Identification of regulatory and catalytic domains in the apoptosis nuclease DFF40/CAD.
The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290-345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1-83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian and Drosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway.
Inohara
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Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Koseki
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Chen
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Benedict
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MA
Núñez
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G
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D000075223:Poly-ADP-Ribose Binding Proteins; D017386:Sequence Homology, Amino Acid
D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; C109664:caspase-activated DNase inhibitor; C113696:DFFB protein, human; D003851:Deoxyribonucleases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases