One of the gaps in our understanding of cellular regulation is the detailed mechanisms through which eukaryotic cells detect nutrients in their environment and respond to them in an integrated manner. This is particularly important with respect to the Target of Rapamycin Complex 1 (TorC1), a global regulator that integrates multiple environmental signals and controls a wide range of basic cellular processes in response to them. The growing clinical potential of rapamycin-family drugs for treating tissue rejection, a variety of cancers, progeria and aging-related diseases requires that we accurately understand the mechanistic details of mTorC1 regulation and in turn its control of downstream processes. For example, an incomplete understanding of how mTorC1 regulates downstream events (increases Tissue Factor production which triggers the coagulation cascade) has raised high concern about late stent thrombosis in rapamycin-derivative eluting stents used to treat acute myocardial infarction. The proposed experiments in this application seek to identify such missed or incompletely understood Tor regulatory mechanisms. The plan first utilizes the powerful genetics and well understood cellular and molecular biology of S. cerevisiae to gain a more accurate and in depth understanding of TorC1 regulation by investigating TorC1-dependent (rapamycin-inducible) and -independent mechanisms regulating the GATA transcription activators Gln3 and Gat1. They are among the most widely used reporters of TorC1 activity in non-pathogenic and pathogenic yeast. The mechanistic principles discovered will then be applied to investigate analogous central questions of mTorC1 nutrient sensing and responses to it in mammalian cells in collaboration with a recognized mammalian cell biologist and an expert in the field of mTor regulation. Specifically, the 1st and 2nd Specific Aims test the hypothesis that Gln3 and Gat1 are individually controlled by TorC1-dependent and -independent regulatory pathways with different nitrogen inputs, protein phosphatase (Sit4 and PP2A) and kinase requirements. The phosphatase and kinase requirements for each pathway will be established. The research strategy achieves this goal using gln3 amino acid substitution mutants that genetically isolate the hypothesized pathways from one another, thereby permitting one pathway's components and regulatory mechanisms to be rigorously analyzed without input or interference from the second pathway. Pivotal, proof-of-principle mutants demonstrating the success of this approach have already been isolated and preliminarily characterized. The 3rd Aim challenges and analyzes fundamental assumptions upon which most TorC1/mTorC1 investigations depend and tests predictions that emanate from the exciting new model describing Vam6-Gtr1/2-Ego1/3 activation of TorC1. This information is then used to design and perform experiments that investigate these questions in mammalian cells. If the proposed experiments substantiate the conclusions derived from recently acquired data by my group, significant reconsideration of existing TorC1 &mTorC1 data will be required, and new pathways of nutrient-responsive regulation will likely be identified.
The importance and increasing clinical use of rapamycin-family drugs to treat tissue rejection in transplant patients, multiple types of cancer and potentially progeria make it imperative that we accurately and completely understand how the target of these drugs, the mammalian Target Of Rapamycin Complex 1 (mTorC1), is regulated and in turn regulates downstream cellular processes. Despite impressive advances, one of the least well understood aspects of mTorC1 regulation are the mechanisms through which the presence of nitrogenous nutrients, such as amino acids, are detected and appropriate, integrated responses to them subsequently implemented. The proposed research will significantly contribute to elucidating these mechanisms.
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