Pro-caspase-3 Is a Major Physiologic Target of Caspase-8

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (k cat/K m) of 8.7 × 105 and 2.8 × 105m−1 s−1, respectively. These rates are of sufficient magnitude to indicate direct processingin vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors. The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (k cat/K m) of 8.7 × 105 and 2.8 × 105m−1 s−1, respectively. These rates are of sufficient magnitude to indicate direct processingin vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors. tumor necrosis factor receptor 1 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate 7-amino-4-trifluoromethyl coumarin p-nitroanilide polyacrylamide gel electrophoresis bovine poly(ADP-ribose)polymerase tumor necrosis factor carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin carbobenzoxy-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethyl coumarin acetyl 1,4-piperazinediethanesulfonic acid polymerase chain reaction. Regulation of apoptosis is vital to the development and long term survival of metazoan animals. Apoptosis is required to maintain the balance between cell proliferation and cell death and, therefore, disruptions in the apoptotic program are associated with pathologies such as cancer, where there is too little cell death, and degenerative diseases, where there is too much cell death. Apoptosis can be initiated by at least three types of signals: (i) specific ligation of members on the tumor necrosis factor receptor (TNFR-1)1family, which includes Fas/Apo-1/CD95; (ii) cellular stress, which includes genotoxic damage and anti-neoplastic drugs; and (iii) delivery of granule-associated serine proteases from cytotoxic lymphocytes into target cells. Key mediators that initiate and execute the apoptotic program are members of the caspase family of cysteine proteases whose activation is believed to be essential for virtually all forms of apoptosis (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar, 2Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4105) Google Scholar, 3Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar). Caspases-3, -6, and -7 are involved in the execution of cells in response to many apoptotic stimuli including ligation of death receptors of the TNFR-1 receptor family, resulting in cleavage of a number of proteins whose limited proteolysis is definitive of apoptosis. However, these executioner caspases are not directly activated by receptor ligation, but rely on the proteolytic activity of upstream initiator caspases-8 and -10 (4Muzio 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, 5Boldin 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, 6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar). In the case of caspase-8 the activation occurs by recruitment of the zymogen to the cytosolic face of the death receptor, such that the initial proteolytic signal originates by autoprocessing of the clustered zymogen (7Muzio 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, 8Yang X. Chang H.Y. Baltimore D. Mol Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar).At the execution phase, caspase-3 seems to be upstream of caspases-6 and -7 and, therefore, its activation represents a key point in transmission of the proteolytic signal (9Hirata H. Takahashi A. Kobayashi S. Yonehara S. Sawai H. Okazaki T. Yamamoto K. Sasada M. J. Exp. Med. 1998; 187: 587-600Crossref PubMed Scopus (398) Google Scholar). However, the exact mechanism of how the death signal is conveyed from caspase-8 to caspase-3 remains unresolved. Is the apoptotic signal transmitted by direct activation of the executioners by the initiators, thus constituting a minimal two-step cascade that serves to mediate the apoptotic signals, or is the signal by the of as by Scaffidi C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google these have a of the activation of pro-caspase-3 by caspases-8 and -10 and proteases in a system, and to the activation of the zymogen by caspases-8 and -10 in cytosolic to the of that in transmission of the proteolytic death signal from the initiator caspases to the and on the of in vitro the that and of the apoptotic are required in are into and executioner (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar, A.M. Dixit V.M. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar). at which caspases initiate apoptotic is at the cell where members of the TNFR-1 family of death receptors a signal the cell receptor The point of the of X. J. R. X. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google G. 1997; Full Text PDF PubMed Scopus Google and pathway is is how the the apoptotic anti-neoplastic genotoxic and of cellular signal all to on the Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). point of the is specific caspase activation, and point In the death receptor the caspase-8 a proteolytic signal at the cytosolic face of the receptor (4Muzio 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, 5Boldin 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, 6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar, T. R. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, C. Dixit V.