The Polypeptide Chain-releasing Factor GSPT1/eRF3 Is Proteolytically Processed into an IAP-binding Protein
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m303179200
ISSN1083-351X
AutoresR.P. Hegde, Srinivasa M. Srinivasula, Pinaki Datta, Muniswamy Madesh, Richard Wassell, Zhijia Zhang, Naeun Cheong, Julie Nejmeh, Teresa Fernandes‐Alnemri, S. Hoshino, Emad S. Alnemri,
Tópico(s)Mitochondrial Function and Pathology
ResumoSmac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation. Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation. The inhibitors of apoptosis proteins (IAPs) 1The abbreviations used are: IAP, inhibitor of apoptosis protein; IBM, IAP-binding motif; Ub, ubiquitin; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; GST, glutathione S-transferase; TRAIL, TNF-related apoptosis-inducing ligand. constitute a family of proteins that interact with active caspases, inhibit their protease activity and prevent programmed cell death or apoptosis (1Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1579) Google Scholar). 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In this report we describe the characterization of a third human IBM-containing protein. This protein is a proteolytically processed form of a previously identified protein known as GSPT1 (G1 to S phase transition protein), which functions in translation through interaction with poly(A)-binding protein PABP and as a polypeptide chain release factor 3 (eRF3) (36Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Uchida N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Like Smac and HtrA2, the processed GSPT1 contains a conserved IBM (AKPF) at its N terminus, which is exposed after proteolytic cleavage of a 69-residue leader sequence. Our results suggest that the processed GSPT1 could participate in the apoptotic pathways by binding to IAPs and inhibiting their activity or target them for proteasome-mediated degradation. Affinity Purification of IAP-binding Proteins from 293 Cells—IAP binding proteins in 293 cellular extracts were purified on GST-BIR3-bound beads as described in detail previously (31Hegde 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). cDNA Cloning and Expression of Recombinant Proteins—The full-length human GSPT1 cDNA clone was obtained from the IMAGE Consortium (GenBank™ accession number AL516539). Constructs encoding full-length GSPT1 or truncated mutants were generated by PCR using modified complementary PCR adapter-primers. C-terminal FLAG-epitope tagging was done by cloning the PCR generated GSPT1 cDNAs in-frame into the mammalian expression vector FLAG-C-pcDNA3. C-terminal His6-epitope tagged GSPT1 variants (processed GSPT1, residues 70–634; GSPT1-ΔAKPF, residues 74–634) were generated by cloning the PCR generated GSPT1 cDNAs in the bacterial expression vector pET21A. The N7-GSPT1-GST was constructed by fusing the first 7 residues of processed GSPT1 (AKPFVPN) to the N terminus of GST. This was done by PCR using an oligonucleotide primer encoding the first 7 residues of processed GSPT1 followed by the first 7 residues of GST (5′-primer) and a second GST termination primer (3′-primer). The PCR product was cloned into the bacterial expression vector pET21A. The ubiquitin-GSPT1-Δ69-FLAG construct was made by fusing PCR generated human ubiquitin cDNA (encoding residues 1–76) to the 5′-end of pGSPT1 cDNA (encoding residues 70–634) using overlapping PCR. The ubiquitin-pGSPT1 PCR product was then cloned in-frame into the mammalian expression vector FLAG-C-pcDNA3. The plasmid encoding C-terminal GFP-tagged Drosophila Reaper protein was constructed by fusing the Reaper cDNA to the GFP cDNA using pEGFP-N1 (Clontech). Full-length XIAP and its BIR3-RING (residues 243–497) domain were overexpressed in Escherichia coli strain DH5α as N-terminally GST-tagged proteins using a pGEX 4T vector (Amersham Biosciences). The bacterial expression vector GST-c-IAP1 was a kind gift of Jonathan Ashwell. The bacterial expression vector GST-c-IAP1-ΔRING was constructed in pGEX 4T. Full-length XIAP, c-IAP1, and c-IAP2 were in vitro translated in the presence of [35S]methionine in reticulocyte lysates using MYC-pcDNA3 constructs. Mature Smac or HtrA2 and its mutants were overexpressed in E. coli strain BL21(DE3) as C-terminally GST- or His6-tagged proteins using a pET-28-GST or pET-28 vectors, respectively. In Vitro Interaction Assays—All in vitro interactions were performed as described previously (20Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (860) Google Scholar, 38Srinivasula 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, 39Wu G. Chai J. Suber T.L. Wu J.W. Du C. Wang X. Shi Y. Nature. 