Elucidating the mechanism of mTORC1 activation by amino acids

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 DESCRIPTION (provided by applicant): Novel treatments for breast cancer are urgently needed to limit the impact of this deadly disease. In depth biological knowledge of signaling pathways is critical to the selection of appropriate targets for drug development. To that end, the goal of this work is to elucidate the biological contributions of the vacuolar H+- ATPase (V-ATPase) rotary proton pump to mTORC1 signaling. mTORC1 is a hub of cellular growth control that is often deregulated in cancer. These experiments will elucidate new ways to alter mTORC1 signaling and combat breast cancer. Specifically, the proposed study will evaluate the mechanism by which the V-ATPase participates in amino acid-induced mTORC1 activation. We hypothesize that reversible disassembly of the V-ATPase is a key event in amino acid signal transduction, and that this mechanism is altered in breast cancer. We will assess V-ATPase assembly in response to amino acid availability in both immortalized, but untransformed, cell lines and in a panel of breast cancer cell lines using both a cell fractionation approach and fluorescence microscopy. We expect that amino acid depletion will result in a reversible increase in assembly of the V-ATPase, which then leads to a decrease in mTORC1 signaling. The full network of factors necessary for amino acid sensing and subsequent mTORC1 activation is unknown. We hypothesize that the amino acid sensing machinery is contained within the lysosome, and will seek to identify the proteins that contribute to this process using a shRNA screening approach. We will screen a library shRNAs targeting lysosomal genes for the ability to modulate amino acid sensing. To facilitate screening in high throughput, we will employ a split luciferase system, consisting of subunits of the V-ATPase and the Ragulator (a key mediator of mTORC1 activation by amino acids) known to associate in an amino acid dependent manner, each tagged with a portion of the luciferase enzyme. Knockdown of a factor necessary for amino acid sensing would mimic the amino acid starved state, leading to constitutive association of the V- ATPase with the Ragulator and sustained luciferase activity. We will validate candidates by co- immunoprecipitation of the V-ATPase and Ragulator and assessment of downstream markers of mTORC1 activity after shRNA knockdown. These studies will identify the other lysosomal factors that are necessary for amino acid sensing. Together, accomplishment of this study will deepen our knowledge of events critical to breast tumor initiation and progression, and reveal new ways to target the mTOR pathway in cancer.