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Conversion of Procaspase-3 to an Autoactivating Caspase by Fusion to the Caspase-2 Prodomain

1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês

10.1074/jbc.273.41.26566

ISSN

1083-351X

Autores

Paul A. Colussi, Natasha L. Harvey, Linda Shearwin‐Whyatt, Sharad Kumar,

Tópico(s)

ATP Synthase and ATPases Research

Resumo

Caspases are cysteine proteases that play an essential role in apoptosis. Initial activation of caspases defines the key step in apoptotic execution. Based on primary structure, caspases can be divided into two groups, those with long amino-terminal prodomains (class I), and those with relatively short prodomains (class II). On overexpression in mammalian cells, class I caspases can induce cell death that is dependent on their autocatalytic activity. Recent studies suggest that the long prodomains in some class I caspases are able to mediate dimerization of procaspase molecules, thereby promoting autoprocessing. In this communication, we demonstrate that fusion of the prodomain of a class I caspase (Nedd2/caspase-2) with procaspase-3 greatly augments autocatalysis and apoptosis induction by the chimeric caspase-3 molecule. The chimeric caspase-3 molecules were able to form homodimers in Saccharomyces cerevisiae and were efficiently processed in transfected mammalian cells. These results provide direct evidence for a role of a class I caspase prodomain in caspase autoactivation and processing and establish a basis for functional hierarchy among the two classes of caspases. Caspases are cysteine proteases that play an essential role in apoptosis. Initial activation of caspases defines the key step in apoptotic execution. Based on primary structure, caspases can be divided into two groups, those with long amino-terminal prodomains (class I), and those with relatively short prodomains (class II). On overexpression in mammalian cells, class I caspases can induce cell death that is dependent on their autocatalytic activity. Recent studies suggest that the long prodomains in some class I caspases are able to mediate dimerization of procaspase molecules, thereby promoting autoprocessing. In this communication, we demonstrate that fusion of the prodomain of a class I caspase (Nedd2/caspase-2) with procaspase-3 greatly augments autocatalysis and apoptosis induction by the chimeric caspase-3 molecule. The chimeric caspase-3 molecules were able to form homodimers in Saccharomyces cerevisiae and were efficiently processed in transfected mammalian cells. These results provide direct evidence for a role of a class I caspase prodomain in caspase autoactivation and processing and establish a basis for functional hierarchy among the two classes of caspases. prodomain of caspase-2/Nedd2 Aequorea victoria green fluorescent protein activation domain DNA-binding domain 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. Caspases are a family of cysteine proteases that cleave their target protein following an Asp residue. Of the eleven published caspases, caspase-1, -4, -5, and -11 seem primarily involved in the processing of proinflammatory cytokines, whereas others play crucial roles in one or more pathways of apoptosis (reviewed in Refs. 1Kumar S. Trends Biochem. Sci. 1995; 20: 198-202Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 2Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 3Kumar S. Lavin M.F. Cell Death Diff. 1996; 3: 255-267PubMed Google Scholar, 4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 230-299Abstract Full Text PDF Scopus (2185) Google Scholar, 5Chinnaiyan A.C. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar). Caspases are produced as precursor molecules that require processing into two subunits to produce a fully active enzyme (1Kumar S. Trends Biochem. Sci. 1995; 20: 198-202Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 2Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 3Kumar S. Lavin M.F. Cell Death Diff. 1996; 3: 255-267PubMed Google Scholar, 4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 230-299Abstract Full Text PDF Scopus (2185) Google Scholar, 5Chinnaiyan A.C. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar). On the basis of primary structure, proapoptotic caspases can be divided into two classes, class I including caspase-2, -8, -9, and -10 that contain a long amino-terminal prodomain, and class II such as caspase-3, -6, and -7 with a short or absent prodomain. Although numerous studies have provided data on the activation of multiple caspases in apoptosis, the mechanism of activation of the initial caspases in a particular apoptotic pathway is poorly understood. The long prodomain in class I caspases such as caspase-2, -8, and -10 can interact with adaptor molecules that are directly or indirectly recruited to specific death receptors. For example, the prodomains of caspase-8 and -10 contain two death effector domains that interact with the death effector domains in the adaptor molecule FADD, which recruits these caspases to the activated Fas receptor (4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 230-299Abstract Full Text PDF Scopus (2185) Google Scholar, 5Chinnaiyan A.C. