Amino acids control cell growth via activation of the highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans display two stable yet epigenetically distinct states of pluripotency, naïve and primed. We now show that nicotinamide-N-methyl transferase (NNMT) and metabolic state regulate pluripotency in hESCs. Specifically, in naïve hESCs NNMT and its enzymatic product 1-methylnicotinamide (1-MNA) are highly upregulated, and NNMT is required for low SAM levels and H3K27me3 repressive state. NNMT consumes SAM in naïve cells, making it unavailable for histone methylation that represses Wnt and activates HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.

The Ser-Thr kinase mammalian target of rapamycin (mTOR) controls cell growth and metabolism by stimulating glycolysis and synthesis of proteins and lipids. To further understand the central role of mTOR in cell physiology, we used quantitative phosphoproteomics to identify substrates or downstream effectors of the two mTOR complexes. mTOR controlled the phosphorylation of 335 proteins, including CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase). CAD catalyzes the first three steps in de novo pyrimidine synthesis. mTORC1 indirectly phosphorylated CAD-S1859 through S6 kinase (S6K). CAD-S1859 phosphorylation promoted CAD oligomerization and thereby stimulated de novo synthesis of pyrimidines and progression through S phase of the cell cycle in mammalian cells. Thus, mTORC1 also stimulates the synthesis of nucleotides to control cell proliferation.

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mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.The PIKK family conserved TOR (target of rapamycin) kinase, originally discovered in Saccharomyces cerevisiae (9), is a central regulator of cell growth in response to nutrient availability and other environmental cues (see references 4 and 44 for reviews). TOR is part of two complexes, TORC1 and TORC2, of which the first is inhibited by low concentrations of the macrolide rapamycin. Inhibition of TORC1 by nutrient deprivation or rapamycin has broad consequences, among them the inhibition of ribosome biogenesis and protein translation. This inhibition is mediated in part through transcriptional repression of genes required for these processes such as the RNA polymerase (pol) I-transcribed large rRNA genes, the pol II-transcribed ribosomal protein genes, and a number of pol III-transcribed genes, including, for example, tRNA genes (4, 44).In yeast, repression of pol III transcription in response to nutrient deprivation, rapamycin treatment, or other stresses such as DNA damage and secretory pathway defects requires the repressor Maf1 (38) (see 6 and 42 for reviews). The protein is regulated by phosphorylation/dephosphorylation events, which control nuclear/cytoplasmic transport as well as the pol III repression function of the protein. The two processes are, however, at least partially independent (20,23,28,36,40). Several kinases have been implicated, in particular PKA and Sch9, the second of which appears to be the main Maf1 kinase (11,17,20,41). Recently, TORC1 was also described as a kinase that weakly phosphorylates yeast Maf1 on unknown residues within the nucleus (40).Human MAF1, like yeast Maf1, is a phosphoprotein. It is unclear, however, whether human MAF1 is indispensable for repression of pol III transcription in response to various stresses, and neither the function of MAF1 phosphorylation nor the MAF1 kinases have been identified. Here we show that mammalian cells lacking the MAF1 gene do not repress pol III transcript...