AMP-activated protein kinase (AMPK) senses energetic stress and, in turn, promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance. We found that a diverse array of AMPK activators increased mTOR complex 2 (mTORC2) signaling in an AMPK-dependent manner in cultured cells. Activation of AMPK with the type 2 diabetes drug metformin (GlucoPhage) also increased mTORC2 signaling in liver in vivo and in primary hepatocytes in an AMPK-dependent manner. AMPK-mediated activation of mTORC2 did not result from AMPK-mediated suppression of mTORC1 and thus reduced negative feedback on PI3K flux. Rather, AMPK associated with and directly phosphorylated mTORC2 (mTOR in complex with rictor). As determined by two-stage in vitro kinase assay, phosphorylation of mTORC2 by recombinant AMPK was sufficient to increase mTORC2 catalytic activity toward Akt. Hence, AMPK phosphorylated mTORC2 components directly to increase mTORC2 activity and downstream signaling. Functionally, inactivation of AMPK, mTORC2, and Akt increased apoptosis during acute energetic stress. By showing that AMPK activates mTORC2 to increase cell survival, these data provide a potential mechanism for how AMPK paradoxically promotes tumorigenesis in certain contexts despite its tumor-suppressive function through inhibition of growth-promoting mTORC1. Collectively, these data unveil mTORC2 as a target of AMPK and the AMPK-mTORC2 axis as a promoter of cell survival during energetic stress.

The Ras/mitogen-activated protein kinase (MAPK) pathway regulates a variety of cellular processes by activating specific transcriptional and translational programs. Ras/MAPK signaling promotes mRNA translation and protein synthesis, but the exact molecular mechanisms underlying this regulation remain poorly understood. Increasing evidence suggests that the mammalian target of rapamycin (mTOR) plays an essential role in this process. Here, we show that Raptor, an essential scaffolding protein of the mTOR complex 1 (mTORC1), becomes phosphorylated on proline-directed sites following activation of the Ras/MAPK pathway. We found that ERK1 and ERK2 interact with Raptor in cells and mediate its phosphorylation in vivo and in vitro. Using mass spectrometry and phosphospecific antibodies, we found three proline-directed residues within Raptor, Ser 8 , Ser 696 , and Ser 863 , which are directly phosphorylated by ERK1/2. Expression of phosphorylationdeficient alleles of Raptor revealed that phosphorylation of these sites by ERK1/2 normally promotes mTORC1 activity and signaling to downstream substrates, such as 4E-BP1. Our data provide a novel regulatory mechanism by which mitogenic and oncogenic activation of the Ras/MAPK pathway promotes mTOR signaling.The Ras/mitogen-activated protein kinase (MAPK) pathway regulates a variety of cellular processes, including cell growth, proliferation, survival, and motility, through the activation of specific transcriptional and translational programs (1-3). The first evidence linking Ras/MAPK signaling to the regulation of mRNA translation stemmed from the finding that inhibition of MEK1/2 blocks growth factor-stimulated global protein synthesis (4). Subsequently, the MAPK-activated protein kinases MNK1/2 were found to phosphorylate the translation initiation factor eIF4E (5, 6), providing a potential mechanism by which MAPK signaling regulates mRNA translation (reviewed in 7, 8). However, more recent evidence indicates that the MNKs are dispensable for global protein synthesis (9). Thus, the functional impact of eIF4E phosphorylation remains unclear and a matter of debate (8, 10).Increasing evidence suggests that Ras/MAPK signaling largely promotes protein synthesis through the regulation of the mammalian target of rapamycin (mTOR), 6 an important regulator of cell growth and proliferation (reviewed in 11-13). mTOR, a member of the phosphoinositide 3-kinase-related protein kinase family, senses and integrates signals from diverse environmental cues to regulate crucial biosynthetic processes involved in cell growth and proliferation (reviewed in 14 -16). mTOR associates with different protein partners to form two functionally distinct signaling complexes, the rapamycin-sensitive and rapamycin-insensitive mTOR complex (mTORC) 1 and 2, respectively (17-19). mTORC1 contains the catalytic subunit mTOR, mLST8/G␤L, PRAS40, and Raptor (17,[20][21][22][23][24]. Raptor directly binds mTOR and is thought to function as a scaffolding protein that recruits mTORC1 substrates through their TOR sig...

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTORinteracting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (

The mammalian target of rapamycin (mTOR) Ser/Thr kinase signals in at least two multiprotein complexes distinguished by their different partners and sensitivities to rapamycin. Acute rapamycin inhibits signaling by mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), which both promote cell growth, proliferation, and survival. Although mTORC2 regulation remains poorly defined, diverse cellular mitogens activate mTORC1 signaling in a manner that requires sufficient levels of amino acids and cellular energy. Before the identification of distinct mTOR complexes, mTOR was reported to autophosphorylate on Ser-2481 in vivo in a rapamycin-and amino acid-insensitive manner. These results suggested that modulation of mTOR intrinsic catalytic activity does not universally underlie mTOR regulation. Here we re-examine the regulation of mTOR Ser-2481 autophosphorylation (Ser(P)-2481) in vivo by studying mTORC-specific Ser(P)-2481 in mTORC1 and mTORC2, with a primary focus on mTORC1. In contrast to previous work, we find that acute rapamycin and amino acid withdrawal markedly attenuate mTORC1-associated mTOR Ser(P)-2481 in cycling cells. Although insulin stimulates both mTORC1-and mTORC2-associated mTOR Ser(P)-2481 in a phosphatidylinositol 3-kinase-dependent manner, rapamycin acutely inhibits insulin-stimulated mTOR Ser(P)-2481 in mTORC1 but not mTORC2. By interrogating diverse mTORC1 regulatory input, we find that without exception mTORC1-activating signals promote, whereas mTORC1-inhibitory signals decrease mTORC1-associated mTOR Ser(P)-2481. These data suggest that mTORC1-and likely mTORC2-associated mTOR Ser-2481 autophosphorylation directly monitors intrinsic mTORC-specific catalytic activity and reveal that rapamycin inhibits mTORC1 signaling in vivo by reducing mTORC1 catalytic activity.

. Tee (2011) ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding, Autophagy,7:7,[737][738][739][740][741][742][743][744][745][746][747]