Molecular and Biochemical Basis of mTORC1-mediated Feedback Loops

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 DESCRIPTION (provided by applicant): The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved serine/threonine kinase complex. It is a central regulator of cellular anabolic processes by integrating a variety of upstream inputs, including growth factors, nutrients, energy and oxygen status. It is being increasingly appreciated that downstream targets of mTORC1 can communicate with its upstream regulators, through various feedback loops. These feedback mechanisms play a critical role in stabilizing the entire network. They also have great significance in a variety of human diseases. For example, aberrant mTORC1 activation (e.g. as a result of chronic overfeeding) can cause insulin resistance, a hallmark of type 2 diabetes. The feedback loops are also particularly relevant in cancer biology. mTORC1 is hyperactivated in a major faction of human cancers. Unfortunately, inhibition of mTORC1 using rapamycin analogs (rapalogs) as single agents is cytostatic but not cytotoxic. This is mainly due to the activation of upstream, pro-survival kinases (e.g. Akt) upon mTORC1 inhibition. Understanding how to avoid the relief of feedback loops will be critical to overcome rapamycin resistance for treating human cancer. To this end, we recently used a quantitative phosphoproteomic approach to systematically characterizing the mTOR-regulated phosphoproteome. Detailed biochemical characterization of a novel mTORC1 substrate, Grb10, showed that mTORC1-mediated phosphorylation stabilized this protein, which led to its accumulation, and subsequent inhibition of insulin and IGF1 signaling. Our unpublished results further indicate that cells with hyperactive mTORC1 also secrete a protein(s) that is able to block IGF1 signaling. In this proposal, we will investigate how mTORC1 may inhibit the function of IGF1 through remodeling the secretome. In Aim 1, we will use a quantitative mass spectrometry approach to comprehensively characterizing the mTORC1-regulated secretome. In Aim 2, we will elucidate the molecular mechanism by which mTORC1 regulates the expression of this secreted protein, and determine its role in mTORC1-mediated feedback inhibition of IGF1 signaling. In Aim3, we will investigate the non-cell autonomous function of this secreted protein, and its interplay with Grb10 in vivo. We will accomplish our goals with a multi- disciplinary approach, utilizing tools including proteomics, biochemistry, molecular biology and animal models. The results will greatly facilitate our understanding of mTORC1-mediated feedback loops. Knowledge we garner in this proposal will also be necessary to further explore how we might better target these feedback mechanisms for treating various human diseases.