Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.     

Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein

The serine/threonine protein kinase Akt is a critical regulator of cell growth and survival in response to growth factors. A key step in Akt activation is phosphorylation at Ser-473 by the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Although Rictor is required for the stability and activity of mTORC2, little is known about functional regions or post-translational modifications within Rictor that are responsible for regulating mTORC2. Here, we demonstrate that Rictor contains two distinct central regions critical for mTORC2 function. One we refer to as the stability region because it is critical for interaction with Sin1.1 and LST8, and a second adjacent region is required for multisite acetylation. p300-mediated acetylation of Rictor increases mTORC2 activity toward Akt, whereas site-directed mutants within the acetylation region of Rictor exhibit reduced insulin-like growth factor 1 (IGF-1)-stimulated mTORC2 kinase activity. Inhibition of deacetylases, including the NAD+-dependent sirtuins, promotes Rictor acetylation and IGF-1-mediated Akt phosphorylation. These results suggest that multiple-site acetylation of Rictor signals for increased activation of mTORC2, providing a critical link between nutrient-sensitive deacetylases and mTORC2 signaling to Akt.     

The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.     

mTOR complex 2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobic motif

The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.     

Chemokine CXCL12 uses CXCR4 and a signaling core formed by bifunctional Akt, extracellular signal-regulated kinase (ERK)1/2, and mammalian target of rapamycin complex 1 (mTORC1) proteins to control chemotaxis and survival simultaneously in mature dendritic cells

Chemokines control several cell functions in addition to chemotaxis. Although much information is available on the involvement of specific signaling molecules in the control of single functions controlled by chemokines, especially chemotaxis, the mechanisms used by these ligands to regulate several cell functions simultaneously are completely unknown. Mature dendritic cells (maDCs) migrate through the afferent lymphatic vessels to the lymph nodes, where they regulate the initiation of the immune response. As maDCs are exposed to chemokine CXCL12 (receptors CXCR4 and CXCR7) during their migration, its functions are amenable to be regulated by this ligand. We have used maDCs as a model system to analyze the mechanisms whereby CXCL12 simultaneously controls chemotaxis and survival in maDCs. We show that CXCL12 uses CXCR4, but not CXCR7, and the components of a signaling core that includes G(i)/Gβγ, PI3K-α/-δ/-γ, Akt, ERK1/2 and mammalian target of rapamycin complex 1 (mTORC1), which organize hierarchically to control both functions. Downstream of Akt, Forkhead box class O (FOXO) regulates CXCL12-dependent survival, but not chemotaxis, suggesting that downstream of the aforementioned signaling core, additional signaling molecules may control more selectively CXCL12-dependent chemotaxis or survival. Finally, the data obtained also show that CXCR4 uses a signaling signature that is different from that used by CCR7 to control similar functions.     

Phosphorylation of Raptor by p38beta participates in arsenite-induced mammalian target of rapamycin complex 1 (mTORC1) activation

Cell growth is influenced by environmental stress. Mammalian target of rapamycin (mTOR), the central regulator of cell growth, can be positively or negatively regulated by various stresses through different mechanisms. The p38 MAP kinase pathway is essential in cellular stress responses. Activation of MK2, a downstream kinase of p38α, enhances mTOR complex 1 (mTORC1) activity by preventing TSC2 from inhibiting mTOR activation. The p38β-PRAK cascade targets Rheb to inhibit mTORC1 activity upon glucose depletion. Here we show the activation of p38β participates in activation of mTOR complex 1 (mTORC1) induced by arsenite but not insulin, nutrients, anisomycin, or H(2)O(2). Arsenite treatment of cells activates p38β and induces interaction between p38β and Raptor, a regulatory component of mTORC1, resulting in phosphorylation of Raptor on Ser(863) and Ser(771). The phosphorylation of Raptor on these sites enhances mTORC1 activity, and contributes largely to arsenite-induced mTORC1 activation. Our results shown here and in previous work demonstrate that the p38 pathway can regulate different components of the mTORC1 pathway, and that p38β can target different substrates to either positively or negatively regulate mTORC1 activation when a cell encounters different environmental stresses.     

Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway

Mammalian target of rapamycin (mTOR) is a kinase that plays a key role in a wide array of cellular processes and exists in two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although mTORC2 is primarily activated by growth factors, mTORC1 is regulated by numerous extracellular and intracellular signals such as nutrients, growth factors, and cellular redox. Previous study has shown that cysteine oxidants sufficiently activate mTORC1 activity under amino acid-depleted conditions and that a reducing agent effectively suppresses amino acid-induced mTORC1 activity, thereby raising the possibility that redox-sensitive mechanisms underlie amino acid-dependent mTORC1 regulation. However, the molecular mechanism by which redox regulates mTORC1 activity is not well understood. In this study, we show that the redox-sensitive regulation of mTORC1 occurs via Rheb but not the Rag small GTPase. Enhancing cellular redox potential with cysteine oxidants significantly increases Rheb GTP levels. Importantly, modulation of the cellular redox potential with a cysteine oxidant or reducing agent failed to alter mTORC1 activity in TSC1(-/-) or TSC2(-/-) mouse embryonic fibroblast cells. Furthermore, a cysteine oxidant has little effect on mTOR localization but sufficiently activates mTORC1 activity in both p18(-/-) and control mouse embryonic fibroblast cells, suggesting that the redox-sensitive regulation of mTORC1 occurs independent of the Ragulator·Rag complex. Taken together, our results suggest that the TSC complex plays an important role in redox-sensitive mTORC1 regulation and argues for the activation of mTORC1 in places other than the lysosome upon inhibition of the TSC complex.     

