Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable approximately 3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1-Atg13-FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1-Atg13-FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the approximately 3-MDa ULK1-Atg13-FIP200 complex.

Maintenance of skeletal muscle mass relies on the dynamic balance between anabolic and catabolic processes and is important for motility, systemic energy homeostasis, and viability. We identified direct target genes of the glucocorticoid receptor (GR) in skeletal muscle, i.e., REDD1 and KLF15. As well as REDD1, KLF15 inhibits mTOR activity, but via a distinct mechanism involving BCAT2 gene activation. Moreover, KLF15 upregulates the expression of the E3 ubiquitin ligases atrogin-1 and MuRF1 genes and negatively modulates myofiber size. Thus, GR is a liaison involving a variety of downstream molecular cascades toward muscle atrophy. Notably, mTOR activation inhibits GR transcription function and efficiently counteracts the catabolic processes provoked by glucocorticoids. This mutually exclusive crosstalk between GR and mTOR, a highly coordinated interaction between the catabolic hormone signal and the anabolic machinery, may be a rational mechanism for fine-tuning of muscle volume and a potential therapeutic target for muscle wasting.

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser 183 ). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe 129 to Ala). Immediately carboxyl-terminal to Phe 129 are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser 183 , to Asp. PRAS40 (Ser 183 ) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser 183 ) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser 183 ) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.The mammalian target of rapamycin (mTOR) 3 is the founding member of the PI-3 kinase-related family of protein (Ser/ Thr) kinases (PIKKs) and controls many important aspects of the cellular response to nutrient sufficiency and growth factors (1). The mTOR polypeptide is now known to function in two distinct, independently regulated hetero-oligomeric complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes contain mTOR and the polypeptide mLST8/G␤L; mTORC1 in addition contains raptor (an ortholog of Saccharomyces cerevisiae KOG1), which binds directly to the known mTORC1 substrates S6K1 and 4E-BP1 and is indispensable for their phosphorylation by mTOR in vivo and in vitro. mTORC2 lacks raptor but contains the polypeptides rictor (an ortholog to ScAVO3) and mSin1 (an ortholog of ScAVO1). mTORC2 is one of the activating kinases for Akt, previously called PDK2, and also regulates the actin cytoskeleton through as yet unidentified effectors. Although rapamycin, in complex with FKBP12, binds directly to mTOR in a segment just amino-terminal to the catalytic domain, only mTORC1 binds the FKBP12-rapamycin complex, and thus only mTORC1 is directly susceptible to inhibition by rapamycin.Rapamycin is among the most selective kinase inhibitors k...

Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.

Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and is a major regulator of translation, cell growth, and autophagy. mTOR exists in two distinct complexes, mTORC1 and mTORC2, that differ in their subunit composition. In this study, we identified KIAA0406 as a novel mTOR-interacting protein. Because it has sequence homology with Schizosaccharomyces pombe Tti1, we named it mammalian Tti1. Tti1 constitutively interacts with mTOR in both mTORC1 and mTORC2. Knockdown of Tti1 suppresses phosphorylation of both mTORC1 substrates (S6K1 and 4E-BP1) and an mTORC2 substrate (Akt) and also induces autophagy. S. pombe Tti1 binds to Tel2, a protein whose mammalian homolog was recently reported to regulate the stability of PIKKs. We confirmed that Tti1 binds to Tel2 also in mammalian cells, and Tti1 interacts with and stabilizes all six members of the PIKK family of proteins (mTOR, ATM, ATR, DNA-PKcs, SMG-1, and TRRAP). Furthermore, using immunoprecipitation and size-exclusion chromatography analyses, we found that knockdown of either Tti1 or Tel2 causes disassembly of mTORC1 and mTORC2. These results indicate that Tti1 and Tel2 are important not only for mTOR stability but also for assembly of the mTOR complexes to maintain their activities. Mammalian target of rapamycin (mTOR)4 is a member of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, which also includes ataxia telangiectasia mutated (ATM), ATM-and Rad3-related (ATR), DNA-PKcs, suppressor with morphological effect on genitalia 1 (SMG-1), and the catalytically inactive transformation/transcription domainassociated protein (TRRAP) (1-3). mTOR forms two distinct complexes that differ in their subunit composition, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 includes mTOR, mLST8 (also known as G␤L), and Raptor, whereas mTORC2 includes mTOR, mLST8, mSin1, and Rictor (4 -7). mTORC1 is activated by several factors, including amino acids. It promotes protein translation through phosphorylation of S6K1 and 4E-BP1 (7), and inhibits autophagy through phosphorylation of ULK1 and Atg13 (8-10). On the other hand, mTORC2 is activated by growth factors and phosphorylates Akt at Ser-473, and plays key roles in cell survival, metabolism, proliferation, and organization of the cytoskeleton (7).Although growing numbers of mTOR-interacting proteins have been identified, little is known about the assembly of these mTOR complexes. Deletion of the mTORC2 components such as Rictor, mSin1, and mLST8 induces disassembly of mTORC2, impairing Akt phosphorylation (at Ser-473) (11-13), whereas mLST8 is not required for mTOR-Raptor interaction and S6K1 activation (11). Prolonged treatment with rapamycin, which is an mTORC1 inhibitor, inhibits mTORC2 assembly (14). Recently, it was proposed that phosphatidic acid is required for the assembly of mTOR complexes, because depletion of phosphatidic acid caused by treatment with 1-butanol and expression of dominant negative mutant phospholipase D results...