Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis

Background: Programmed cell death or apoptosis is a key feature of normal development, tissue homeostasis and disease progression in metazoans. Genetic studies in the nematode C. elegans have identified three key genes involved in apoptosis, ced-3, ced-4 and ced-9. Expression of ced-3 and ced-4 is required for the induction of cell death, whereas expression of ced-9 is necessary to inhibit cell death. The precise mechanism by which these genes influence the life or death decision of a cell is not known. In this study, we have expressed the genes in an insect cell line to explore their role in the apoptotic pathway. Results: Go-expression of ced-4 with ced-3 in insect cells stimulated both the induction and the level of CED-3-mediated apoptosis. Stimulation of CED-3-dependent apoptosis by CED-4 was accompanied by accelerated processing of CED-3, which was dependent on the presence of a wild-type CED-3 pro-domain and a conserved lysine residue within a putative ATP/GTP-binding motif of CED-4. Go-expression of ced-9 with ced-4 and ced-3 inhibited the ability of CED-4 to stimulate CED-3 processing and CED-9-dependent apoptosis, Although a temperature-sensitive CED-9 mutant was unable to block CED-4 activity and failed to associate with CED-4, a deletion mutant of CED-9 lacking the carboxy-terminal hydrophobic domain could associate with CED-4 and block CED-4 activity. Conclusions: Our results establish a role for CED-4 in the processing of CED-3 and the stimulation of CED-3-induced apoptosis, Furthermore, we show that CED-9 achieves its anti-apoptotic effect by associating with CED-4 and blocking the ability of CED-4 to process CED-3.