the interaction of with caspase-9. Mutation in the binding pocket of abrogates interaction with Previously, we demonstrated that mutating aspartates 120 and 138 to alanine in abolishes its anti-apoptotic activity (15). The effect of these mutations on interaction with was characterized. As an additional control we expressed a double glutamate mutant (E87,88A) that possessed equivalent anti-apoptotic activity to wild type. immunoprecipitated and E87,88A ML- IAP but no interaction was observed between and the D120A or D138A mutants (Fig. 6A). The same was true in the inverse experiment where and E87,88A mutant, but not D138A mutant, immunoprecipitated (Fig. 6B). To examine the interaction of endogenous with, we generated stably-transfected MCF-7 cell lines expressing Flag-tagged or the D138A mutant. Consistent with our earlier results (Fig. 6A and B), endogenous was coimmunoprecipitated from cells expressing, but not D138A mutant (Fig. 6C). To determine if peptide can bind IAPs expressed in cells, lysates prepared from 293T cells transfected with X-IAP, or D138A mutant were incubated with biotinylated peptide or control peptide. Immunoblotting following peptide 12

13 precipitation revealed that peptide precipitated X-IAP and but not the D138A mutant (Fig. 6D). Therefore, aspartate 138 in the BIR domain of ML- IAP is a critical residue for the binding of. Inhibition of caspases by IAPs occurs at the core of the apoptotic machinery and thus regulation of IAPs by and -like proteins represents a key control point in deciding cell fate. We have shown that is regulated by since physically associates with and abrogates the anti-apoptotic activity of. Interaction with is mediated through the BIR domain of and aminoterminal residues of active. Three-dimensional modeling together with protein binding studies demonstrated that binds the BIR of with high affinity and in a manner similar to which it binds the X-IAP BIR3 domain. Further highlighting the similarity of these interactions, mutation of residues in BIR that correspond to functionally important amino acids in X-IAP BIR3 (41) interrupted binding to. Aspartate 138 of is predicted to be in contact with alanine at the amino terminus of active peptide and, therefore, is critical for the interaction of with. Previous studies have shown the importance of the amino-terminus of active for binding X-IAP (33,40,41). We have demonstrated that this short region is important for binding and antagonizing its anti-apoptotic activity. We have also demonstrated that peptides can specifically bind and inhibit IAPs. peptide 13

14 bound purified BIR domains and X-IAP and from cell lysates, and in doing so, nullified IAP s ability to inhibit apoptosis. To our knowledge, this is the first report in which the functional potential of or -like peptides has been explored in a cellular context. Such peptides have obvious therapeutic potential in the treatment of cancer cells that resist conventional cytotoxic therapies. IAPs do contribute to the resistance of cancers to chemotherapeutic agents since they are widely and, in some cases like, specifically expressed in human malignancies (15,19). Acknowledgments We thank Karen O Rourke and Kim Newton for critically reading the manuscript, Grace Mausisa for input in the flourescence polarization assays, sequencing lab for help with sequencing and the members of the Dixit lab for helpful discussions. REFERENCES 1. Steller, H. (1995) Science 267, Thompson, C. B. (1995) Science 267, Ashkenazi, A., and Dixit, V. M. (1998) Science 281, Budihardjo, I., Oliver, H., Lutter, M., Luo, X., and Wang, X. (1999) Annu Rev Cell Dev Biol 15, Vaux, D. L., and Korsmeyer, S. J. (1999) Cell 96,

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19 45. Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trends Cell Biol 8, Ekert, P. G., Silke, J., Hawkins, C. J., Verhagen, A. M., and Vaux, D. L. (2001) J Cell Biol 152, FIGURE LEGENDS Figure 1. physically interacts with. A, 293T cells were transiently transfected with and X-IAP, or vector. After 40 h, cells were lysed in NP-40 lysis buffer and lysates were immunoprecipitated (IP) with anti-myc antibody. Samples were then immunoblotted (W) with anti-flag and anti-myc antibodies. B, 293T cells were transiently transfected with and indicated constructs or vector. After 40 h, cells were lysed in NP-40 lysis buffer and samples were processed as in A. FL- designates full-length. Figure 2. A, Amino acid sequence alignment of the nine amino-terminal residues of active of human, HtrA2 and caspases-9, and fly RPR, HID and GRIM. B, Model of the / complex, showing the binding of the amino-terminal four residues of (in yellow) onto the binding groove (green). The crystal structure of X-IAP used for the modeling is shown in gray. Peptide and protein residues 19

