Abstract
We report here the purification of the third protein factor, Apaf-3, that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuated the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Apoptosis / physiology*
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Apoptotic Protease-Activating Factor 1
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Binding Sites
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Breast Neoplasms
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Caspase 3
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Caspase 9
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Caspases*
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Cell Line
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / isolation & purification
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Cysteine Endopeptidases / metabolism*
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Cytochrome c Group / metabolism*
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Deoxyadenine Nucleotides / metabolism*
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Enzyme Activation
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Epithelial Cells
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HeLa Cells
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Humans
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Kidney
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Models, Chemical
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Molecular Sequence Data
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Multienzyme Complexes / metabolism
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Mutation
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Protein Binding
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Proteins / metabolism*
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Tumor Cells, Cultured
Substances
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APAF1 protein, human
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Apoptotic Protease-Activating Factor 1
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Cytochrome c Group
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Deoxyadenine Nucleotides
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Multienzyme Complexes
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Proteins
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CASP3 protein, human
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CASP9 protein, human
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Caspase 3
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Caspase 9
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Caspases
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Cysteine Endopeptidases
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2'-deoxyadenosine triphosphate