The tuberous sclerosis complex genes (TSC1) and 2-(TSC2) encode proteins that form a complex with tumor suppressor function. The TSC1/TSC2 complex functions to integrate a variety of signals with mammalian target of rapamycin complex 1 (mTORCI;generically referred to as mTOR), a critical regulator of protein translation. Signals from growth factors and energy stores regulate mTOR through a process that requires the TSC1/TSC2 complex and which involves changes in the phosphorylation status of the TSC2 protein. We have discovered that the TSC1/TSC2 complex is required for mTOR inhibition by hypoxia and that hypoxia results in a phosphatase-sensitive mobility shift in the TSC2 protein. The simplest explanation for these data is that in response to hypoxia a kinase is activated, which phosphorylates TSC2 and thereby inhibits mTOR. To uncover the kinase responsible for TSC2 phosphorylation by hypoxia, we have undertaken an unbiased genetic screen using RNAi. The goal of this screen is to identify a kinase that like TSC2, when knocked down, blocks mTOR inhibition by hypoxia. Because of the efficiency of RNAi and lesser complexity of the kinome we sought to conduct this screen in D. melanogaster cells. We have established that as in mammalian cells, in Drosophila cells TOR is inhibited in response to hypoxia and that this inhibition depends upon an intact TSCirrSC2 complex. An RNAi screen of the Drosophila kinome involving 638 redundant dsRNAs has been conducted and we have selected 40 candidates for further evaluation.
The specific aims for this proposal are: (1) to validate of kinases identified in primary RNAi screen, (2) to map and to evaluate the site(s) of phosphorylation on TSC2 in response to hypoxia, and (3) to evaluate of the link between the putative hypoxia signaling kinase(s) and TSC2 phosphorylation. The identification of a hypoxia activated TSC2 kinase that regulates mTOR would have profound implications in our understanding of how mTOR is regulated during development and in tumors.