2000; 408: 1008-1012Crossref PubMed Scopus (710) Google Scholar). In Vitro Potentiation of Caspase Activity by IAP-binding Proteins— These procedures were performed as described previously (31Hegde 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, 38Srinivasula 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). Cell Death and Caspase Activity Assays—The ability of processed GSPT1 (pGSPT1) to potentiate tunicamycin, Brefeldin A, or TRAIL-induced apoptosis (Fig. 4C) was assayed by transfecting human HeLa cells (1 × 105 cells/well) in 6-well plates with 0.3 μg of pEGFP-N1 reporter plasmid (Clontech), 1.2 μg of empty vector or constructs encoding human ubiquitin or a C-terminally FLAG-tagged Ubiquitin-GSPT1-Δ69 (UB-GSPT1-Δ69-FLAG) using the LipofectAMINE™ method. Cells were treated with tunicamycin (2.5 μg/ml), Brefeldin A (5 μg/ml), or TRAIL (1 μg/ml) for 7 h. Cells were stained with propidium iodide and DAPI stains. Normal and apoptotic GFP-expressing cells were counted using fluorescence microscopy. The percentage of apoptotic cells in each experiment was expressed as the mean percentage of apoptotic cells as a fraction of the total number of GFP-expressing cells. The ability of processed GSPT1 (pGSPT1) to enhance the caspase activity of transfected cells (Fig. 4B) was assayed after transfecting 293 cells with empty vector or constructs encoding human ubiquitin or a C-terminally FLAG-tagged ubiquitin-GSPT1-Δ69 (UB-GSPT1-Δ69-FLAG) using the LipofectAMINE™ method. 24 h after transfection the cells were lysed in buffer A (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 250 mm sucrose) and S100 extracts were prepared by centrifuging the lysates at 100,000 × g. The caspase activity in the S100 extracts was assayed after stimulation with cytochrome c (5 μg/ml) and dATP (1 mm) over a 90-min period of time using the caspase substrate DEVD-AMC. The release of AMC from the DEVD-AMC substrate was measured by fluorometry. In Vitro Ubiquitination Reactions—In vitro ubiquitination assays were carried out as described below. Briefly, bacterially expressed GST-c-IAP1, or GST-c-IAP1-ΔRING were incubated with pGSPT1-His6, pG-SPT1-ΔAKPF, N7-GSPT1-GST, or Reaper-GST for 30 min. After the brief incubation, glutathione-Sepharose beads were added to the reaction mixtures for 1 h. The protein-bound beads were then washed with ubiquitination reaction buffer (50 mm Tris-HCl, pH 7.4, 2.5 mm MgCl2, and 0.5 mm dithiothreitol). Approximately 3–5 μg of bound GST fusion proteins was incubated in ubiquitination reaction buffer (50 mm Tris, pH 7.4, 2 mm ATP, 2.5 mm MgCl2, 0.5 mm dithiothreitol, 1 mm creatine phosphate, 15 units of creatine phosphokinase) with or without 100 ng of E1, 100 ng of E2 (UbcH5b), 1 μg of Ub, and 40 ng of Ub aldehyde at 30 °C for 1 h. The reactions were terminated by adding SDS-PAGE sample buffer. The samples were then fractionated onto a 7.5% SDS-polyacrylamide gel and Western blotted for ubiquitinated protein products using monoclonal antibodies to ubiquitin or c-IAP1. Immunostaining and Confocal Microscopy—Cells grown on coverslips were fixed with 3.7% paraformaldehyde and then stained with polyclonal anti-GSPT1 raised against human GSPT1 peptide 179–190 and a mouse anti-human hemoxygenase (HO-1) monoclonal antibody (Stressgen Biotechnologies). Fluorescein isothiocyanate-conjugated anti-rabbit and rhodamine-conjugated anti-mouse antibodies were used as secondary antibodies. In some experiments FLAG monoclonal antibody (Sigma) and anti-S3A ribosomal protein polyclonal antibody were used. After staining, the coverslips were mounted on slides and observed using Confocal Microscopy. Subcellular Fractionation–These procedures were performed as described previously (31Hegde 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). Identification