Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes
2004; Springer Nature; Volume: 23; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7600210
ISSN1460-2075
AutoresMichelle M. Hill, Colin Adrain, Patrick J. Duriez, Emma M. Creagh, Séamus J. Martin,
Tópico(s)Autophagy in Disease and Therapy
ResumoArticle22 April 2004free access Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes Michelle M Hill Michelle M Hill Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, IrelandPresent address: Institute of Molecular Bioscience, University of Queensland, St. Lucia QLD 4072, Australia Search for more papers by this author Colin Adrain Colin Adrain Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Patrick J Duriez Patrick J Duriez Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Emma M Creagh Emma M Creagh Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Seamus J Martin Corresponding Author Seamus J Martin Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Michelle M Hill Michelle M Hill Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, IrelandPresent address: Institute of Molecular Bioscience, University of Queensland, St. Lucia QLD 4072, Australia Search for more papers by this author Colin Adrain Colin Adrain Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Patrick J Duriez Patrick J Duriez Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Emma M Creagh Emma M Creagh Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Seamus J Martin Corresponding Author Seamus J Martin Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland Search for more papers by this author Author Information Michelle M Hill1, Colin Adrain1, Patrick J Duriez1, Emma M Creagh1 and Seamus J Martin 1 1Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland *Corresponding author. Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland. Tel.: +353 1 608 1289; Fax: +353 1 679 8558; E-mail: [email protected] The EMBO Journal (2004)23:2134-2145https://doi.org/10.1038/sj.emboj.7600210 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Apaf-1 apoptosome is a multi-subunit caspase-activating scaffold that is assembled in response to diverse forms of cellular stress that culminate in apoptosis. To date, most studies on apoptosome composition and function have used apoptosomes reassembled from recombinant or purified proteins. Thus, the precise composition of native apoptosomes remains unresolved. Here, we have used a one-step immunopurification approach to isolate catalytically active Apaf-1/caspase-9 apoptosomes, and have identified the major constituents of these complexes using mass spectrometry methods. Using this approach, we have also assessed the ability of putative apoptosome regulatory proteins, such as Smac/DIABLO and PHAPI, to regulate the activity of native apoptosomes. We show that Apaf-1, caspase-9, caspase-3 and XIAP are the major constituents of native apoptosomes and that cytochrome c is not stably associated with the active complex. We also demonstrate that the IAP-neutralizing protein Smac/DIABLO and the tumor-suppressor protein PHAPI can enhance the catalytic activity of apoptosome complexes in distinct ways. Surprisingly, PHAPI also enhanced the activity of purified caspase-3, suggesting that it may act as a co-factor for this protease. Introduction The caspase family of cysteine proteases plays a critical role in apoptosis by coordinating the controlled destruction of the cell from within (reviewed in Martin and Green, 1995; Cohen, 1997). Caspases are initially synthesized as inactive zymogens that require further post-translational processing for full activity (Stennicke and Salvesen, 1999). During apoptosis, caspase activation appears to be achieved in two main ways: through recruitment of apical caspases to activation scaffolds or through direct proteolytic processing of downstream caspases by other caspases (or non-caspase proteases such as granzyme B). The Apaf-1 apoptosome is a multi-subunit caspase-activating scaffold that is assembled in response to diverse forms of cellular stress (Li et al, 