L-threonine regulates G1/S phase transition of mouse embryonic stem cells via PI3K/Akt, MAPKs, and mTORC pathways

Although amino acids can function as signaling molecules in the regulation of many cellular processes, mechanisms surrounding L-threonine involvement in embryonic stem cell (ESC) functions have not been explored. Thus, we investigated the effect of L-threonine on regulation of mouse (m)ESC self-renewal and related signaling pathways. In L-threonine-depleted mESC culture media mRNA of self-renewal marker genes, [(3)H]thymidine incorporation, expression of c-Myc, Oct4, and cyclins protein was attenuated. In addition, resupplying L-threonine (500 μM) after depletion restores/maintains the mESC proliferation. Disruption of the lipid raft/caveolae microdomain through treatment with methyl-β-cyclodextrin or transfection with caveolin-1 specific small interfering RNA blocked L-threonine-induced proliferation of mESCs. Addition of L-threonine induced phosphorylation of Akt, ERK, p38, JNK/SAPK, and mTOR in a time-dependent manner. This activity was blocked by LY 294002 (PI3K inhibitor), wortmannin (PI3K inhibitor), or an Akt inhibitor. L-threonine-induced activation of mTOR, p70S6K, and 4E-BP1 as well as cyclins and Oct4 were blocked by PD 98059 (ERK inhibitor), SB 203580 (p38 inhibitor) or SP 600125 (JNK inhibitor). Furthermore, L-threonine induced phosphorylation of raptor and rictor binding to mTOR was completely inhibited by 24 h treatment with rapamycin (mTOR inhibitor); however, a 10 min treatment with rapamycin only partially inhibited rictor phosphorylation. L-threonine induced translocation of rictor from the membrane to the cytosol/nuclear, which blocked by pretreatment with rapamycin. In addition, rapamycin blocked L-threonine-induced increases in mRNA expressions of trophoectoderm and mesoderm marker genes and mESC proliferation. In conclusion, L-threonine stimulated ESC G(1)/S transition through lipid raft/caveolae-dependent PI3K/Akt, MAPKs, mTOR, p70S6K, and 4E-BP1 signaling pathways.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) and 2 (mTORC2) Control the Dendritic Arbor Morphology of Hippocampal Neurons

Dendrites are the main site of information input into neurons. Their development is a multistep process controlled by mammalian target of rapamycin (mTOR) among other proteins. mTOR is a serine/threonine protein kinase that forms two functionally distinct complexes in mammalian cells: mTORC1 and mTORC2. However, the one that contributes to mammalian neuron development remains unknown. This work used short hairpin RNA against Raptor and Rictor, unique components of mTORC1 and mTORC2, respectively, to dissect mTORC involvement in this process. We provide evidence that both mTOR complexes are crucial for the proper dendritic arbor morphology of hippocampal neurons. These two complexes are required for dendritic development both under basal conditions and upon the induction of mTOR-dependent dendritic growth. We also identified Akt as a downstream effector of mTORC2 needed for proper dendritic arbor morphology, the action of which required mTORC1 and p70S6K1.     

Osmotic stress regulates mammalian target of rapamycin (mTOR) complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor protein phosphorylation

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.     

Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling

Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.     

Phosphatidic acid activates mammalian target of rapamycin complex 1 (mTORC1) kinase by displacing FK506 binding protein 38 (FKBP38) and exerting an allosteric effect

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro. Furthermore, we show that PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Phospholipase D 1, a PA-generating enzyme that is an established upstream regulator of mTORC1, is found to negatively affect mTOR-FKBP38 interaction, confirming the role of endogenous PA in this regulation. Interestingly, removal of FKBP38 alone is insufficient to activate mTORC1 kinase and signaling, which require PA even when the FKBP38 level is drastically reduced by RNAi. In conclusion, we propose a dual mechanism for PA activation of mTORC1: PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1.     

Differential Contribution of Insulin and Amino Acids to the mTORC1-Autophagy Pathway in the Liver and Muscle

Autophagy is a highly inducible intracellular degradation process. It is generally induced by nutrient starvation and suppressed by food intake. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is considered to be the major regulator of autophagy, but the precise mechanism of in vivo regulation remains to be fully characterized. Here, we examined the autophagy-suppressive effect of glucose, insulin, and amino acids in the liver and muscle in mice starved for 1 day. Refeeding after starvation with a standard mouse chow rapidly suppressed autophagy in both tissues, and this suppression was inhibited by rapamycin administration almost completely in the liver and partially in muscle, confirming that mTORC1 is indeed a crucial regulator in vivo. As glucose administration showed no major suppressive effect on autophagy, we examined the role of insulin and amino acids using hyperinsulinemic-euglycemic clamp and intravenous amino acid infusion techniques. Insulin administration showed a clear effect on the mTORC1-autophagy pathway in muscle, but had only a very weak effect in the liver. By contrast, amino acids were able to regulate the mTORC1-autophagy pathway in the liver, but less effectively in muscle. These results suggest that autophagy is differentially regulated by insulin and amino acids in a tissue-dependent manner.     

