Quantitative phosphoproteomics reveal that mTOR regulates cell growth and proliferation by phosphorylating a functionally diverse set of substrates

The atypical Ser/Thr kinase target of rapamycin (TOR) is a central controller of cell growth and proliferation. TOR forms two distinct multiprotein complexes, TORC1 and TORC2, which are structurally and functionally conserved from yeast to humans. Four major inputs control mammalian TOR (mTOR): growth factors, such as insulin; cellular energy levels, such as the AMP:ATP ratio; stress, such as hypoxia; and nutrients, such as amino acids. mTOR controls cell growth by the positive and negative regulation of several anabolic and catabolic processes, respectively, that collectively regulate cell size and proliferation. These cellular processes include autophagy, cytoskeleton rearrangement, glycolysis, lipogenesis, nutrient transport, ribosome biogenesis, and translation. Dysregulation of the mTOR signaling network has been associated with aging, and a multitude of diseases including cancer, cardiovascular disease, diabetes, inflammation, immune dysfunctions, and neurodegeneration. However, relatively few direct substrates of either one of the two mTOR complexes, mTORC1 and mTORC2, are known. \n\tTo determine downstream effectors of mammalian TOR (mTOR), we applied a functional, quantitative phosphoproteomics workflow to identify novel mTORC1 or mTORC2 regulated phosphorylations. Raptor and Rictor are essential components of mTORC1 and mTORC2, respectively. To distinguish phosphorylations regulated by mTORC1 or mTORC2, we specifically deleted Raptor or Rictor using an inducible gene knockout system in mouse embryonic fiberblasts (MEFs). We detected 4584 phosphorylation sites on 1398 proteins, and identified 335 novel mTOR effectors. Many of the novel effectors are implicated in cancer and metabolic diseases, but have no known links to mTOR. To distinguish direct mTOR substrates from indirect effectors, we combined peptide array in vitro kinase assays with phosphorylation motif analysis. This revealed that mTORC1 phosphorylates CAD in vivo and in vitro. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) is the initial and rate limiting enzyme in de novo pyrimidine synthesis. The macrolide rapamycin, which forms a complex with FKBP12, binds and acutely inhibits mTORC1 but not mTORC2. Rapamycin treatment inhibited growth factor stimulated CAD phosphorylation and oligomerization, decreased de novo pyrimidine synthesis, and delayed progression of S-phase where CAD activity is essential. Thus mTORC1 phosphorylates CAD and thereby stimulates de novo pyrimidine synthesis to promote cell proliferation. \n\tSeparately, we characterize the autophosphorylation of mTOR on Ser2481. Insulin stimulates the phosphorylation of mTOR at Ser2481 specifically in mTORC2. Knockout of Rictor, but not Raptor, abolished mTOR autophosphorylation at Ser2481. Prolonged treatment with rapamycin, which indirectly inhibits mTORC2 complex formation, inhibited Ser2481 phosphorylation. Surprisingly, mTORC2 autophosphorylation at Ser2481 temporally occurs after the insulin-induced phosphorylation of Akt/PKB and the SGK1 substrate NDGR1. Mutation of Ser2481 to aspartic acid rendered mTOR unable to phosphorylate Akt/PKB in vitro. However the function of mTOR-Ser2481 phosphorylation in vivo remains elusive, as mutation of mTOR-Ser2481 did not alter Akt/PKB phosphorylation in vivo. \n\tIn summary, mTORC1 and mTORC2 regulate the phosphorylation of a functionally diverse set of substrates to control several anabolic and catabolic processes that determine cell size and proliferation. As a central controller of cell growth and proliferation, mTOR plays a key role in regulating development, whereas dysregulation of mTOR signaling has been linked to aging and diseases such as cancer and metabolic disorders.