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The signal for activation to be of that activity in its zymogen to proteolytic (7Muzio 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, 8Yang X. Chang H.Y. Baltimore D. Mol Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In the specific or delivery of D. S.M. Ahmad M. Alnemri E.S. X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar, T. G. J. Exp. Med. 1996; PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, H. X. A. X. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google to the in recruitment and activation of D. S.M. Ahmad M. Alnemri E.S. X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-8 and have to on in vitro and the executioner caspases whose activation with apoptosis. there are to activate the executioner and in caspases-8 and can be of as whose at the execution of caspases-8 and -10 can activate pro-caspase-3 in to the caspases directly or previous studies have on in vitro or cytosolic extracts S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar, M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, K. Chinnaiyan A.M. M. Dixit V.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We here demonstrate that caspase-8 and -10 can activate on caspase-8 that activation with the same rate in a cytosolic a requirement for an intermediary of from the on caspase activation by and cells in caspase-3 to of of in death apoptosis the and activation of caspase-3 which a to a role in caspase that are required to the apoptotic signal generated by of cells by but in a of cell C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar). that the caspase of the a In of the of caspase activity initiated by of caspase-8 to cytosolic extracts of is in the of T. M. Dixit S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of to is is not a and the of activation of a caspase-3 or the caspase activity in extracts is to such an have not The data in not requirement for an intermediary between caspase-8 and the is the occurs in and caspase-8 its signal to the executioners in vivo. the for a role of in apoptosis triggered by death receptor ligation from studies of or which is to by apoptosis in Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). that apoptosis triggered by genotoxic and but there are in the data the of apoptosis induced by death receptors C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar, A.M. Dixit V.M. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar, A. A. R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Krammer P.H. S. J. PubMed Scopus Google Scholar). In a of cell Scaffidi C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google that are not by from apoptosis triggered by However, the of and cell not be the to a role for apoptosis in which is on ligation, is in A. Krammer P.H. S. J. PubMed Scopus Google Scholar). In death of in was in A. M. T. A. A. D. A. Med. 1996; PubMed Scopus Google Scholar). These studies can be cells direct transmission of caspase-8 to a C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google is not processed by the upstream of the and, therefore, upstream of the the signal is activated by the of caspase-8 on or a that for apoptosis. The of the in death is not that a of cell to the caspase-8 direct transmission of the signal from caspase-8 to the executioner caspase-3 occurs in cell is the of a of are to as by the of the is not cells by a of to the death but studies on that cells in the that are to apoptosis during of the have death and the caspase-8 to direct transmission of the death and that pro-caspase-3 is a of caspase-8 Regulation of apoptosis is vital to the development and long term survival of metazoan animals. Apoptosis is required to maintain the balance between cell proliferation and cell death and, therefore, disruptions in the apoptotic program are associated with pathologies such as cancer, where there is too little cell death, and degenerative diseases, where there is too much cell death. Apoptosis can be initiated by at least three types of signals: (i) specific ligation of members on the tumor necrosis factor receptor (TNFR-1)1family, which includes Fas/Apo-1/CD95; (ii) cellular stress, which includes genotoxic damage and anti-neoplastic drugs; and (iii) delivery of granule-associated serine proteases from cytotoxic lymphocytes into target cells. Key mediators that initiate and execute the apoptotic program are members of the caspase family of cysteine proteases whose activation is believed to be essential for virtually all forms of apoptosis (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar, 2Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4105) Google Scholar, 3Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2176) Google Scholar). Caspases-3, -6, and -7 are involved in the execution of cells in response to many apoptotic stimuli including ligation of death receptors of the TNFR-1 receptor family, resulting in cleavage of a number of proteins whose limited proteolysis is definitive of apoptosis. However, these executioner caspases are not directly activated by receptor ligation, but rely on the proteolytic activity of upstream initiator caspases-8 and -10 (4Muzio 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, 5Boldin 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, 6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar). In the case of caspase-8 the activation occurs by recruitment of the zymogen to the cytosolic face of the death receptor, such