THE DEATH INHIBITORY MOLECULES CED-9 AND CED-4L USE A COMMON MECHANISM TO INHIBIT THE CED-3 DEATH PROTEASE

The apoptotic machinery of Caenorhabditis elegans includes three core interacting components: CED-3, CED-4, and CED-9, CED-3 is a death prot ease composed of a prodomain and a protease domain. CED-4 is a P-loop- containing, nucleotide-binding molecule that complexes with the single polypeptide zymogen form of CED-3, promoting its activation by autopr ocessing, CED-9 blocks death by complexing with CED-4 and suppressing its ability to promote CED-3 activation. A naturally occurring alterna tively spliced form of CED-4 that contains an insertion within the nuc leotide-binding region (CED-4L) functions as a dominant negative inhib itor of CED-3 processing and attenuates cell death. Domain mapping stu dies revealed that distinct regions within CED-4 bind to the CED-3 pro domain and protease domain. Importantly, the CED-4 P-loop was involved in prodomain binding, Disruption of P-loop geometry because of mutati on of a critical lysine (K165R) or insertional inactivation (CED-4L) a bolished prodomain binding. Regardless, K165R and CED-4L still retaine d CED-3 binding through the protease domain but were unable to initiat e CED-3 processing. Therefore, the P-loop-prodomain interaction is cri tical for triggering CED-4-mediated CED-3 processing. Underscoring the importance of this interaction was the finding that CED-9 contacted t he P-loop and selectively inhibited its interaction with the CED-3 pro domain. These results provide a simple mechanism for how CED-9 functio ns to block CED-4-mediated CED-3 processing and cell death.