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
Co-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 prodomain and a conserved lysine residue within a putative ATP/GTP-binding motif of CED-4. Co-expression of ced-9 with ced-4 and ced-3 inhibited the ability of CED-4 to stimulate CED-3 processing and CED-3-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.
Seshagiri
S
S
Department of Entomology, University of Georgia, Athens, Georgia 30602, USA.
Miller
L K
LK
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000235:genetics*; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D015801:Helminth Proteins / Q000235:genetics*; D016296:Mutagenesis; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D018411:Spodoptera / Q000166:cytology
D051017:Apoptosis Regulatory Proteins; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C089224:Ced-4 protein, C elegans; C087233:Ced-9 protein, C elegans; D015801:Helminth Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D020169:Caspases; D003546:Cysteine Endopeptidases; C089223:ced-3 protein, C elegans