1997; Cain et al, 1999, 2000; Zou et al, 1999). Assembly of the apoptosome is provoked by the release of cytochrome c from the mitochondrial intermembrane space, an event that is both positively and negatively regulated by members of the Bcl-2 family (Kluck et al, 1997; Yang et al, 1997; Kuwana et al, 2002). Upon entry into the cytosol, cytochrome c associates with monomeric Apaf-1 and promotes a conformational change that permits oligomerization of the latter and recruitment of caspase-9 to the complex (Adrain et al, 1999; Zou et al, 1999; Jiang and Wang, 2000). Electron cryomicroscopy studies have established that apoptosomes assembled from recombinant components adopt a wheel-like configuration and are composed of approximately seven Apaf-1 molecules in complex with an unknown number of caspase-9 dimers (Acehan et al, 2002). To date, the majority of studies on apoptosome activity, structure and composition have utilized reconstituted apoptosomes prepared from recombinant or purified proteins. Partially purified native apoptosomes have been successfully isolated by gel filtration approaches, but further purification of these complexes has been hampered by instability in high-salt buffers (Cain et al, 1999, 2000; Rodriguez and Lazebnik, 1999). For these reasons, while it is clear that Apaf-1 and caspase-9 form the core components of the apoptosome, the precise composition of native apoptosome complexes remains unclear. Using overexpression analysis, several additional proteins have been implicated as apoptosome constitutents, but the presence of these proteins in native apoptosomes remains to be confirmed. Thus, in addition to Apaf-1/caspase-9, proteins that have been implicated as constitutents of the apoptosome include caspase-3, caspase-7, XIAP, Aven, NAC, Bcl-2 and Bcl-XL (Pan et al, 1998; Cain et al, 1999; Chau et al, 2000; Bratton et al, 2001; Chu et al, 2001). To explore the protein composition of native apoptosomes, we have used a one-step immunoprecipitation approach to isolate active caspase-9/Apaf-1 apoptosomes. To identify their constituents, these complexes were analyzed by analytical as well as preparative two-dimensional (2D)-PAGE coupled with MALDI mass spectrometry. Using this approach, we have also assessed the ability of putative apoptosome regulatory proteins, such as Smac/Diablo and PHAPI, to regulate the catalytic activity of the apoptosome. Here we describe the major constituents of native apoptosomes and show that the PHAPI tumor-suppressor protein enhances the catalytic activity of these complexes, possibly by acting directly on caspase-3. Results To explore the composition of native Apaf-1/caspase-9 apoptosomes, we initially attempted to immunoprecipitate Apaf-1 from cell-free extracts derived from Jurkat T cells. To do this, we used a panel of Apaf-1 antibodies, several of which successfully immunoprecipitated Apaf-1 in the absence of triggers for apoptosome assembly (data not shown). However, when apoptosome assembly was initiated by addition of cytochrome c/dATP to the cell extracts, all Apaf-1 antibodies tested failed to immunoprecipitate Apaf-1/caspase-9 complexes (data not shown). This approach was unsuccessful, presumably because the epitope(s) recognized by these antibodies became inaccessible upon oligomerization of Apaf-1 and recruitment of caspase-9 into the apoptosome. Differential immunoprecipitation of Apaf-1 via caspase-9 As an alternative approach, we explored whether a monoclonal anti-caspase-9 antibody could differentially co-immunoprecipitate Apaf-1 in the presence or absence of cytochrome c/dATP. As Figure 1A illustrates, this approach was successful as Apaf-1 was readily co-precipitated with caspase-9 in the presence of cytochrome c/dATP, but not in the absence of these co-factors for apoptosome assembly. To explore whether Apaf-1/caspase-9 complexes retained catalytic activity after the extensive washing steps associated with the immunoprecipitation procedure, we assessed the ability of these complexes to cleave synthetic tetrapeptide caspase substrates (Figure 