The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains

Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.     

PI3K/Akt/mTOR信号通路及临床相关肿瘤抑制剂

哺乳动物雷帕霉素靶（mTOR）和蛋白激酶B（Akt/PKB）与肿瘤发生的密切关系已被广泛地认可.mTOR是一种丝/苏氨酸激酶,可以通过影响mRNA转录、代谢、自噬等方式调控细胞的生长.它既是PI3K的效应分子,也可以是PI3K的反馈调控因子.mTORC1 和mTORC2是mTOR的两种不同复合物. 对雷帕霉素敏感的mTORC1受到营养、生长因子、能量和应激4种因素的影响.生长因子通过PI3K/Akt信号通路调控mTORC1是最具特征性调节路径.而mTORC2最为人熟知的是作为Akt473磷酸化位点的上游激酶. 同样,Akt/PKB在细胞增殖分化、迁移生长过程中发挥着重要作用. 随着Thr308和Ser473两个位点激活,Akt/PKB也得以全面活化.因此,mTORC2-Akt-mTORC1的信号通路在肿瘤形成和生长中是可以存在的.目前临床肿瘤治疗中,PI3K/Akt/mTOR是重要的靶向治疗信号通路.然而,仅抑制mTORC1活性,不是所有的肿瘤都能得到预期控制.雷帕霉素虽然能抑制mTORC1,但也能反馈性地增加PI3K信号活跃度,从而影响治疗预后.近来发现的第二代抑制剂可以同时抑制mTORC1/2和PI3K活性,这种抑制剂被认为在肿瘤治疗上颇具前景.本综述着重阐述了PI3K/Akt/mTOR信号通路的传导、各因子之间的相互调控以及相关抑制剂的发展.     

Hyperactivation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Promotes Breast Cancer Progression through Enhancing Glucose Starvation-induced Autophagy and Akt Signaling

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation. Recent studies have suggested that constitutive activation of mTORC1 in normal cells could lead to malignant tumor development in several tissues. However, the mechanisms of mTORC1 hyperactivation to promote the growth and metastasis of breast or other cancers are still not well characterized. Here, using a new inducible deletion system, we show that deletion of Tsc1 in mouse primary mammary tumor cells, either before or after their transplantation, significantly increased their growth in vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activation in vivo.     

Proteasome-dependent Activation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Is Essential for Autophagy Suppression and Muscle Remodeling Following Denervation

Drastic protein degradation occurs during muscle atrophy induced by denervation, fasting, immobility, and various systemic diseases. Although the ubiquitin-proteasome system is highly up-regulated in denervated muscles, the involvement of autophagy and protein synthesis has been controversial. Here, we report that autophagy is rather suppressed in denervated muscles even under autophagy-inducible starvation conditions. This is due to a constitutive activation of mammalian target of rapamycin complex 1 (mTORC1). We further reveal that denervation-induced mTORC1 activation is dependent on the proteasome, which is likely mediated by amino acids generated from proteasomal degradation. Protein synthesis and ribosome biogenesis are paradoxically increased in denervated muscles in an mTORC1-dependent manner, and mTORC1 activation plays an anabolic role against denervation-induced muscle atrophy. These results suggest that denervation induces not only muscle degradation but also adaptive muscle response in a proteasome- and mTORC1-dependent manner.     

The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C

The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.     

Phospholipase D mediates nutrient input to mammalian target of rapamycin complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.     

Prolonged deprivation of arginine or leucine induces PI3K/Akt-dependent reactivation of mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase complex that promotes anabolic processes including protein, lipid, and nucleotide synthesis, while suppressing catabolic processes such as macroautophagy. mTORC1 activity is regulated by growth factors and amino acids, which signal through distinct but integrated molecular pathways: growth factors largely signal through the PI3K/Akt-dependent pathway, whereas the availabilities of amino acids leucine and arginine are communicated to mTORC1 by the Rag-GTPase pathway. While it is relatively well described how acute changes in leucine and arginine levels affect mTORC1 signaling, the effects of prolonged amino acid deprivation remain less well understood. Here, we demonstrate that prolonged deprivation of arginine and/or leucine leads to reactivation of mTORC1 activity, which reaches activation levels similar to those observed in nutrient-rich conditions. Surprisingly, we find that this reactivation is independent of the regeneration of amino acids by canonical autophagy or proteasomal degradation but is dependent on PI3K/Akt signaling. Together, our data identify a novel crosstalk between the amino acid and PI3K/Akt signaling pathways upstream of mTORC1. These observations extend our understanding of the role of mTORC1 in growth-related diseases and indicate that dietary intervention by removal of leucine and/or arginine may be an ineffective therapeutic approach.