of a Processed Form of GSPT1 as a BIR3-binding Protein—Using a GST-BIR3 fusion protein as an affinity reagent to purify new IAP-binding proteins from extracts of human cells and mouse tissues, we previously isolated 3 proteins of molecular masses 23, 38, and 80 kDa (31Hegde 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). The 23 and 38 kDa proteins were identified as mitochondrial mature Smac and HtrA2 proteins, respectively. To identify the third 80 kDa protein, a large-scale preparation of GST-BIR3-affinity purified proteins from 293 cells was fractionated by SDS-PAGE and Coomassie stained bands were cut and subjected to microsequencing and mass spectrometric peptide mass fingerprinting as described before (31Hegde 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). N-terminal sequence analysis of the 80-kDa band revealed an N-terminal sequence AKPFVPNVHAAEFVP. A PSI-BLAST analysis of the NCBI protein data base revealed that this sequence is found only in human GSPT1 and GSPT2 proteins. Mass spectrometric data of a tryptic peptide digest of the 80-kDa band confirmed that it is a processed form of the human GSPT1 protein, lacking the first 69 residues (Fig. 1) (36Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). As the N-terminal sequence indicates, the processed BIR3-affinity purified GSPT1 protein starts at residue 70 with an N-terminal AKPF sequence, which represent a conserved IBM (Fig. 1, A and B). A BLAST search of the GenBank™ data base revealed that the AKPF motif is conserved in all vertebrate and invertebrate GSPT1 homologues (Fig. 1B). To confirm that the 80-kDa GST-BIR3-binding protein is a processed form of GSPT1, we bound extracts from 293 cells to wild type GST-BIR3 or the GST-BIR3-(E314S) mutant, which does not bind to the IBM, and then analyzed the bound proteins by Western blotting with a GSPT1-specific antibody. As shown in Fig. 1C, the GSPT1-specific antibody detected an 80-kDa band in the GST-BIR3 lane but not in the GST-BIR3-(E314S) lane. The above results were further confirmed by stable expression of a C-terminal FLAG-tagged full-length GSPT1 protein in 293 cells (Fig. 1D). Western blot analysis of lysates made by solubilizing the transfected cells in SDS-sample buffer showed two bands corresponding to the full-length (lane 2, upper band) and the processed (lane 2, lower band) GSPT1 proteins. The amount of the processed GSPT1 isoform in the cellular lysate were estimated to be ∼10–20% of the amount of the full-length GSPT1 protein. As expected, only the processed GSPT1 isoform was able to bind to GST-BIR3. Taken together, our data indicate that GSPT1 undergoes limited proteolytic processing in 293 cells to generate an IAP-binding isoform of GSPT1. Based on the deduced amino acid sequence of the cloned full-length GSPT1 protein, the IBM is located at residue 70, suggesting that the IAP-binding isoform of GSPT1 is generated by proteolytic processing of the full-length GSPT1 at residue 69. To determine whether a recombinant GSPT1 lacking the first 69 residues (processed GSPT1) could interact with the GST-BIR3 fusion protein, we expressed this protein with a C-terminal His6 tag in bacteria and purified it to apparent homogeneity (Fig. 1E). Similarly, Smac and HtrA2 proteins were expressed in bacteria and used as positive controls. Like Smac and HtrA2, the processed GSPT1 was able to interact with the wild type GST-BIR3 protein but not with the GST-BIR3-E314S mutant. Processed GSPT1 did not significantly interact with GST-BIR1,2 fusion protein, whereas Smac and HtrA2 interacted, though relatively less compared with their interaction with GST-BIR3. However, deletion of the AKPF motif in GSPT1 completely abolished binding to GST-BIR fusion proteins, indicating that the AKPF motif is necessary for binding of GSPT1 to the BIR domain. Processed GSPT1 Interacts with Full-length IAPs—To determine whether processed GSPT1 could interact with human full-length IAPs, we performed in vitro interaction assays with recombinant processed GSPT1 proteins and in vitro translated human XIAP, cIAP1, and cIAP2. As shown in Fig. 2A, the recombinant processed GSPT1 was able to interact with XIAP, cIAP1, and