The importance of the PI3Kinase-Akt-Tsd/Tsc2-mTOR signaling pathway in the regulation of both cell sizeand cell growth has become apparent in recent years. The seminal observations that initiated this explosiveincrease in our understanding were made in Drosophila 4 years ago. Although much progress has beenmade in mammalian systems on this pathway as well, Drosophila continues to offer major advantages andinsights due to the ease of genetic manipulation, and relatively low costs. In addition, recently thetechnological approach of whole genome RNAi screens in Drosophila has provided the opportunity forpathway analysis that cannot be approached in mammalian systems. This approach is robust and reliable,and is the primary tool we will use to explore critical questions on Tsc1/Tsc2-Tor-S6K signaling in fourspecific aims.
In Aim 1 and Aim 2, two small scale - hypothesis driven - RNAi screen will be performed toidentify a crucial missing links in this pathway: The GEF(s) for Rheb and kinases/phosphatases affectingAMPK_ phosphorylation and activity.
In Aim 3, we will identify core components involved in Aktphosphorylation and its regulation by a feedback mechanism. We will distinguish those that affect both S6Kand Akt,and those that affect Akt only.
In Aim 4, a comprehensive genome-wide RNAi screen will beperformed to identify regulators acting downstream of Tsc1/Tsc2 on S6K phosphorylation. Findings will beexplored and confirmed through biochemical and genetic analyses in vivo in the fly,and also translated tomammalian systems in concert with the other projects of this PPG. The improved understanding of thewiring of this signaling pathway will provide both fundamental insight and the opportunity for therapeuticintervention.
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