Caspase-9 Can Be Activated without Proteolytic Processing

The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coliis processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP. Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms. To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen. These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors. The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells. Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity. The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coliis processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP. Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms. To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen. These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors. The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells. Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity. Apoptosis, the ordered dismantling of animal cells that results in their death and removal from the organism, requires specific proteolysis of a subset of cellular proteins, mediated by caspases. Many apoptotic responses are initiated by activation of the apical caspases-8 or -9; the former by recruitment to ligated cell surface receptors belonging to tumor necrosis factor receptor-1 family (1Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar, 2Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2723) Google Scholar), and the latter by recruitment to Apaf-1, in the presence of ATP or dATP, following delivery of cytochrome c(cyto-c) 1The abbreviations used are:cyto-c , cytochrome c ; Ac, acetyl; Z, carbobenzoxy; AFC, 7-amino-4-trifluoromethyl coumarin; FMK, fluoromethyl ketone; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; casp, caspase(s); tPA, tissue plasminogen activator; MALDI-TOF, matrix-assisted laser desorption-time-of-flight.1The abbreviations used are:cyto-c , cytochrome c ; Ac, acetyl; Z, carbobenzoxy; AFC, 7-amino-4-trifluoromethyl coumarin; FMK, fluoromethyl ketone; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; casp, caspase(s); tPA, tissue plasminogen activator; MALDI-TOF, matrix-assisted laser desorption-time-of-flight. from mitochondria (3Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2726) Google Scholar). Activation of either of these two initiator caspases can lead to activation of the executioner caspase-3 (casp-3), by direct proteolysis of the casp-3 precursor (4Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar, 5Stennicke H.R. Salvesen G.S. Biochim. Biophys. Acta. 1998; 1387: 17-31Crossref PubMed Scopus (355) Google Scholar). Indeed all caspase zymogens are thought to be activated by limited proteolysis within a linker segment which results in generation of the characteristic large and small subunits of the catalytically active enzyme. Thus it has been specifically demonstrated that casp-1, -3, -7, and -8 gain marked increases in activity after proteolytic processing (6Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.T. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2185) Google Scholar, 7Zhou Q. Salvesen G.S. Biochem. J. 1997; 324: 361-364Crossref PubMed Scopus (122) Google Scholar, 8Stennicke H.R. Jürgenmeier J.M. Shin H. Deveraux Q. Wolf B.B. Yang X. Zhou Q. Ellerby H.M. Ellerby L.M. Bredesen D. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: 27084-27090Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 9Muzio 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 (882) Google Scholar). This is typical of proteolytic enzymes from all families and catalytic classes. As pointed out by Neurath (10Neurath H. Trends Biochem. Sci. 1989; 14: 268-271Abstract Full Text PDF PubMed Scopus (156) Google Scholar), the zymogens are stored in an inactive form to prevent adventitious proteolysis before it is needed at the required site of action. Frequently protease zymogens are activated by other proteases, in a cascade mechanism that results in either amplification or localization to specific sites. There are few exceptions to the rule that zymogen activation requires proteolysis, and these exceptional zymogens, such as tissue plasminogen activator (tPA), usually have specific allosteric modulators that allow them to become active at the required site, without proteolysis.The initiator casp-8 has some of these exceptional properties. Casp-8 initiates apoptotic signaling induced by specific ligation of death receptors (reviewed in Refs. 11Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar and 12Ware C.F. Santee S. Glass A. Thomson A. The Cytokine Handbook. 3rd Ed. Academic Press Ltd., San Diego, CA1998Google Scholar). Recruitment of procasp-8 to the cytosolic face of ligated Fas or tumor necrosis factor receptor-1 results in its autoactivation by a mechanism involving clustering of zymogen molecules (9Muzio 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 (882) Google Scholar, 13Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The zymogen possesses ∼1% of the activity of the activated enzyme, and it is hypothesized that this small amount of activity of the zymogen is sufficient to drive cleavage of clustered casp-8 zymogen molecules to the active form. Thus casp-8 has a zymogenicity ratio of 100 (defined as the ratio of the activity of the enzyme to the zymogen). Nevertheless, proteolysis of the casp-8 zymogen is thought to be required for its activation (9Muzio 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 (882) Google Scholar, 13Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar).In light of the casp-8 activation studies, it has been hypothesized that the initiator caspases, including caspases-8 and -9 (14Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar) andCaenorhabditis elegans CED-3, share a common mechanism of activation (15Yang X. Chang H.Y. