Genetic studies of PI3'K/PKB/GSK-3 signaling in Drosophila

The PI3'K/PKB/GSK-3 signaling pathway is involved in the regulation of a diverse array of cellular functions including cell growth, proliferation, survival and differentiation. Consequentially, a plethora of diseases, ranging from cancer to metabolic disorders, are significantly influenced by the activity of many members of this signaling cascade. The determination of novel PKB substrates and regulatory elements is therefore of significance as this expands the current understanding of this pathway towards the design of therapeutics for treatment of many diseases. Using Drosophila as a model system, I present herein the analysis of two genes identified in a genetic screen for factors which interact with the Drosophila homolog of PKB, Dakt1. I show that the PI3'K/PKB pathway regulates the activity of the bHLH-PAS domain protein Trachealess (Trh), through direct phosphorylation on S665, thus identifying it as a novel target of PKB in vivo. PKB phosphorylation on this residue results in the translocation of Trh into the nucleus resulting in its transcriptional activation. These findings implicate the PI3'K/PKB pathway as a key regulator of tubulogenesis in Drosophila. This study also provides evidence for PKB as a potential regulator of many other bHLH-PAS domain proteins in vertebrates. A number have been identified which show significant homology to Trh including hypoxia inducible factor-1alpha (HIF-1alpha) and neuronal PAS domain protein 3 (NPAS3), molecules which provide a prospective linkage between PKB and the regulation of angiogenesis and neurogenesis.;I also present the genetic analysis of a second gene identified in this screen, shaggy (sgg), which is the Drosophila homolog of glycogen synthase kinase-3 (GSK-3). I show that sgg mutant larvae are obese, exhibit fasting state hyperglycemia, accumulate metabolites such as glycogen and lipids, have increased insulin production, undergo accelerated cell growth and have a disruption in glucose homeostasis. These animals also exhibit a feedback between Sgg and Dakt1 on S505, the homologous residue of S473 on PKB, which has been shown to be required for activity. This reduction in Dakt1 activity also coincides with a decrease in glucose transport within the larval fat body, which does not respond properly to insulin stimulation, effectively demonstrating insulin resistance within this tissue. Thus, reductions in Sgg/GSK-3 in Drosophila broadly disrupt metabolic homeostasis with many phenotypes mimicking those observed in type 2 diabetic patients. I present this as a model system for the study of the involvement of PI3'K/PKB/GSK signaling in the etiology of metabolic disorders.