Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase that regulates numerous cellular processes. mTOR Complex 1 (mTORC1), one of two mTOR-containing complexes in the cell, controls cell growth and metabolism, and is regulated by environmental factors such as growth factors, glucose availability, and oxygen content. mTORC1 activation is estimated in greater than 70% of many cancers, highlighting the importance of understanding how mTORC1 senses its environment and communicates this to the cell growth machinery. While growth factor, glucose, and oxygen availability signals are largely integrated through the TSC1/2 tumor suppressor, amino acid deprivation inhibits mTORC1 through mechanisms independent of TSC1/2. Amino acid availability is sensed through a mechanism dependent on the Rag family of GTPases, which exist in heterodimers of either RagA or RagB and either RagC or RagD. The Rag heterodimers localize to lysosomal membranes through interactions with the trimeric "Ragulator" complex which is constitutively localized to lysosomal membranes due to myristoylation and palmitoylation of one of its components. Amino acids regulate the GTP/GDP-binding of the Rag GTPases which regulates binding of the Rag heterodimer to mTORC1. The result is that mTORC1 is diffuse in the absence of amino acids, whereas amino acids induce localization of mTORC1 to lysosomal membranes where its activator, Rheb, resides. Although the current model proposes that the Rag proteins localize to the lysosomal membrane constitutively, our data demonstrate that the Rags diffuse away from the lysosomes following amino acid stimulation. Due to the timing of mTORC1 localization vs. mTORC1 activation, the data suggest a role for the Rags beyond recruitment of mTORC1 to the lysosomal membrane, and that the Rags potentially "transport" mTORC1 to its substrates following activation at the lysosome or the Rags must leave the lysosomes and become "recharged" as part of the sustained activation mechanism of mTORC1. Published data as well as our preliminary data suggest that individual amino acids such as leucine affect mTORC1 activation through mechanisms separate from that of total amino acids, which we hypothesize to be through regulation of Rag localization. For these reasons, the goals of the proposed research are to test the hypothesis that Rag movement is required for mTORC1 regulation by amino acids, and to delineate the roles of total amino acid concentration versus individual amino acid availability in regulation of the Rag protein signaling. To address these questions, we plan to employ a variety of approaches including expression of mutant forms of the Rag proteins, interaction studies, and GTP/GDP binding assays to assess the requirement for Rag movement in mTORC1 regulation and to elucidate the mechanisms by which total and individual amino acids regulate mTORC1. Developing a solid understanding of the mechanisms behind amino acid regulation of mTORC1 should aid in our understanding of mTORC1-driven cancers and in the development of more effective therapeutics.
The goals of the proposed research aim to investigate how cells sense availability of nutrients in their environment, specifically amino acid availability, ad communicate this to the cellular growth machinery. A common feature of cancer cells is a disregulation of growth via pathways that are normally tightly regulated by environmental cues. Elucidation of the mechanisms by which cells regulate activity of mTORC1, a central regulator of cell growth, in response to amino acids availability, will ultimately aid in the development of more effective cancer therapeutics.