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar). This would require clustering zymogens that possess a small amount of activity in their single chain forms, with production of the active protease dependent on proteolytic processing of the zymogen. In turn, this would mean that preventing proteolysis of the zymogen in the interdomain linker should inactivate apoptotic signal transmission. We tested this hypothesis for casp-9 by utilizing a cell-free system (16Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4433) Google Scholar) that allows dissection of the requirements for zymogen activation. Apoptosis, the ordered dismantling of animal cells that results in their death and removal from the organism, requires specific proteolysis of a subset of cellular proteins, mediated by caspases. Many apoptotic responses are initiated by activation of the apical caspases-8 or -9; the former by recruitment to ligated cell surface receptors belonging to tumor necrosis factor receptor-1 family (1Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar, 2Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2723) Google Scholar), and the latter by recruitment to Apaf-1, in the presence of ATP or dATP, following delivery of cytochrome c(cyto-c) 1The abbreviations used are:cyto-c , cytochrome c ; Ac, acetyl; Z, carbobenzoxy; AFC, 7-amino-4-trifluoromethyl coumarin; FMK, fluoromethyl ketone; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; casp, caspase(s); tPA, tissue plasminogen activator; MALDI-TOF, matrix-assisted laser desorption-time-of-flight.1The abbreviations used are:cyto-c , cytochrome c ; Ac, acetyl; Z, carbobenzoxy; AFC, 7-amino-4-trifluoromethyl coumarin; FMK, fluoromethyl ketone; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; casp, caspase(s); tPA, tissue plasminogen activator; MALDI-TOF, matrix-assisted laser desorption-time-of-flight. from mitochondria (3Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2726) Google Scholar). Activation of either of these two initiator caspases can lead to activation of the executioner caspase-3 (casp-3), by direct proteolysis of the casp-3 precursor (4Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar, 5Stennicke H.R. Salvesen G.S. Biochim. Biophys. Acta. 1998; 1387: 17-31Crossref PubMed Scopus (355) Google Scholar). Indeed all caspase zymogens are thought to be activated by limited proteolysis within a linker segment which results in generation of the characteristic large and small subunits of the catalytically active enzyme. Thus it has been specifically demonstrated that casp-1, -3, -7, and -8 gain marked increases in activity after proteolytic processing (6Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.T. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2185) Google Scholar, 7Zhou Q. Salvesen G.S. Biochem. J. 1997; 324: 361-364Crossref PubMed Scopus (122) Google Scholar, 8Stennicke H.R. Jürgenmeier J.M. Shin H. Deveraux Q. Wolf B.B. Yang X. Zhou Q. Ellerby H.M. Ellerby L.M. Bredesen D. Green D.R. Reed J.C. Froelich C.J. Salvesen G.S. J. Biol. Chem. 1998; 273: 27084-27090Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar, 9Muzio 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 (882) Google Scholar). This is typical of proteolytic enzymes from all families and catalytic classes. As pointed out by Neurath (10Neurath H. Trends Biochem. Sci. 1989; 14: 268-271Abstract Full Text PDF PubMed Scopus (156) Google Scholar), the zymogens are stored in an inactive form to prevent adventitious proteolysis before it is needed at the required site of action. Frequently protease zymogens are activated by other proteases, in a cascade mechanism that results in either amplification or localization to specific sites. There are few exceptions to the rule that zymogen activation requires proteolysis, and these exceptional zymogens, such as tissue plasminogen activator (tPA), usually have specific allosteric modulators that allow them to become active at the required site, without proteolysis. The initiator casp-8 has some of these exceptional properties. Casp-8 initiates apoptotic signaling induced by specific ligation of death receptors (reviewed in Refs. 11Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar and 12Ware C.F. Santee S. Glass A. Thomson A. The Cytokine Handbook. 3rd Ed. Academic Press Ltd., San Diego, CA1998Google Scholar). Recruitment of procasp-8 to the cytosolic face of ligated Fas or tumor necrosis factor receptor-1 results in its autoactivation by a mechanism involving clustering of zymogen molecules (9Muzio 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 (882) Google Scholar, 13Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The zymogen possesses ∼1% of the activity of the activated enzyme, and it is hypothesized that this small amount of activity of the zymogen is sufficient to drive cleavage of clustered casp-8 zymogen molecules to the active form. Thus casp-8 has a zymogenicity ratio of 100 (defined as the ratio of the activity of the enzyme to the zymogen). Nevertheless, proteolysis of the casp-8 zymogen is thought to be required for its activation (9Muzio 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 (882) Google Scholar, 13Yang X. Chang H.Y. Baltimore D. Mol. Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). In light of the casp-8 activation studies, it has been hypothesized that the initiator caspases, including caspases-8 and -9 (14Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar) andCaenorhabditis elegans CED-3, share a common mechanism of activation (15Yang X. Chang H.Y. Baltimore D. Science. 1998; 281: 1355-1357Crossref PubMed Scopus (234) Google Scholar). This would require clustering zymogens that possess a small amount of activity in their single chain forms, with production of the active protease dependent on proteolytic processing of the zymogen. In turn, this would mean that preventing proteolysis of the zymogen in the interdomain linker should inactivate apoptotic signal transmission. We tested this hypothesis for casp-9 by utilizing a cell-free system (16Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4433) Google Scholar) that allows dissection of the requirements for zymogen activation. We thank Scott Snipas, Annamarie Price, and Qiao Zhou for assistance with expression constructs. We also thank Martin Renatus for helpful discussion of the manuscript.