A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster.
Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.
Yokoyama
H
H
Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Mukae
N
N
Sakahira
H
H
Okawa
K
K
Iwamatsu
A
A
Nagata
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases