Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: Implicated mechanisms for caspase activation

CED-4 protein plays an important role in the induction of programmed cell d eath in Caenorhabditis elegans through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To i nvestigate the conservation of CED-4 function in evolution, transgenic Dros ophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death throu gh caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165 R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Dro sophila S2 cells. Immunoprecipitation analysis showed that both CED-4 and C ED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpress ion of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially preve nted cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 f unction is evolutionarily conserved in Drosophila, and the molecular mechan isms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.