Apoptosis, or programmed cell death, is an evolutionarily conserved, genetically regulated process, whereby cells that are no longer needed undergo self-destruction through the activation of a cell suicide program [1, 2]. This cell death program is associated with characteristic morphological alterations, such as condensation of the nucleus and cytoplasm, fragmentation of nuclear DNA, reorganization of the cytoskeleton, and reduction of the cell into apoptotic bodies that can be phagocytosed by neighbouring epithelial cells or phagocytes [1, 3].
Autophagy is also an evolutionarily conserved mechanism that degrades unnecessary long-lived proteins and organelles. During autophagy, cellular components are sequestered by double-membrane structures called autophagosomes. These autophagosomes then fuse with lysosomes to form autolysosomes, where degradation occurs . The autophagy acts as a cellular response against extracellular stresses, such as nutrient starvation, hypoxia, and overcrowding and against intracellar stresses, such as formation of damaged or redundant organelles and cytoplasmic components . Even though autophagy can induce a cell-survival response to some conditions, autophagic structures, especially the autophagic vacuoles, are associated with cell death. This cell death phenomenon is classified as type II cell death and called autophagic cell death. In Drosophila, loss of function of the Atg genes leads to lethality in the transition from the larval to pupal stages, because autophagic cell death is essential for puparium formation .
The caspases are a family of ubiquitously expressed cysteine proteases whose prototypic member is the Caenorhabditis elegans death effector, CED-3 . Activation of caspases typically leads to the selective cleavage of a restricted set of target proteins, generally resulting in inactivation of the target proteins. Normally present in cells as inactive precursors, caspases are proteolytically activated following upstream pro-apoptotic signals. Activated caspases cleave their substrates at an aspartic acid residue, and substrate specificity is determined by a four-residue motif N-terminal to the cleavage site [6, 7]. The "initiator" caspases primarily activate the downstream "effector" caspases whose proteolytic activity is directed toward the deconstruction of the cellular machinery during apoptosis.
In Drosophila, a normally functioning apoptotic pathway depends critically on caspases, seven of which have been identified in the Drosophila melanogaster genome [8, 9]. These Drosophila caspase genes include three initiators and four effectors. The initiators are Dronc (Drosophila Nedd-2-like caspase), Dredd (Death-related ced-3/Nedd2-like), and strica/dream. The effectors are Dcp-1 (Death caspase-1), Drice (Drosophila ice), Damm, and decay (Death executioner caspase related to Apopain/Yama) [10–13]. These caspases are expected to have functions that lead to apoptosis. Many recent reports have described non-apoptotic functions of caspases, such as the cell proliferation function of Dronc [14, 15], spermatid individualization by Drice [14, 16], and activation of the Drosophila immune system, toll receptor signaling, by Dredd .
Dcp-1 proteins cleave cysteine protease substrates and are important for development and oogenesis [13, 17]. Dcp-1 deletion mutants display a lack of germline cell death phenotype during mid-stage oogenesis in response to nutrient deprivation , whereas in normal flies, cell death occurs during mid-stage oogenesis under nutrient-deprived conditions (stage 7 to 8) [3, 19–21]. In contrast, over-expression of a single copy of the truncated N-terminal region of Dcp-1 (constitutively active Dcp-1), specifically in the eye using the Glass Multimer Reporter (GMR) promoter, results in a slightly rough and reduced pigment eye phenotype . In addition to the critical roles of Dcp-1 and caspase 3 in apoptosis, recent studies in mammals and flies suggest that these caspases have many important non-apoptotic roles [13, 23], although how these caspases act in these non-apoptotic responses are incompletely understood.
We hypothesized the existence of unknown effectors for Dcp-1. Identification and characterization of such proteins would allow us to better understand apoptotic pathways and Dcp-1-related non-apoptotic pathways. Since a large-scale genetic screen to identify components of the Dcp-1 pathway had not been preformed, either in vivo or in vitro, we screened ~15,000 GenExel EP fly lines. Interestingly, we noticed that autophagy-related genes specifically suppressed the rough eye phenotype caused by Dcp-1 expression. In addition to eight autophagy genes and two genes reported to be related to autophagy, we identified five Insulin/IGF and TOR signaling genes, six MAP kinase and Jun N-terminal kinase (JNK) signaling, four Ecdysone genes, and others (Additional file 1). There were several interesting novel genes among the 72 Dcp-1 genetic interactors. The identification of many new Dcp-1-interacting genes will help clarify the molecular mechanisms by which Dcp-1 regulates apoptosis and other non-apoptotic cellular processes. In addition, our findings that autophagy genes influence the roles of the Dcp-1 caspase in autophagy suggest a relationship between autophagy and apoptosis. Finally, our findings that signaling pathways such as MAP kinase, JNK, and ecdysone signaling regulate Dcp-1 indicate that there are other regulatory pathways for caspase functions.