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar, 6Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Scaffidi C. Bretz J.D. Zhang M. Ni J. Gentz R. Mann N. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar, 7Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar). In a similar manner, caspase-2 is thought to be recruited to death receptors by binding through the adaptor RAIDD (8Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (469) Google Scholar). The prodomain of caspase-9 shares a degree of homology with the amino terminus of Apaf-1, a CED-4-like mammalian molecule that can interact with caspase-9 (9Li P. Nijhawan D. Budhihardjo I. Srinivasula S. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6257) Google Scholar). Several recent studies demonstrate that procaspases can be activated through dimerization/oligomerization mediated through their prodomains (10Butt A.J. Harvey N.L. Parasivam G. Kumar S. J. Biol. Chem. 1998; 273: 6763-6768Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) or induced artificially (11Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar, 12Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). This suggests that the primary role of adaptor molecules that recruit class I caspases may be to bring procaspase molecules into close proximity with each other to enable dimerization. In vitro cleavage experiments suggest that class II caspases require activated class I caspases for their proteolytic processing (reviewed in Refs. 3Kumar S. Lavin M.F. Cell Death Diff. 1996; 3: 255-267PubMed Google Scholar, 4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 230-299Abstract Full Text PDF Scopus (2185) Google Scholar, 5Chinnaiyan A.C. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar). We have recently demonstrated that caspase-2, a class I caspase, can dimerize and autoprocess in Saccharomyces cerevisiae and mammalian cells (10Butt A.J. Harvey N.L. Parasivam G. Kumar S. J. Biol. Chem. 1998; 273: 6763-6768Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The prodomain of caspase-2 is essential for the dimerization of the precursor molecule and caspase-2 lacking the prodomain is poorly processed in S. cerevisiae cells (10Butt A.J. Harvey N.L. Parasivam G. Kumar S. J. Biol. Chem. 1998; 273: 6763-6768Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Overexpression of procaspase-2 in mammalian cells induces apoptosis because of its autoprocessing activity (13Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (588) Google Scholar, 14Wang L. Miura M. Bergeron L. Zhu H. Yuan J. Cell. 1994; 78: 739-750Abstract Full Text PDF PubMed Scopus (801) Google Scholar, 15Kumar S. Kinoshita M. Dorstyn L. Noda M. Cell Death Differ. 1997; 4: 378-387Crossref Scopus (28) Google Scholar, 16Dorstyn L. Kumar S. Cell Death Differ. 1997; 4: 570-579Crossref PubMed Scopus (26) Google Scholar). Caspase-3, a class II effector caspase, lacks a long prodomain (17Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2279) Google Scholar, 18Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.A. Smulson M.E. Yamin T.-T., Yu, V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3802) Google Scholar). Unlike procaspase-2, procaspase-3 is a poor inducer of cell death when transfected in mammalian cells (16Dorstyn L. Kumar S. Cell Death Differ. 1997; 4: 570-579Crossref PubMed Scopus (26) Google Scholar, 17Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2279) Google Scholar, 18Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.A. Smulson M.E. Yamin T.-T., Yu, V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3802) Google Scholar), presumably because of its inability to autoactivate. We reasoned that if the prodomain of a class I caspase was essential for the activation of a procaspase, fusing a class I caspase prodomain onto a class II caspase would generate a procaspase molecule able to dimerize and mediate autocatalytic processing. In this communication we show that amino-terminal fusion of the caspase-2 prodomain (C2P)1 to procaspase-3 results in remarkable enhancement of its apoptotic activity. We further demonstrate that fusion of C2P to caspase-3 enables the chimeric molecule to dimerize in S. cerevisiaeand to autoprocess in vivo. These data provide direct evidence that the primary