that the initial proteolytic signal originates by autoprocessing of the clustered zymogen (7Muzio 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, 8Yang X. Chang H.Y. Baltimore D. Mol Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). the execution phase, caspase-3 seems to be upstream of caspases-6 and -7 and, therefore, its activation represents a key point in transmission of the proteolytic signal (9Hirata H. Takahashi A. Kobayashi S. Yonehara S. Sawai H. Okazaki T. Yamamoto K. Sasada M. J. Exp. Med. 1998; 187: 587-600Crossref PubMed Scopus (398) Google Scholar). However, the exact mechanism of how the death signal is conveyed from caspase-8 to caspase-3 remains unresolved. Is the apoptotic signal transmitted by direct activation of the executioners by the initiators, thus constituting a minimal two-step cascade that serves to mediate the apoptotic signals, or is the signal by the of as by Scaffidi C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar). these have a of the activation of pro-caspase-3 by caspases-8 and -10 and proteases in a system, and to the activation of the zymogen by caspases-8 and -10 in cytosolic to the of that in transmission of the proteolytic death signal from the initiator caspases to the and on the of in vitro the that and of the apoptotic are required in vivo. are into and executioner (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar, A.M. Dixit V.M. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar). at which caspases initiate apoptotic is at the cell where members of the TNFR-1 family of death receptors a signal the cell receptor The point of the of X. J. R. X. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google G. 1997; Full Text PDF PubMed Scopus Google and pathway is is how the the apoptotic anti-neoplastic genotoxic and of cellular signal all to on the Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). point of the is specific caspase activation, and point In the death receptor the caspase-8 a proteolytic signal at the cytosolic face of the receptor (4Muzio 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, 5Boldin 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, 6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar, T. R. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, C. Dixit V.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The signal for activation to be of that activity in its zymogen to proteolytic (7Muzio 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, 8Yang X. Chang H.Y. Baltimore D. Mol Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In the specific or delivery of D. S.M. Ahmad M. Alnemri E.S. X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar, T. G. J. Exp. Med. 1996; PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, H. X. A. X. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google to the in recruitment and activation of D. S.M. Ahmad M. Alnemri E.S. X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-8 and have to on in vitro and the executioner caspases whose activation with apoptosis. there are to activate the executioner and in caspases-8 and can be of as whose at the execution of caspases-8 and -10 can activate pro-caspase-3 in to the caspases directly or previous studies have on in vitro or cytosolic extracts S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar, M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, K. Chinnaiyan A.M. M. Dixit V.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We here demonstrate that caspase-8 and -10 can activate on caspase-8 that activation with the same rate in a cytosolic a requirement for an intermediary of from the on caspase activation by and cells in caspase-3 to of of in death apoptosis the and activation of caspase-3 which a to a role in caspase that are required to the apoptotic signal generated by of cells by but in a of cell C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar). that the caspase of the a In of the of caspase activity initiated by of caspase-8 to cytosolic extracts of is in the of T. M. Dixit S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of to is is not a and the of activation of a caspase-3 or the caspase activity in extracts is to such an have not The data in not requirement for an intermediary between caspase-8 and the is the occurs in and caspase-8 its signal to the executioners in vivo. the for a role of in apoptosis triggered by death receptor ligation from studies of or which is to by apoptosis in Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). that apoptosis triggered by genotoxic and but there are in the data the of apoptosis induced by death receptors C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar, A.M. Dixit V.M. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar, A. A. R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Krammer P.H. S. J. PubMed Scopus Google Scholar). In a of cell Scaffidi C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google that are not by from apoptosis triggered by However, the of and cell not be the to a role for apoptosis in which is on ligation, is in A. Krammer P.H. S. J. PubMed Scopus Google Scholar). In death of in was in A. M. T. A. A. D. A. Med. 1996; PubMed Scopus Google Scholar). These studies can be cells direct transmission of caspase-8 to a C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google is not processed by the upstream of the and, therefore, upstream of the the signal is activated by the of caspase-8 on or a that for apoptosis. The of the in death is not that a of cell to the caspase-8 direct transmission of the signal from caspase-8 to the executioner caspase-3 occurs in cell is the of a of are to as by the of the is not cells by a of to the death but studies on that cells in the that are to apoptosis during of the have death and the caspase-8 to direct transmission of the death and that pro-caspase-3 is a of caspase-8 are into and executioner (1Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar, A.M. Dixit V.M. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar). at which caspases initiate apoptotic is at the cell where members of the TNFR-1 family of death receptors a signal the cell receptor The point of the of X. J. R. X. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google G. 1997; Full Text PDF PubMed Scopus Google and pathway is is how the the apoptotic anti-neoplastic genotoxic and of cellular signal all to on the Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). point of the is specific caspase activation, and point In the death receptor the caspase-8 a proteolytic signal at the cytosolic face of the receptor (4Muzio 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, 5Boldin 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, 6Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar, T. R. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, C. Dixit V.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The signal for activation to be of that activity in its zymogen to proteolytic (7Muzio 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, 8Yang X. Chang H.Y. Baltimore D. Mol Cell. 1998; 1: 319-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In the specific or delivery of D. S.M. Ahmad M. Alnemri E.S. X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar, T. G. J. Exp. Med. 1996; PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, H. X. A. X. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google to the in recruitment and activation of D. S.M. Ahmad M. Alnemri E.S. X. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-8 and have to on in vitro and the executioner caspases whose activation with apoptosis. there are to activate the executioner and in caspases-8 and can be of as whose at the execution of apoptosis. caspases-8 and -10 can activate pro-caspase-3 in to the caspases directly or previous studies have on in vitro or cytosolic extracts S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (480) Google Scholar, M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. J. S.M. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, K. Chinnaiyan A.M. M. Dixit V.M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We here demonstrate that caspase-8 and -10 can activate on caspase-8 that activation with the same rate in a cytosolic a requirement for an intermediary of from the on caspase activation by and cells in caspase-3 to of of in death apoptosis the and activation of caspase-3 which a to a role in caspase that are required to the apoptotic signal generated by of cells by but in a of cell C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar). that the caspase of the a In of the of caspase activity initiated by of caspase-8 to cytosolic extracts of is in the of T. M. Dixit S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the of to is is not a and the of activation of a caspase-3 or the caspase activity in extracts is to such an have not The data in not requirement for an intermediary between caspase-8 and the is the occurs in and caspase-8 its signal to the executioners in vivo. the for a role of in apoptosis triggered by death receptor ligation from studies of or which is to by apoptosis in Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). that apoptosis triggered by genotoxic and but there are in the data the of apoptosis induced by death receptors C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar, A.M. Dixit V.M. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar, A. A. R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Krammer P.H. S. J. PubMed Scopus Google Scholar). In a of cell Scaffidi C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google that are not by from apoptosis triggered by However, the of and cell not be the to a role for apoptosis in which is on ligation, is in A. Krammer P.H. S. J. PubMed Scopus Google Scholar). In death of in was in A. M. T. A. A. D. A. Med. 1996; PubMed Scopus Google Scholar). These studies can be cells direct transmission of caspase-8 to a C. S. A. C. Krammer P.H. Peter M.E. J. 1998; PubMed Scopus Google Scholar). is not processed by the upstream of the and, therefore, upstream of the the signal is activated by the of caspase-8 on or a that for apoptosis. The of the in death is not that a of cell to the caspase-8 direct transmission of the signal from caspase-8 to the executioner caspase-3 occurs in cell is the of a of are to as by the of the is not cells by a of to the death but studies on that cells in the that are to apoptosis during of the have death and the caspase-8 to direct transmission of the death and that pro-caspase-3 is a of caspase-8 We Dixit for and and for