cIAP2. As expected, deletion of the AKPF motif abolished the interaction of GSPT1 with these IAPs. To determine whether the endogenous processed GSPT1 is present in a complex with the endogenous XIAP in 293 cellular extracts, we performed co-immunoprecipitation experiments using an antibody specific for GSPT1. These experiments demonstrated that the endogenous processed GSPT1 isoform can indeed associate with endogenous XIAP (Fig. 2B). Combined, these data indicate that processed GSPT1 interacts specifically with cellular IAPs. Processed GSPT1 Can Potentiate Caspase Activation—Previous studies demonstrated that the IAP-binding proteins such as Smac and HtrA2, could potentiate caspase activation by preventing binding of caspases to IAPs or disrupting already formed caspase-IAP complexes. Thus, we performed in vitro caspase activation assays to determine whether the processed GSPT1 protein could promote caspase-9 activity in HEK293 S100 extracts in the presence of inhibitory concentration of XIAP (Fig. 3). To measure the caspase-9 activity in these extracts, we added 35S-labeled procaspase-3 to the S100 extracts and stimulated the extracts with cytochrome c and dATP. Previously we have shown that both Smac and HtrA2 could potentiate caspase cleavage under these conditions (10Hays R. Wickline L. Cagan R. Nat. Cell Biol. 2002; 4: 425-431Crossref PubMed Scopus (109) Google Scholar). As shown in Fig. 3A, the processed GSPT1 protein was able to neutralize the XIAP inhibition of the caspase activity in the S100 extracts, though at a slightly lower potency than Smac or HtrA2. This activity was dependent on an intact IAP-binding motif (AKPF), as deletion of this motif abolished the caspase-promoting activity of GSPT1. Furthermore, the first N-terminal 7 residues of GSPT1 coupled to GST was sufficient to neutralize XIAP inhibition to the same extent, as does the processed GSPT1 at identical concentrations. This indicates that the IBM of GSPT1 is responsible for both its IAP-binding and caspase promoting activities. The potency of the caspase promoting activity of GSPT1 was further confirmed by assaying the DEVD cleaving activity of S100 extracts stimulated with cytochrome c and dATP under similar conditions as above (Fig. 3B). The data shows that the DEVD cleaving activity in the S100 extracts upon incubation with Smac, HtrA2, or the GSPT1 proteins, correlates very well with the extent of caspase-3 cleavage shown in Fig. 3A, under the same conditions. Ectopic Expression of Processed GSPT1 in Human Cells Enhances Caspase Activation and Apoptosis—To express the processed IAP-binding form of GSPT1 we engineered a construct encoding an N-terminal Ub-tagged GSPT1-fusion protein, which lacks the first 69 residues (UB-GSPT1-Δ69)(Fig. 4A). The engineered protein also contains a C-terminal FLAG tag to allow detection by Western blotting. The Ub fusion protein approach is based on the observation that the covalent bond between Ub and other proteins can be cleaved by multiple ATP-dependent proteases, which recognize a Ub moiety and cleave at the Ub-adduct junction (13Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar, 14MacFarlane M. Merrison W. Bratton S.B. Cohen G.M. J. Biol. Chem. 2002; 277: 36611-36616Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 15Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-347Crossref PubMed Scopus (394) Google Scholar). As expected, ectopic expression of the UB-GSPT1-Δ69 fusion protein in 293 cells resulted in generation of the processed GSPT1, which was able to bind GST-BIR3 (Fig. 4B, inset). To determine whether transient expression of the processed GSPT1 in 293 cells enhances caspase activity, we incubated extracts from the transfected 293 cells with DEVD-AMC. As shown in Fig 4B, the DEVD cleaving activity in the extracts derived from the processed GSPT1-transfected cells was significantly higher than the activity in the extracts from the Ub- or vector-transfected cells. These data indicate that binding of the processed GSPT1 to cellular IAPs can augment caspase activity in these cells. The ability of the transiently expressed processed GSPT1 to bind to IAPs and augment caspase activation in human cells suggests that it could also