function of a class I caspase prodomain is to mediate dimerization and that this dimerization is necessary and sufficient for autoprocessing of the caspase precursor. Our results also establish a structural basis for the functional hierarchy among two classes of caspases. Details of the caspase-GFP fusion constructs used in this study have been described elsewhere (19Colussi P.A. Harvey N.L. Kumar S. J. Biol. Chem. 1998; 273: 24535-24542Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, caspase coding regions were amplified by polymerase chain reaction using Pfu polymerase and fused in frame to the amino terminus of the mammalian codon optimized GFP in pEGFP-N1 (CLONTECH). Caspase-2-GFP contains the entire coding region of mouse Nedd2 (452 amino acid), whereas caspase-2(C320G)-GFP is a catalytically inactive mutant of caspase-2. Caspase-3-GFP contains the full-length coding region of wild-type caspase-3. C2P-caspase-3-GFP and C2P-caspase-3Δ9-GFP are chimeric constructs in which the prodomain of caspase-2 (169 residues in the wild-type or 166 residues in Δ9 chimeric construct) was fused at the amino terminus of caspase-3-GFP. C2P-caspase-3(C163G)-GFP and C2P-caspase-3Δ9(C163G)-GFP constructs were generated from C2P-caspase-3-GFP and C2P-caspase-3Δ9-GFP plasmids, respectively, by site-directed mutagenesis. The C2P-caspase-3Δ9-GFP and C2P-caspase-3Δ9(C163G)-GFP constructs lack the first 9 amino-terminal residues of the caspase-3 precursor (19Colussi P.A. Harvey N.L. Kumar S. J. Biol. Chem. 1998; 273: 24535-24542Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. For cell death assays, 2 × 105 cells were plated per 35-mm dish the day before transfection. Expression vectors (2 μg of total DNA) were transfected into cells using the Fugene reagent (Boehringer Mannheim) according to the manufacturer's protocol. Cells were observed, scored for apoptotic morphology and photographed using a fluorescence microscope (Olympus BH2-RFCA) 18 h post-transfection. For immunoblotting, cells were plated at a density of 6 × 105 cells per 60-mm dish and transfected with a total of 6 μg of plasmid DNA. Cells were harvested 18 h post-transfection. In some cases caspase-GFP expression vectors were cotransfected with either CrmA, P35, MIHA, or Bcl-2 expression constructs at a ratio of 1:3. Construction of CrmA, P35, and MIHA expression vectors has been described previously (16Dorstyn L. Kumar S. Cell Death Differ. 1997; 4: 570-579Crossref PubMed Scopus (26) Google Scholar). The Bcl-2 expression vector was kindly provided by Dr David Vaux. Proteins were resolved on SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidine difluoride membrane (DuPont). Blots were probed with an anti-GFP monoclonal antibody (Boehringer Mannheim). Following incubation with appropriate horseradish peroxidase-coupled secondary antibodies, signals were detected using the ECL system (Amersham Pharmacia Biotech). The coding regions of caspase-3 and the caspase-3(C163G) mutant were amplified from the caspase-3-GFP and C2P-caspase-3(C163G)-GFP vectors, respectively, by 30 cycles of polymerase chain reaction using Pfu polymerase (Stratagene). For cloning into yeast Gal4 DNA binding domain (Gal4BD) vector pAS2.1 (CLONTECH), an upstream oligonucleotide containing an EcoRI site (primer A) and a downstream primer containing a BamHI site (primer B) were used, and the amplified product cloned in frame intoEcoRI/BamHI digested pAS2.1. For cloning into the Gal4 activation domain (Gal4AD) vector pACT2 (CLONTECH), an upstream primer containing aBamHI restriction site (primer C) and the downstream primer B were used, and the amplified product cloned in frame intoBamHI digested pACT2. Primer A, 5′-CCGGAATTCGAGAACACTGAAAACTCA; primer B, 5′-GGTGGATCCCGGTGATAAAAATAGAGTTCTTT; primer C, 5′-CGCGGATCCGAGAGAACACTGAAAACTCA (regions complementary to the caspase-3 sequence are underlined). The coding regions of the chimeric caspase-3 molecules containing the caspase-2 prodomain were amplified from the previously described vectors2 C2P-caspase-3(C163G)-GFP, C2P-caspase-3Δ9-GFP and C2P-caspase-3(C163G)Δ9-GFP using the following oligonucleotide primers: primer D, 5′-GGAATTCCATATGGCGGCGCCGAGCGGGA(regions complementary to the caspase-2 sequence are underlined) and primer B described above. The amplified products were digested withBamHI and cloned in frame intoSmaI/BamHI-digested pAS2.1 and pACT2. These assays were essentially carried out as described previously (10Butt A.J. Harvey N.L. Parasivam G. Kumar S. J. Biol. Chem. 1998; 273: 6763-6768Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Caspase-3 constructs in Gal4AD and Gal4BD vectors were cotransformed into S. cerevisiae strain Y190 and colonies containing both vectors were selected on SD medium lacking leucine and tryptophan. Transformed colonies were directly screened for β-galactosidase activity in a colony-lift filter assay using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Overexpression of procaspase-2 results in apoptosis in many cell types, whereas overexpression of procaspase-3 leads to relatively little cell death (13Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (588) Google Scholar, 14Wang L. Miura M. Bergeron L. Zhu H. Yuan J. Cell. 1994; 78: 739-750Abstract Full Text PDF PubMed Scopus (801) Google Scholar, 15Kumar S. Kinoshita M. Dorstyn L. Noda M. Cell Death Differ. 