Identity, regulation, and function of mTOR phosphorylation sites

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DESCRIPTION (provided by applicant): The goal of this proposal, entitled "Identity, regulation, and function of mTOR phosphorylation sites", is to understand the mechanisms by which mTOR, the mammalian target of rapamycin, is regulated by physiological signals in cultured cells. mTOR is an evolutionary conserved protein kinase that associates with numerous cellular proteins to form distinct signaling complexes. TOR complex 1 (TORC1) is the best characterized mTOR complex and is inhibited by the immunosuppressive drug rapamycin. TORC1 integrates signals derived from nutrients, growth factors, and cellular stress to control fundamental cellular processes such as protein biosynthesis and cell growth/size. While less is known about the more recently described rapamycin-insensitive TOR complex 2 (TORC2), this complex regulates the organization of the actin cytoskeleton. While mTOR signaling is tightly regulated, the mechanisms by which physiological signals directly modulate mTOR activity in either TORC1 or TORC2 are not known. As phosphorylation controls the activity of many TORC1 regulatory proteins, the central hypothesis of this proposal is that phosphorylation of mTOR itself in response to cellular signals regulates mTOR signaling and biological function. Indeed, we have identified seven novel serine/threonine (Ser/Thr) phosphorylation sites (P-sites) on mTOR by tandem mass spectrometry (MS2). In this proposal we will identify the major in vivo phosphorylation sites on mTOR isolated from intact cells using MS2 and perform experiments directed towards understanding the regulation and function of these P-sites. We propose the following specific aims: 1. Identify additional phosphorylation sites on mTOR. 2. Determine the regulation of mTOR phosphorylation. 3. Determine the function of mTOR phosphorylation. Dysregulated mTOR signaling is implicated in the pathophysiology of several human diseases including type II diabetes, obesity, cardiovascular disease, and cancer. Indeed, mTOR-inhibitory drugs are currently employed to prevent transplant rejection and coronary restenosis and are being tested as anti-tumor and anti-diabetic agents. Given the therapeutic potential mTOR inhibitors, understanding the regulation of mTOR represents a problem of major therapeutic value as well as biological importance.