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 sub units (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 (2 2% identity). Mammalian CADs carry a nuclear localization signal at the C t erminus. In contrast, dCAD lacked the corresponding sequence, and the purif ied dCAD did not cause DNA fragmentation in nuclei in a cell-free system. W hen dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dI CAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (dr ICE), 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 i n gel filtration. When a Drosophila neuronal cell line was induced to apopt osis 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.