1997; 4: 378-387Crossref Scopus (28) Google Scholar, 16Dorstyn L. Kumar S. Cell Death Differ. 1997; 4: 570-579Crossref PubMed Scopus (26) Google Scholar, 17Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2279) Google Scholar, 18Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.A. Smulson M.E. Yamin T.-T., Yu, V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3802) Google Scholar). To check if a class I prodomain fused to procaspase-3 can enhance its cell killing activity, we generated expression constructs in which the C2P was placed in frame at the amino terminus of procaspase-3 (Fig. 1 A). We constructed two chimeric expression vectors in which either the full-length procaspase-3 or procaspase-3 lacking the first 9 amino-terminal residues (Δ9) was fused to the prodomain of caspase-2. Because one of the cleavage sites in procaspase-3 occurs following Asp-9 (20Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (439) Google Scholar), the small prodomain region in caspase-3 and 3 amino acid residues at the carboxyl terminus of the C2P were deleted in the caspase-3Δ9 construct to avoid removal of C2P upon processing in transfected cells. To aid direct visualization of transfected cells we also placed the coding region of GFP at the carboxyl terminus of caspase-3-prodomain chimeric molecules (Fig. 1 A). All control and chimeric expression constructs were transfected into NIH-3T3 cells and visualized by fluorescence microscopy 18 h later. Most cells transfected with the GFP vector control showed normal flat morphology (Figs. 1 B and 2 A). As expected, procaspase-2-GFP expression killed almost all transfected cells (>95%), whereas caspase-3-GFP induced apoptosis in around 15% cells (Fig. 1 B). Most of the caspase-3-GFP transfected cells showed normal morphology and, as reported by us recently (19Colussi P.A. Harvey N.L. Kumar S. J. Biol. Chem. 1998; 273: 24535-24542Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). the caspase-3-GFP fusion protein was localized largely in the cytoplasm (Fig. 2 C). The cells transfected with the procaspase-2-GFP construct showed very weak fluorescence, presumably because of destruction of GFP in apoptotic cells (Fig. 2 B). Although caspase-3-GFP induced apoptosis in some of the transfected cells, apoptotic cells retained fluorescence suggesting that these cells represent early apoptotic cells (data not shown). The chimeric C2P-caspase-3-GFP molecules, with or without the first 9 amino acids of procaspase-3, were potent killers of transfected NIH-3T3 cells when compared with wild-type procaspase-3 with an apoptotic activity comparable with procaspase-2 (Figs. 1 B, 2 D, and 2 E). C2P-caspase-3(C163G)-GFP, the catalytically inactive caspase-3 mutant fused to the caspase-2 prodomain, was unable to induce cell death, suggesting that autocatalytic activity of C2P-caspase-3 was necessary for cell killing (Figs. 1 B and 2 F). As reported recently (19Colussi P.A. Harvey N.L. Kumar S. J. Biol. Chem. 1998; 273: 24535-24542Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), the C2P-caspase-3(C163G)-GFP mutant chimeric protein localized to discreet "dot-like" structures in both cytoplasmic and nuclear compartments of the transfected cells (Fig. 2 F). These results suggest that addition of the caspase-2 prodomain to the amino terminus of caspase-3 can confer strong apoptosis-inducing activity onto caspase-3. However, we do not yet know whether concentration in specific dot-like regions contributes to the apoptotic activity of the chimeric C2P-caspase-3 by promoting oligomerization and processing.Figure 2Immunofluorescence microscopy of various caspase-GFP fusion construct-transfected NIH-3T3 cells. NIH-3T3 cells were transfected with various expression constructs. At 18 h post-transfection, cells were fixed and photographed using a fluorescence microscope. A, GFP; B, caspase-2-GFP; C, caspase-3-GFP; D, C2P-caspase-3-GFP; E, C2P-caspase-3Δ9-GFP; F, C2P-caspase-3(C163G)-GFP. The relative exposures times forA–F were 16, 60, 32, 60, 60, and 32 s, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Many viral and cellular proteins have distinct inhibitory effects on biological and biochemical activities of caspases (1Kumar S. Trends Biochem. Sci. 1995; 20: 198-202Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 2Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 3Kumar S. Lavin M.F. Cell Death Diff. 1996; 3: 255-267PubMed Google Scholar, 4Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 230-299Abstract Full Text PDF Scopus (2185) Google Scholar, 5Chinnaiyan A.C. