Investigating the effect of altered nutrition on signalling via Drosophila class 1A phosphatidylinositol-3 kinase (PI3K), and functional analysis of PI3K mutated within its Ras-binding domain

Adult Drosophila size is determined during larval life, by the growth of internal epithelial structures known as imaginal discs. These discs metamorphose during pupal development to form the adult epidermal structures. The insulin/PI3K signalling pathway in Drosophila has been shown to regulate disc, final organ and organism size. Disruption of this signalling pathway inhibits Drosophila growth and generates smaller than wild-type flies whereas increasing PI3K signalling increases growth, generating larger flies. As the nutritional state of larvae also has a bearing on final organism size, with malnourished larvae generating small adults, my initial aim was to evaluate whether nutrition acts through the PI3K signalling pathway to affect growth. Levels of PI3K signalling were monitored in whole-larval lysates via the phosphorylation and activation of the PI3K target dAkt. Preliminary experiments showed that dAkt phosphorylation decreased with reduced nutritional input. Kinase assays confirmed that dAkt activity was reduced co-ordinately. However, despite extensive optimisation, the reproducibility of these results was poor. I concluded that whole larval extracts were not a suitable system for monitoring physiological changes in dAkt activity. Drosophila embryos lacking the class IA PI3K catalytic sub-unit. Dp110, arrest development late in larval life and contain no visible imaginal discs. A similar phenotype is observed in Ras/MAPK pathway loss of function mutants. Further to this, many biochemical data from mammalian cell systems suggest that Ras-family GTPases interact with and activate PI3K catalytic sub-units via a Ras binding domain (RBD). However, the biological importance of this interaction is unclear. My second aim was to establish the functional significance of the RBD in Dp110. Larvae with only one copy of the Dp110 gene containing a nonconservative mutation within the RBD (6N3) remain small and fail to pupate, raising the possibility that RBD integrity is essential for Dp110 function. However, the mutation may equally reduce protein stability, binding to the adapter, p60, or catalytic activity. These possibilities were tested in turn, using a cell culture system. Other RBD mutants of Dp110 were tested in parallel. Results suggest that the 6N3 mutation partially disrupts Dp110 catalytic activity. However, this and other properties of Dp110 remain unimpaired in other RBD mutants, indicating that these proteins could be used in further studies to establish the significance of this domain in vivo