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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK078135-02
Application #
7367096
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Blondel, Olivier
Project Start
2007-03-01
Project End
2012-01-31
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
2
Fiscal Year
2008
Total Cost
$275,576
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Huang, Kezhen; Fingar, Diane C (2014) Growing knowledge of the mTOR signaling network. Semin Cell Dev Biol 36:79-90
Fingar, Diane C; Inoki, Ken (2012) Deconvolution of mTORC2 ""in Silico"". Sci Signal 5:pe12
Magnuson, Brian; Ekim, Bilgen; Fingar, Diane C (2012) Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J 441:1-21
Ray, Bridgette N; Kweon, Hye Kyong; Argetsinger, Lawrence S et al. (2012) Research resource: identification of novel growth hormone-regulated phosphorylation sites by quantitative phosphoproteomics. Mol Endocrinol 26:1056-73
Dunlop, Elaine A; Hunt, David K; Acosta-Jaquez, Hugo A et al. (2011) ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding. Autophagy 7:737-47
Ekim, Bilgen; Magnuson, Brian; Acosta-Jaquez, Hugo A et al. (2011) mTOR kinase domain phosphorylation promotes mTORC1 signaling, cell growth, and cell cycle progression. Mol Cell Biol 31:2787-801
Carriere, Audrey; Romeo, Yves; Acosta-Jaquez, Hugo A et al. (2011) ERK1/2 phosphorylate Raptor to promote Ras-dependent activation of mTOR complex 1 (mTORC1). J Biol Chem 286:567-77
Foster, Kathryn G; Acosta-Jaquez, Hugo A; Romeo, Yves et al. (2010) Regulation of mTOR complex 1 (mTORC1) by raptor Ser863 and multisite phosphorylation. J Biol Chem 285:80-94
Foster, Kathryn G; Fingar, Diane C (2010) Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem 285:14071-7
Soliman, Ghada A; Acosta-Jaquez, Hugo A; Fingar, Diane C (2010) mTORC1 inhibition via rapamycin promotes triacylglycerol lipolysis and release of free fatty acids in 3T3-L1 adipocytes. Lipids 45:1089-100

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