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar). Therefore, it was of interest to determine whether fusion of the caspase-2 prodomain to caspase-3 would affect interaction of the chimeric molecule with cellular and viral inhibitors of apoptosis. We compared the inhibitory effects of three caspase inhibitors (CrmA, P35, and MIHA/XIAP) and Bcl-2 on apoptosis induced by caspase-2-GFP and caspase-3-GFP containing the caspase-2 prodomain (C2P-caspase-3-GFP). Caspase expression constructs were cotransfected with expression constructs containing one of the inhibitors at a ratio of 1:3, and transfected NIH-3T3 cells scored 18 h later. As shown in Fig. 3, only P35 significantly inhibited caspase-2-GFP induced apoptosis (around 20%). However, C2P-caspase-3-GFP induced apoptosis was strongly inhibited by MIHA and P35, and partially inhibited by Bcl-2 and CrmA (Fig. 3). The observation that C2P-caspase-3-GFP-mediated apoptosis is suppressed by P35 and XIAP, but not by CrmA, is consistent with previously reported inhibitory profiles of caspase-3 (16Dorstyn L. Kumar S. Cell Death Differ. 1997; 4: 570-579Crossref PubMed Scopus (26) Google Scholar, 17Tewari M. Quan L.T. O'Rourke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2279) Google Scholar, 18Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.A. Smulson M.E. Yamin T.-T., Yu, V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3802) Google Scholar, 20Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (439) Google Scholar, 21Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 301-304Crossref Scopus (1724) Google Scholar), suggesting that the C2P-domain fusion does not alter the basic kinetic characteristics of caspase-3. To check whether the addition of the caspase-2 prodomain can mediate in vivo processing of caspase-3, we carried out immunoblotting of the extracts from transfected NIH-3T3 cells. As shown in Fig. 4, this protein did not show any appreciable processing in NIH-3T3 cells transfected with caspase-3-GFP (59 kDa). However, in cells transfected with C2P-caspase-3-GFP, no full-length (77 kDa) protein was detected. Instead, a band of 39 kDa, representing processed p12-GFP protein, was present (Fig. 4), demonstrating that the chimeric molecule containing the caspase-2 prodomain was much more readily processed when compared with wild-type caspase-3. On the other hand, the catalytically inactive C2P-caspase-3(C163G)-GFP protein was detected as a single unprocessed band of 77 kDa (Fig. 4) suggesting that autocatalytic activity of the chimeric caspase is required for its processing. Interestingly, in all transfection experiments, we noticed that the intensity of the processed protein band (39 kDa) representing the p12-GFP protein was always much lower than that of the unprocessed protein, suggesting that the processed caspase subunits may be unstable. This may explain, at least in part, why the GFP fluorescence is very weak in apoptotic NIH-3T3 cells transfected with C2P-caspase-3-GFP or caspase-2-GFP (Fig. 2). In NIH-3T3 cells cotransfected with C2P-caspase-3-GFP and either MIHA or P35, almost complete suppression of processing of the chimeric caspase molecule was seen (Fig. 4), suggesting that MIHA and P35 inhibit C2P-caspase-3-GFP-induced apoptosis by inhibiting procaspase processing. Bcl-2 is known to act upstream of caspase-3 by inhibiting the activation of caspase-9, the class I caspase required for the processing of caspase-3 (9Li P. Nijhawan D. Budhihardjo I. Srinivasula S. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6257) Google Scholar, 22Chinnaiyan A.M. Orth K. O'Rourke K. Duan H. Poirier G.G. Dixit V.M. J. Biol. Chem. 1996; 271: 4573-4576Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 23Pan G. O'Rourke K. Dixit V.M. J. Biol. Chem. 1998; 273: 5841-5845Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 24Srinivasula S.M. Ahmed M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar). However, Bcl-2 did not inhibit the processing of the C2P-caspase-3 chimeric molecule (Fig. 4). CrmA, which has only a marginal effect on C2P-caspase-3-mediated apoptosis (Fig. 3), also did not inhibit the processing of the chimeric molecule. We also compared the effects of Bcl-2, CrmA, MIHA, and P35 on caspase-2-GFP processing. Consistent with cell