Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis.
The ICE/CED-3 family of proteases has been implicated in playing a fundamental role in programmed cell death. Bcl-2 protein represses a number of apoptotic death programs, but the biochemical mechanism of its action is not known. We investigated the activation of ICE/CED-3 proteases induced by three apoptotic stimuli (staurosporine, ceramide, and serum withdrawal) in the neuronal cell line GT1-7 and in cells overexpressing Bcl-2. Rapid activation of a 17 kDa subunit of an activated member of the ICE/CED-3 family is demonstrated by affinity-labeling GT1-7 extracts from apoptotic controls cells with a biotinylated ICE/CED-3 inhibitor. This activation corresponds to an increased ICE/CED-3-like protease activity in extracts measured by a fluorogenic substrate assay. In a cell-free system, these extracts induce apoptotic morphological changes in intact nuclei. All three activities are readily inhibited by treatment of control extracts with ICE/CED-3-like protease inhibitors. Overexpressed Bcl-2 inhibits the activation of the 17 kDa protein, the ICE/CED-3-like protease activity in the fluorogenic assay, and the induction of apoptotic morphological changes in HeLa nuclei in the cell-free system, similar to results obtained with ICE/CED-3 protease inhibitors. At the mRNA level, overexpression of Bcl-2 did not alter expression of five members of the ICE/CED-3 family: CPP32, ICE, Mch 2, Nedd 2, and TX. Overexpression of Bcl-2 prevented the apoptosis-induced processing of pro-Nedd 2 to the cleaved form. These data suggest that Bcl-2 participates upstream from the function of ICE/CED-3 proteases and may inhibit apoptosis by preventing the post-translational activation of ICE/CED-3 proteases.
Srinivasan
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A
Program on Aging, Burnham Institute, La Jolla, California 92037, USA.
Foster
L M
LM
Testa
M P
MP
Ord
T
T
Keane
R W
RW
Bredesen
D E
DE
Kayalar
C
C
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001790:Blood Physiological Phenomena; D029742:Caenorhabditis elegans Proteins; D020170:Caspase 1; D020169:Caspases; D002460:Cell Line; D002518:Ceramides / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015801:Helminth Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002518:Ceramides; D004791:Enzyme Inhibitors; D015801:Helminth Proteins; D015345:Oligonucleotide Probes; D019253:Proto-Oncogene Proteins c-bcl-2; D020169:Caspases; D003546:Cysteine Endopeptidases; C089223:ced-3 protein, C elegans; D020170:Caspase 1; D019311:Staurosporine