1B). Apoptosomes prepared in this way displayed LEHDase (caspase-9-like), as well as DEVDase (caspase-3-like) activities, but failed to hydrolyze the caspase-1-selective substrate YVAD, as expected (Figure 1B). Importantly, caspase-9 immunoprecipitated in the absence of cytochrome c/dATP failed to display any proteolytic activity (Figure 1B). We also asked whether purified apoptosomes were capable of cleaving natural caspase substrates such as BID, Vimentin and caspase-3 (Figure 1C). As a specificity control, pro-IL-1β was chosen as this cytokine is an established substrate for caspase-1 but not for caspases that are activated during apoptosis. These experiments revealed that apoptosomes purified in this manner retained robust and specific catalytic activity towards natural substrate proteins (Figure 1C). Figure 1.One-step isolation of native apoptosomes using a differential immunoprecipitation strategy. (A) Jurkat cell-free extracts were incubated in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP for 10 min at 37°C. Reactions were adjusted to 50 mM NaCl and 0.3% CHAPS and precleared by incubating with protein A/G agarose beads. Protein complexes immunoprecipitated with a monoclonal anti-caspase-9 antibody (Upstate Biotechnology) were analyzed for the presence of Apaf-1 and caspase-9 by immunoblotting. Equivalent amounts of the input (6.5 μl of the reaction), the IP supernatant (Sup) and protein binding to the preclearing beads (preclear) were loaded to aid comparison. (B) Jurkat cell-free extracts were incubated for 15 min at 37°C in the presence (filled circles) or absence (open circles) of 50 μg/ml cytochrome c/1 mM dATP. Caspase-9 complexes isolated from 100 μl reactions were washed extensively and then incubated for 1 h at 37°C with 50 μM of the indicated fluorogenic peptides. (C) Caspase-9 complexes, isolated from Jurkat cell-free extract incubated for 15 min at 37°C in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP, were incubated with in vitro translated Bid, caspase-3, vimentin or pro-interleukin-1β for 2 h at 37°C. Proteolysis of radiolabeled substrate proteins was analyzed by SDS—PAGE, followed by autoradiography. Download figure Download PowerPoint Kinetics of apoptosome assembly We then explored the kinetics of Apaf-1 and caspase-9 association upon addition of cytochrome c/dATP to the cell extracts. As Figure 2A illustrates, in the absence of a trigger for apoptosome assembly, Apaf-1 failed to associate with caspase-9, as expected. However, in the presence of cytochrome c/dATP, Apaf-1 was very rapidly recruited to pro-caspase-9 (within minutes) and this reproducibly occurred during assembly of the reactions at 4°C (Figure 2A). Caspase-9 was observed to undergo rapid proteolytic maturation within the apoptosome and this was routinely complete within 10 min of addition of cytochrome c/dATP to the extracts (Figure 2A). Stable recruitment of caspase-3, but not caspase-6 or -7, to apoptosomes was also observed, providing an explanation for the significant DEVDase activity displayed by these complexes (Figures 2A and 1B). Figure 2.Cytochrome c triggers the rapid and stable association of Apaf-1, caspase-9 and caspase-3. (A) Jurkat cell-free extracts were incubated at 37°C in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP for the times indicated and then brought to 4°C. Caspase-9 was then immunoprecipitated from each reaction as described in the legend to Figure 1A. Caspase-9 immunocomplexes (Casp-9 IP) or 10% of the input reactions (input) were then probed for the presence of Apaf-1, caspase-3, -6, -7 and cytochrome c by immunoblotting. Bands corresponding to immunoglobulin heavy and light chains are indicated by asterisks. (B) Caspase-9 complexes were immunoprecipitated from Jurkat cell-free extracts incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of 50 μg/ml cytochrome c/1 mM dATP. Caspase-9 immunocomplexes were then washed extensively, as described in Materials and methods, and peptide hydrolysis assays were then performed in the presence (triangles) or absence (circles) of 50 μg/ml cytochrome c/1 mM dATP. Note that for DEVD-AFC hydrolysis reactions complexes were prepared from 50 μl cell-free reactions, whereas for LEHD-AFC hydrolysis assays 150 μl cell-free reactions were required. Download figure Download PowerPoint Cytochrome c is not stably associated with apoptosomes Interestingly, we were unable to find stable association of cytochrome c within purified apoptosomes, despite addition of saturating amounts of this protein to cell-free extracts (Figure 2A). This suggests either that cytochrome c acts in a ‘hit-and-run’ manner to trigger apoptosome assembly, or that the interaction between cytochrome c and Apaf-1 was of insufficient affinity to remain bound after washing of immunocomplexes. As apoptosomes prepared in this manner displayed robust catalytic activity toward caspase substrates (Figure 1), we favor a model where the continued presence of cytochrome c, after functional apoptosomes have been assembled, is not required. In support of this model, re-addition of cytochrome c and dATP to purified apoptosomes failed to enhance the catalytic activity of these complexes toward tetrapeptide substrates (Figure 2B). Rapid recruitment of XIAP, but not of other IAPs, to the apoptosome and neutralization by Smac/DIABLO The inhibitors of apoptosis proteins have been implicated as important regulators of apoptosis through their ability to associate with and repress the catalytic activity of mature caspases (Deveraux et al, 1997). In particular, XIAP has been shown to be capable of associating with active caspase-9 and -3, and has previously been implicated as a constituent of the apoptosome by gel filtration and co-immunoprecipitation analysis (Bratton et al, 2001). To explore whether any of the IAPs were recruited to Apaf-1/caspase-9 apoptosomes, we performed similar immunoprecipitation experiments and probed these complexes for the presence of XIAP, cIAP-1 and cIAP-2. We also explored whether the heat-shock proteins, HSP90 or HSP70, could be detected within the apoptosome, as both have been implicated as negative regulators of apoptosome assembly through binding to Apaf-1 (Beere et al, 2000; Pandey et al, 2000; Saleh et al, 2000). As Figure 3A shows, XIAP was rapidly recruited to apoptosomes and was also observed to undergo proteolytic processing within the complex. Addition of the caspase-3-selective inhibitory peptide Ac-DEVD-CHO to cell-free extracts during preparation of apoptosomes abolished XIAP proteolysis, suggesting that caspase-3 was responsible for this effect (Figure 3B). Figure 3.XIAP is a constituent of native apoptosomes. (A) Jurkat cell-free extracts were incubated at 37°C in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP for the times indicated and then brought to 4°C. Caspase-9 was then immunoprecipitated from each reaction as described in the legend to Figure 1A. Caspase-9 immunocomplexes (Casp-9 IP) or 10% of the input reactions (Input) were analyzed for the presence of IAPs, HSP90, HSP70, Bcl-2, Bcl-XL and PHAPI by immunoblotting. Bands corresponding to immunoglobulin light chain are indicated by asterisks. (B) Apoptosome assembly was initiated in Jurkat cell-free extracts in the presence or absence of 1 μM Ac-DEVD-CHO, as indicated. Caspase-9 was then immunoprecipitated from each reaction as described in the legend to Figure 1A. Caspase-9 complexes (Casp-9 IP) or 10% of the input reactions (Input) were analyzed for the presence of Apaf-1, caspase-9, -3 and XIAP by immunoblotting. Cleaved XIAP is indicated with an arrow. (C) Caspase-9 complexes were immunoprecipitated from Jurkat cell-free extracts incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of 50 μg/ml cytochrome c/1 mM dATP. Peptide hydrolysis assays were subsequently performed in the presence of buffer (circles), 0.5 μM (triangles) or 5 μM (squares) recombinant Smac Δ1–55. Download figure Download PowerPoint In contrast to the recruitment of XIAP to apoptosomes, we failed to find any evidence for recruitment of cIAP-1, cIAP-2, HSP70, HSP90, Bcl-2 or Bcl-xL under the same