potentiate apoptosis. To test this hypothesis, we transfected HeLa cells with the expression construct encoding mutant full-length GSPT1-A70G or processed GSPT1 (UB-GSPT1-Δ69), and then tested their sensitivity to apoptotic agents such as tunicamycin, Brefeldin A, and TRAIL. As shown in Fig. 4C, expression of the processed GSPT1 protein, but not the mutant full-length GSPT1-A70G, enhanced the apoptotic effect of tunicamycin, Brefeldin A, and TRAIL. These observations suggest that GSPT1 could potentiate cell death by neutralizing IAPs. Processed GSPT1 Stimulates the Ubiquitination Activity of c-IAP1—Recent studies demonstrated that binding of the Drosophila IAP-binding proteins reaper and Hid to DIAP1 stimulates its ubiquitination activity (9Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (181) Google Scholar, 12Yoo 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, 13Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar), which could lead to degradation of the formed complex by the proteasomal pathway. To determine whether binding of the processed GSPT1 to cellular c-IAP1 in vitro could stimulate its ubiquitination activity, we bound recombinant processed GSPT1 to wild type or RING deleted mutant human GST-c-IAP1 fusion proteins and then incubated the complexes in the presence or absence of E1, E2, and ubiquitin. As shown in Fig. 5A, the processed GSPT1 was able to stimulate the ubiquitination activity of wild type cIAP1 but not the RING deletion mutant. Furthermore, the ability of processed GSPT1 to stimulate c-IAP1 ubiquitination activity was dependent on its AKPF, as deletion of this motif inhibited ubiquitination (Fig. 5B). No ubiquitination was observed when the first N-terminal 7 residues of processed GSPT1 coupled to GST (N7-GST) was used, indicating that other regions of GSPT1 are required for stimulation of c-IAP1 ubiquitination activity. Moreover, processed GSPT1 was not able to induce the auto-ubiquitination activity of XIAP or c-IAP2 (data not shown). GSPT1 Is Associated with the ER—To determine the subcellular localization of GSPT1, we stained MCF-7 cells with a GSPT1-specific antibody. Consistent with its function in translation and its association with the ribosomes, immunoflourescence, confocal microscopy of the stained MCF-7 cells revealed that the endogenous GSPT1 protein exhibits clear perinuclear staining characteristic of endoplasmic reticulum localization (Fig. 6A, middle panel). To confirm the ER localization of GSPT1, we stained the same cells with an anti-human heamoxyginase antibody (Fig. 6A, left panel), which is a known ER marker. As shown in Fig. 6A, right panel, complete overlap between the GSPT1 and heamoxygenase staining was observed. To confirm the ER localization of GSPT1, we transfected MCF-7 cells with a construct encoding C-terminal FLAG-tagged full-length GSPT1 protein and then stained the cells with an anti-FLAG antibody and an anti-human S3A ribosomal protein antibody, which is another known ER marker (Fig. 6B). The ectopically transfected full-length GSPT1 protein exhibited ER-specific fluorescence, which completely overlapped with the S3A ribosomal protein (Fig. 6B, right panel). The localization of GSPT1 was further confirmed by subcellular fractionation and Western blotting using anti-GSPT1 antibody (Fig. 6C). This analysis reveled the presence of a GSPT1-specific band in the microsomal and soluble cytosolic protein fractions but not in the mitochondrial or nuclear fractions. The presence of GSPT1 in the cytosolic fraction indicates that GSPT1 is loosely associated with the ER and its association with ER could be disrupted upon lysis of cells in a mild hypotonic buffer. To determine the subcellular localization of the processed GSPT1, we transfected MCF-7 cells with a construct encoding C-terminal FLAG-tagged processed GSPT1 protein, and then stained the cells with an anti-FLAG antibody (Fig. 6D). Interestingly, deletion of the first 69 residues resulted in expression of GSPT1 in the cytoplasm and nuclear compartments (Fig. 6D, right panel), suggesting that its association with the ER is disrupted by deletion of its N terminus. Combined, our data suggest that proteolytic processing of GSPT1 at residue 