20 involved in the interaction are labeled with residues in bold and X-IAP residues below them. Figure 3. A, Binding of X-IAP BIR3 and BIR to biotinylated, HtrA2, caspase-9 or control (irrelevant) peptides. X-IAP BIR3 and BIR were incubated in NP-40 buffer with indicated peptides (10 µm) for several hours and the complexes precipitated with streptavidin agarose. Following acrylamide gel electrophoresis, precipitated proteins were visualized by silver staining. Binding affinities of 5-FAMlabeled peptides to X-IAP BIR3 (B) and BIR (C) as determined by fluorescence polarization-based assay. Kds of 5-FAM-coupled peptides were 1.5 µm (), 1.8 µm (caspase-9) and 8.0 µm (HtrA2) for X-IAP BIR3, and 0.10 µm (), 0.15 µm (caspase-9) and 0.50 µm (HtrA2) for BIR. Figure 4. blocks the anti-apoptotic activity of. A, Schematic representation of and 55M constructs. Numbers designate the codingregion boundaries expressed in each construct. B, 293T cells were transiently transfected with or 55M and X-IAP, or vector. After 40 h, cells were lysed in NP-40 lysis buffer and samples were processed as in Figure 1A. C, MCF7 cells were transiently transfected with the reporter plasmid pcmv-βgal and either vector control 20

21 alone or plus vector, or 55M. Following transfection, cells were exposed to adriamycin, stained with X-gal and apoptosis assessed as described previously (42). D, MCF7 cells were transiently transfected with the reporter plasmid pcmv-βgal and either vector control, X-IAP or. Following transfection, or M- peptides (50 µm) were added where indicated and cells exposed to adriamycin. Apoptosis was assessed as previously described (42). Figure 5. disrupts binding of to processed caspase-9. A, 293T cells were transiently transfected with caspase-9 and, mutant or vector. After 40 h, cells were lysed in NP-40 lysis buffer and lysates immunoprecipitated (IP) with anti-myc antibody. Samples were then immunoblotted (W) with anti-caspase-9 and anti-myc antibodies. B, 293T cells were transiently transfected with indicated constructs. Vector plasmid was used to keep the amount of transfected DNA equal. After 40 h, cells were lysed in NP-40 lysis buffer and lysates immunoprecipitated (IP) with anti-myc antibody. Samples were then immunoblotted (W) with anti-caspase-9, anti-flag and anti-myc antibodies. IgG designates monoclonal immunoglobulin G antibody. Figure 6. Mutation in the binding pocket of abrogates interaction with. A and B, 293T cells were transiently transfected with and indicated 21

22 constructs. After 40 h, cells were lysed in NP-40 lysis buffer and lysates immunoprecipitated (IP) with anti-flag antibody (A) or anti-myc antibody (B). Samples were then immunoblotted (W) with anti-flag and anti-myc antibodies. C, MCF-7 cells stably expressing Flag-tagged or D138A mutant were lysed in NP-40 lysis buffer and lysates immunoprecipitated (IP) with an anti-flag antibody. Samples were then immunoblotted (W) with anti- antibody. D, 293T cells were transiently transfected with X-IAP, or D138A mutant. After 40 h, cells were lysed in NP-40 lysis buffer and lysates incubated with or control (irrelevant) biotinylated peptides (10 µm) for several hours. Complexes were precipitated with steptavidin agarose, washed extensively and immunoblotted (W) with anti-myc antibody. 22

23 A B vector X-IAP vector ML-BIR ML-RING IP: amyc W: aflag IP: amyc W: aflag FL- X-IAP W: aflag W: amyc FL- ML-BIR ML-RING W: aflag W: amyc Vucic et al. Figure 1

24 A HtrA2 caspase-9 HID GRIM RPR B Vucic et al. Figure 2

25 A Biotinylated peptides input HtrA2 caspase-9 control X-IAP BIR3 BIR B Fraction Bound C XIAP-BIR Free Protein (µm) Casp-9 HtrA2 1.2 ML-BIR Fraction Bound Free Protein (µm) Casp-9 HtrA2 Vucic et al. Figure 3

26 A 55M AVPI MAVPI B C X-IAP vector X-IAP vector FL- 55M X-IAP D IP: αmyc W: αflag W: αflag W: αmyc % APOPTOSIS Adr (0.5 µg/ml) vector vector Adr (0.5 µg/ml) 55M 60 % Apoptosis vector X-IAP vector X-IAP vector X-IAP - M- Antennapedia-fused peptides Vucic et al. Figure 4

27 A vector D138A P35 caspase-9 caspase-9-42 IP: αmyc W: αcaspase-9 caspase-9 P35 caspase-9-42 W: αcaspase-9 B caspase-9 P35 caspase-9 caspase-9 P35 caspase-9 IgG W: αmyc IP: αmyc W: αcaspase-9 W: αcaspase-9 IP: αmyc W: αflag W: αflag W: αmyc Vucic et al. Figure 5

28 A B E87,88A D120A D138A vector E87,88A D138A IP: αflag W: αmyc W: αmyc IP: αmyc W: αflag C D138A W: αflag D X-IAP D138A peptide-biotin control-biotin W: αflag W: αmyc IP: αflag W: α W: α W: αflag X-IAP D138A IP: biotin W: αmyc W: αmyc Vucic et al. Figure 6

29 negatively regulates the anti-apoptotic activity of Domagoj Vucic, Kurt Deshayes, Heidi Ackerly, Maria Teresa Pisabarro, Saloumeh Kadkhodayan, Wayne J. Fairbrother and Vishva M. Dixit J. Biol. Chem. published online January 18, 2002 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts