Discovery of the First Endogenous Gain of Function Mutation in Drosophila RAS1 as a Dominant Suppressor of Apoptosis

The development of multicellular organisms requires a tight coordination of cell proliferation, cell differentiation and cell death in order to correctly specify cell fate and number. According to the trophic theory of survival, this is achieved in part by a competition between cells in a tissue for a limited number of extracellular survival factors. Cells that do not receive sufficient quantities of these survival cues engage a default cell death program and are thereby eliminated. This ‘social control’ of cell survival ensures the integrity of tissues by matching the correct number of different cell types to each other. Apoptosis is one morphologically distinct, genetically programmed form of cell death by which cells in an organism are efficiently and rapidly removed. The proper execution of apoptosis is therefore critical to normal development and homeostasis in metazoans and defects in the regulation of apoptosis is known to contribute to the etiology of several major diseases. Initial insights into the complex molecular networks that regulate apoptosis derived largely from elegant genetic analyses of invertebrate model organisms. These early studies identified several genes critical for the execution of the apoptosis and established an evolutionarily conserved core cell death pathway. To further elucidate the molecular mechanisms underlying the control of apoptosis, we conducted several mutagenesis screens in Drosophila melanogaster to identify genes that can modulate cell death phenotypes. One particularly interesting mutant isolated in these screens was recovered as a strong, specific and dominant suppressor of cell death induced by the RHG protein hid. We demonstrate that this mutant is a gain-of-function allele of ras85D (ras1), the Drosophila homolog of mammalian oncogenes H-ras, K-ras and N-ras. We further establish that this viable allele, rasR68Q, contains a mutation in the Switch II region of Ras and that it produces a GTPase protein with diminished enzymatic activity. RasR68Q is the first endogenous gain-of-function ras1 allele to be identified in Drosophila and represents one of very few hypermorphic Ras mutations compatible with organismal viability to be isolated

A Gain-of-Function Germline Mutation in Drosophila ras1 Affects Apoptosis and Cell Fate during Development

The RAS/MAPK signal transduction pathway is an intracellular signaling cascade that transmits environmental signals from activated receptor tyrosine kinases (RTKs) on the cell surface and other endomembranes to transcription factors in the nucleus, thereby linking extracellular stimuli to changes in gene expression. Largely as a consequence of its role in oncogenesis, RAS signaling has been the subject of intense research efforts for many years. More recently, it has been shown that milder perturbations in Ras signaling during embryogenesis also contribute to the etiology of a group of human diseases. Here we report the identification and characterization of the first gain-of-function germline mutation in Drosophila ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. A single amino acid substitution (R68Q) in the highly conserved switch II region of Ras causes a defective protein with reduced intrinsic GTPase activity, but with normal sensitivity to GAP stimulation. The ras1R68Q mutant is homozygous viable but causes various developmental defects associated with elevated Ras signaling, including cell fate changes and ectopic survival of cells in the nervous system. These biochemical and functional properties are reminiscent of germline Ras mutants found in patients afflicted with Noonan, Costello or cardio-facio-cutaneous syndromes. Finally, we used ras1R68Q to identify novel genes that interact with Ras and suppress cell death