69 could result in release of the processed GSPT1 from the ER-associated ribosomes into the cytosol. The three main structures of apoptotic signaling in the cell are the plasma membrane, where death and survival receptors reside, the mitochondrion, which is home to several proteins that regulate apoptosis and the third is the endoplasmic reticulum, which has the potential to initiate an independent ER stress-mediated apoptotic pathway (40Ferri K.F. Kroemer G. Nat Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1302) Google Scholar). The first two mammalian IAP inhibitors, Smac and HtrA2, reside in the mitochondrial intermembrane space and are released along with cytochrome c, which activates the Apaf-1-dependent apoptotic pathway (31Hegde 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). In this study we show that the ER associated GSPT1 protein could participate in the death pathways of mammalian cells after it undergoes limited proteolytic processing into an IAP-binding protein. Our data demonstrate that processed GSPT1 sensitizes cells to different apoptotic triggers suggesting that processed GSPT1 could function together with the other IAP-inhibitory proteins, Smac and HtrA2, to regulate apoptosis. The data indicate that processed GSPT1 promotes apoptosis by binding to IAPs and disrupting their interaction with the initiator caspase-9 and possibly the effector caspases 3 and 7, allowing the activation of these caspases. Moreover, the processed GSPT1 protein could promote apoptosis by inducing ubiquitination and degradation of c-IAP1. While the exact physiological function of the mammalian GSPT family of proteins is not clear at present, these proteins (GSPT1 and GSPT2) appear to be structural homologues of the yeast GST1 (also known as SUP35) gene product (36Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The yeast GST1 gene product is a GTP-binding protein structurally related to polypeptide elongation factor 1-α and reportedly functions as a polypeptide releasing factor 3 (eRF3) in yeast. Mutations in the GST1 gene were shown to increase the level of translational ambiguity (41Surguchov A.P. Beretetskaya Y.V. Fominykch E.S. Pospelova E.M. SmirnovVn Ter-Avanesyan M.D. Inge-Vechtomov S.G. FEBS Lett. 1980; 111: 175-178Crossref PubMed Scopus (35) Google Scholar, 42Eustice D.C. Wakem L.P. Wilhelm J.M. Sherman F. J. Mol. Biol. 1986; 188: 207-214Crossref PubMed Scopus (60) Google Scholar, 43Inge-Vechtomov S.G. Mironova L.N. Ter-Avanesian M.D. Genetika. 1994; 30: 1022-1035PubMed Google Scholar). The human GSPT1 was shown to be capable of complementing the temperature-sensitive growth of the yeast gst1 mutant (44Hoshino S. Miyazawa H. Enomoto T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 1989; 8: 3807-3814Crossref PubMed Scopus (107) Google Scholar), suggesting that human GSPT1 is a functional homologue of the yeast GST1 gene product and could also function as an eRF3. Recent results suggest that GSPT proteins could function in translation through association with PABP (37Uchida N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Proteolytic processing of GSPT1 into an IAP-binding protein appears to occur during cellular growth by an as yet unidentified protease. Processing of GSPT1 might be triggered by ER stress or other cellular stress conditions. Alternatively, the observation that the endogenous or the stably expressed GSPT1 protein was not completely processed to the IAP-binding form suggests the possibility that processing of GSPT1 could be a regulatory step to modulate the protein levels of GSPT1 during the cell cycle by targeting it for proteosomal degradation via the IAP pathway. This idea is consistent with the observations that processed GSPT-1 can trigger the autoubiquitination activity of c-IAP1, and that expression of GSPT1 is cell cycle dependent and is maximally seen at the G1 to S phase of the cell cycle (44Hoshino S. Miyazawa H. Enomoto T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 1989; 8: 3807-3814Crossref PubMed Scopus (107) Google Scholar). We thank Dr. Colin Duckett for the anti-c-IAP1 antibody and Dr. Jonathan Ashwell for the GST-c-IAP1 construct.
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