killing experiments, none of these molecules completely blocked procaspase-2 processing, as a processed doublet of 39/41 kDa, representing the p12-GFP/p14-GFP was seen in all cases (data not shown). As expected, caspase-2(C320G)-GFP protein was not significantly processed in transfected cells (data not shown). These results provide compelling evidence that the addition of a class I caspase prodomain to a class II caspase can confer autoprocessing ability on class II caspases. Procaspase-2 molecules can dimerize through their prodomains to mediate autocatalysis (10Butt A.J. Harvey N.L. Parasivam G. Kumar S. J. Biol. Chem. 1998; 273: 6763-6768Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To check if the chimeric caspase-3 molecule is able to dimerize, we utilized the yeast two-hybrid system. Caspase-3 constructs with or without the caspase-2 prodomain were fused to Gal4 DNA-binding and activation domains in S. cerevisiae vectors. To avoid processing of the molecules in transformed cells, we also used C163G mutants of caspase-3. As shown in Table I wild-type caspase-3 was unable to homodimerize. However, a small number of colonies ( 95%) colonies transformed with the C2P, or the chimeric proteins C2P-caspase-3(C163G) and C2P-caspase-3Δ9(C163G) exhibited β-galactosidase activity (Table I). The chimera containing the wild-type caspase-3 lacking the first 9 amino acid residues (C2P-caspase-3Δ9) was also able to form homodimers, albeit with a lower efficiency (∼70% colonies positive for β-galactosidase). These results suggest that addition of a caspase-2 prodomain onto caspase-3 can greatly enhance the ability of caspase-3 molecules to dimerize.Table IChimeric C2P-caspase-3 proteins homodimerize in S. cerevisiaeGal4BD construct in pAS2.1 vectorGal4AD construct in pACT2 vectorβ-galactosidase activityEmpty vectorEmpty vector—p53SV40 large T+Caspase-3Caspase-3—Caspase-3(C163G)Caspase-3(C163G)±C2PC2P+C2PEmpty vector—Empty vectorC2P—C2P-caspase-3Δ9C2P-caspase-3Δ9+C2P-caspase-3Δ9Empty vector—Empty vectorC2P-caspase-3Δ9—C2P-caspase-3(C163G)C2P-caspase-3(C163G)+C2P-caspase-3(C163G)Empty vector—Empty vectorC2P-caspase-3(C163G)—C2P-caspase-3Δ9(C163G)C2P-caspase-3Δ9(C163G)+C2P-caspase-3Δ9(C163G)Empty vector—Empty vectorC2P-caspase-3Δ9(C163G)—Various caspase-3 and C2P-caspase-3 constructs in Gal4BD and Gal4AD vectors were cotransformed into S. cerevisiae Y190 strain. Cotransformation of p53 and SV40 large T in Gal4BD and Gal4AD vectors, respectively, served as a positive control. Colonies were assayed for β-galactosidase activity by colony lift filter assay using X-gal as a substrate. In caspase-3(C163G) transformed cells, less than 10% colonies were positive for β-galactosidase (shown as ±) as compared to 70% for C2P-caspase-3Δ9, and >95% for C2P, C2P-caspase-3(C163G) and C2P-caspase-3Δ9(C163G). Open table in a new tab Various caspase-3 and C2P-caspase-3 constructs in Gal4BD and Gal4AD vectors were cotransformed into S. cerevisiae Y190 strain. Cotransformation of p53 and SV40 large T in Gal4BD and Gal4AD vectors, respectively, served as a positive control. Colonies were assayed for β-galactosidase activity by colony lift filter assay using X-gal as a substrate. In caspase-3(C163G) transformed cells, less than 10% colonies were positive for β-galactosidase (shown as ±) as compared to 70% for C2P-caspase-3Δ9, and >95% for C2P, C2P-caspase-3(C163G) and C2P-caspase-3Δ9(C163G). Our results provide strong support for the notion that the primary function of the prodomain in class I caspases is to promote dimerization and that dimerization is necessary and sufficient for autoprocessing. These results also support the idea that recruitment of class I caspases through adaptor molecules mediates dimerization and activation of upstream procaspases. Procaspase-8 has been shown to possess some intrinsic protease activity that probably mediates its autoprocessing (11Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). Because procaspase-3 in transfected cells is not processed, intrinsic protease activity in procaspase-3 may be responsible for its weak apoptosis-inducing activity when overexpressed in mammalian cells. A recently published study shows that artificially induced procaspase-3 oligomerization was sufficient for its activation (25MacCorkle R.A. Freeman K.W. Spencer D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3655-3660Crossref PubMed Scopus (175) Google Scholar), supporting our findings. Because most living cells express moderate levels of many procaspases, the intrinsic procaspase activity is unlikely to have any deleterious effect, until dimerization further augments processing and generation of fully activated caspase.

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