Genetic analysis of programmed cell death in Drosophila melanogaster

Abstract

The correct regulation of programmed cell death, or apoptosis, is critical for proper development and prevention of disease. Components of the molecular mechanisms that govern apoptosis are conserved among organisms as diverse as C. elegans, Drosophila, and mammals. A central step in the execution of cell death is the activation of caspases, a conserved family of cysteine proteases. In Drosophila, the proteins, Reaper (Rpr), Head involution defective (Hid), and Grim, induce cell death via a mechanism that involves caspase activation. In order to further elucidate the mechanisms underlying the control of apoptosis, we conducted screens for genes involved in Rpr- or Hid-induced cell death. The analysis of the mutants isolated led to several new insights. The death inducing activity of Hid is post-transcriptionally down-regulated by the Ras/MAPK pathway. This is consistent with the pro-survival activity of this pathway and is probably mediated by direct phosphorylation of Hid. Furthermore, analysis of mutations in the gene encoding the Drosophila IAP, Diapl, led to a model for how Rpr, Hid and Grim activate caspases and induce cell death. In this model, Diapl binds and inhibits caspases; Rpr, Hid, and Grim induce cell death by binding Diapl and relieving caspases of Diapl-mediated inhibition. In addition, our mutants indicate that Diapl's RING finger domain, a domain found in proteins that function in ubiquitination, is required for inhibition of Rpr- and Grim-induced death but not Hid-induced death.

(cont.) Moreover, we identified a predicted ubiquitin conjugating enzyme, dBRUCE, which also functions to inhibit Rpr and Grim but not Hid. We propose that Diapl and dBRUCE function together to inhibit Rpr- and Grim-induced death by ubiquitinating pro-apoptotic proteins, possibly caspases or Rpr and Grim themselves, and targeting them for degradation by the proteasome. These findings are likely applicable to mammalian systems, since both dBRUCE and Diapl are conserved proteins with close homologs in murine and human genomes.