conditions (Figure 3A). Interestingly, we also failed to find evidence for stable recruitment of PHAPI—recently identified as a regulator of caspase-9 activation within this pathway (Jiang et al, 2003)—within apoptosome complexes (Figure 3A). Smac/DIABLO is a mitochondrial protein that is released from the mitochondrial intermembrane space during apoptosis and has been shown to neutralize IAPs and potentiate caspase activation (Du et al, 2000; Verhagen et al, 2000). As XIAP was readily detected in association with apoptosome complexes, we explored whether addition of recombinant mature Smac/DIABLO (Δ1–55) to purified apoptosomes would enhance the catalytic activity of these complexes. As Figure 3C illustrates, Smac/DIABLO significantly enhanced the activity of native apoptosome complexes, confirming that XIAP was inhibitory to the complexes and could be neutralized by this IAP-binding protein. XIAP acts as a tether for caspase-3 within the apoptosome As XIAP was rapidly and efficiently recruited to apoptosomes, we also explored whether XIAP was required for apoptosome assembly. Thus, we immunodepleted XIAP from Jurkat cell-free extracts and prepared apoptosomes by immunoprecipitating caspase-9 in the presence or absence of cytochrome c/dATP as before. Interestingly, these experiments revealed that caspase-3 failed to be recruited to apoptosome complexes devoid of XIAP (Figure 4A). Consistent with this, apoptosomes prepared under these conditions also failed to exhibit significant DEVDase activity (Figure 4B). In contrast, DEVDase activity in cytochrome c/dATP-treated cell-free extracts depleted of XIAP displayed a net increase relative to mock-depleted extracts, which is consistent with the removal of a caspase inhibitory protein (Figure 4B). Taken together, these data suggest that XIAP is responsible for the stable recruitment of caspase-3 to the apoptosome. Interaction between XIAP and one of the catalytic sites in a mature caspase-3 dimer would be sufficient to tether caspase-3 to the apoptosome, while leaving the other half of the caspase-3 dimer free to act upon substrates. This would explain why significant apoptosome-associated caspase-3 activity is detectable despite interaction between this caspase and its inhibitor within the complex (Figures 1B and 4B). Figure 4.XIAP acts as a tether for recruitment of caspase-3 to the apoptosome. (A) Left panel, mock-depleted or XIAP-immunodepleted cell-free extracts were treated with 50 μg/ml cytochrome c/1 mM dATP for 15 min at 37°C, followed by immunoblotting and probing for the indicated proteins. Right panel, mock-depleted or XIAP-immunodepleted cell-free extracts were treated with 50 μg/ml cytochrome c/1 mM dATP for 15 min at 37°C. Reactions were then brought to 4°C and caspase-9 immunocomplexes were prepared, followed by immunoblotting for the indicated proteins. Arrows indicate mature caspase-3. Asterisks denotes immunoglobulin heavy and light chains. (B) Caspase-9 complexes (left panel) were immunoprecipitated from mock-depleted (circles) or XIAP-depleted (squares) Jurkat cell-free extracts incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of 50 μg/ml cytochrome c/1 mM dATP. Hydrolysis of DEVD-AFC (50 μM) was monitored for 2 h at 37°C. Cell-free reactions (right panel) from the same extracts were incubated in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP for 30 min at 37°C, and assayed for DEVD-AFC hydrolysis for 60 min at 37°C. Download figure Download PowerPoint Analysis of apoptosome composition by preparative 2D-PAGE/mass fingerprinting analysis To seek additional apoptosome constituents, we used a scaled up approach where apoptosomes were immunoprecipitated from cell-free lysates of 109 Jurkat cells (∼60 mg protein) using anti-caspase-9 mAb (50 μg per IP), in the presence or absence of cytochrome c/dATP. Immunoprecipitates were washed extensively, eluted into urea-based sample buffer and preparative 2D-PAGE gels were run. All protein spots that differentially co-immunoprecipitated with caspase-9 under these conditions were excised from the gels and were analyzed by MALDI-TOF mass spectrometry. As Figure 5A shows, a total of 12 new protein spots were reproducibly detected by silver staining when caspase-9 was immunoprecipitated after apoptosome assembly. By mass fingerprinting analysis, these spots were identified as alternative cleavage products of mature caspase-9 (p35, p37), alternative cleavage products of mature caspase-3 (p20, p17, p12) and XIAP (Table I). These assignments were also confirmed by Western blot analysis on similar 2D gels (Figure 5B). Figure 5.Analysis of native Apaf-1 apoptosomes by 2D-PAGE. (A) Caspase-9 complexes, isolated from 2 ml Jurkat cell-free reactions incubated for 15 min at 37°C in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP, were analyzed by 2D gel electrophoresis (first dimension: pH 5–8, second dimension: 12% SDS–PAGE). Top: A representative silver-stained preparative gel is shown. Heavy and light chains of the immunoprecipitating antibody are indicated. Bottom: enlarged areas containing proteins that differentially immunoprecipitate with caspase-9 (areas I and II in the gel above) are shown. (B) Caspase-9 complexes were isolated from Jurkat cell-free extracts incubated for 15 min at 37°C in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP. Proteins co-precipitating with caspase-9 were analyzed by 2D gel electrophoresis (pH 5–8 first dimension and 12% SDS–PAGE second dimension), followed by immunoblotting with the indicated antibodies. Spots corresponding to immunoglobulin heavy and light chains are indicated by asterisks. Download figure Download PowerPoint Table 1. Mass fingerprinting analysis of apoptosome-associated proteins Spot no. Protein ID Accession no. No. of matches Amino-acid coverage Pro-caspase-9 P55211 16 14–409/416 (55%) 1 XIAP P98170 32 11–491/497 (67%) 2 Caspase-9 (p37) P55211 8 16–324/416 (38%) 3 Caspase-9 (p35) P55211 8 57–189/416 (28%) 4 no ID 5 Rho-GDI2 P52566 3 51–164/201 (26%) 6 Rho-GDI2 P52566 5 51–196/201 (56%) 7 No ID 8 No ID 9 No ID 10 Caspase-3 (p20) P42574 9 20–147/277 (34%) 11 Caspase-3 (p17) P42574 9 39–147/277 (30%) 12 Caspase-3 (p10) P42574 7 176–277/277 (26%) Interestingly, two of the novel spots were identified as C-terminal cleavage products of Rho-GDI2 (Table I), a member of the Rho family-specific guanine nucleotide dissociation inhibitors and an established caspase substrate (Martin et al, 1996; Na et al, 1996). Despite repeated attempts, four of the novel protein spots could not be identified by mass spectrometry analysis due to the low abundance of these proteins (Figure 5A and Table I). However, one of these spots (Figure 5A, spot 9) was subsequently identified by Western blot analysis as a caspase-3 cleavage product (Figure 5B). Note that Apaf-1 was not identified using mass spectrometry analysis due to poor penetration of high-molecular-weight proteins into the first dimension immobilized pH gradient strips. Apaf-1 was readily detected by direct immunoblot analysis on similar preparations (Figure 5B), but was not present on 2D gels in sufficient quantities to detect by silver staining. These data suggest that, in addition to Apaf-1 and proteolytically processed caspase-9, the major constituents of native apoptosomes are mature caspase-3, XIAP and additional low-molecular-weight proteins that have yet to be identified. The mass spectrometry-based identification of RhoGDI2 degradation products in apoptosome immunoprecipitates was interesting and suggested that this protein might play a role within the apoptosome. However, using immunoblot analysis, we failed to confirm recruitment of this protein to the apoptosome complex (Figure 6). Moreover, while we readily detected Apaf-1, caspase-9, caspase-3 and XIAP in apoptosomes generated from BJAB and U937 cell-free extracts, we also failed to find evidence for recruitment of RhoGDI2 to these complexes (Figure 6). Thus, it appears likely that RhoGDI2 was nonspecifically co-precipitated with large-scale apoptosome preparations from Jurkat cells. Figure 6.Analysis of apoptosome constituents from Jurkat, BJAB and U937 cells. Cell-free extracts derived from Jurkat, BJAB or U937 cells were incubated at 37°C in the presence or absence of 50 μg/ml cytochrome c/1 mM dATP for the times indicated and then brought to 4°C. Caspase-9 was then immunoprecipitated from each reaction as described in the legend to Figure 1A. Caspase-9 complexes (CASP-9 IP) or 10% of the input reactions (input) were then immunoblotted for Apaf-1, caspase-3, caspase-9, RhoGDI2 and XIAP. Download figure Download PowerPoint PHAPI as an enhancer of caspase activity within apoptosomes Recently, Wang and colleagues have identified the tumor-suppressor protein PHAPI (also known as I1PP2A, Mapmodulin and PP32) as an enhancer of caspase-9 activation in the apoptosome pathway (Jiang et al, 2003). However, it is unclear whether PHAPI acts directly on the apoptosome to stimulate maturation of the caspases within, or whether PHAPI can enhance the catalytic activity of the mature complex (Jiang et al, 2003). As shown in Figure 3A, we failed to detect stable recruitment of PHAPI to the apoptosome, suggesting that this protein is not an integral part of the complex. However, it remained possible that PHAPI could act to enhance apoptosome activity after assembly of the complex. To explore whether this was the case, we added recombinant full-length PHAPI, or a deletion mutant lacking 84 amino acids from the C-terminus (aa 1–163; PHAPI-Δtail), to purified apoptosomes to assess their impact on apoptosome activity (Figure 7). PHAPI had a very significant enhancement effect on the proteolytic activity of apoptosomes, whereas the PHAPI-Δtail deletion mutant failed to display any activity in this assay (Figure 7A, left panel). PHAPI also profoundly enhanced caspase catalytic activity when added to total cell-free extracts in the presence of cytochrome c/dATP, whereas PHAPI-Δtail had no activity in this regard (Figure 7A, right panel). This suggests that the C-terminal region of PHAPI is critical for the activity of this protein. Figure 7.PHAPI enhances the catalytic activity of apoptosomes. (A) Caspase-9 apoptosome complexes (left) and Jurkat cell-free extracts (right), incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of 50 μg/ml cytochrome c/1 mM dATP, were assayed for DEVDase activity in the presence of 0.5 μM recombinant PHAPI (triangles), PHAPI-ΔTail (squares) or buffer alone (circles). (B) Caspase-9 apoptosome complexes (left) and Jurkat cell-free extracts (right), incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of cytochrome c/dATP, were assayed for DEVDase activity in the presence of 0.5 μM recombinant PHAPI (small circles), GST (large triangles), GST-PHAPI-Tail (small triangles), 15–50 kDa polyglutamine polymers (squares) or buffer alone (large circles). The results shown are representative of three separate experiments. (C) Aliquots (750 ng) of purified bacterially expressed PHAPI, PHAPI ΔTail, GST and GST-PHAP-Tail proteins were separated by SDS–PAGE, and visualized by coomassie blue staining. A degradation product of full-length PHAPI is indicated by an asterisk. (D) Jurkat cell-free extracts, incubated for 30 min at 37°C in the presence (filled symbols) or absence (open symbols) of 2 μg/ml cytochrome c/1 mM dATP, were assayed for DEVDase activity in the presence of 1 μM recombinant PHAPI (small circles), 5 μM okadaic acid (triangles), 1 μM calyculin A (squares) or buffer alone (large circles). Download figure Download PowerPoint As the C-terminus of PHAPI is highly acidic, we also generated a GST-PHAPI deletion mutant (aa 164–249; GST-PHAPI-Tail) containing this acidic region to ask whether the acidic C-terminal tail was sufficient to enhance caspase activity in isolated apoptosomes or cell-free extracts. To control for nonspecific effects of acidic polypeptides on caspase activity, we also tested polyglutamic acid in the same assays. However, as shown in Figure 7B, neither the acidic PHAP tail nor polyglutamic acid was sufficient to mimic the stimulatory effects of PHAPI on caspase acti
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