Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway.

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase, which regulates protein translation, cell size, and autophagy. However, the amino acid sensor that directly couples intracellular amino acid-mediated signaling to mTORC1 is unknown. Here we show that leucyl-tRNA synthetase (LRS) plays a critical role in amino acid-induced mTORC1 activation by sensing intracellular leucine concentration and initiating molecular events leading to mTORC1 activation. Mutation of LRS amino acid residues important for leucine binding renders the mTORC1 pathway insensitive to intracellular levels of amino acids. We show that LRS directly binds to Rag GTPase, the mediator of amino acid signaling to mTORC1, in an amino acid-dependent manner and functions as a GTPase-activating protein (GAP) for Rag GTPase to activate mTORC1. This work demonstrates that LRS is a key mediator for amino acid signaling to mTORC1.

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer.

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.

IgGs from patients with amyotrophic lateral sclerosis and diabetes target CaVα2δ1 subunits impairing islet cell function and survival.

Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K+-induced increases in cytosolic free Ca2+ concentration ([Ca2+]i) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALS-T2DM sera. The pathogenic IgGs immunocaptured the voltage-dependent Ca2+ (CaV) channel subunit CaVα2δ1 in the plasma membrane enhancing CaV1 channel-mediated Ca2+ influx and [Ca2+]i, resulting in impaired mitochondrial function. Consequently, impairments in [Ca2+]i dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking CaVα2δ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering from a combination of these diseases.

Direct visualization of single-molecule membrane protein interactions in living cells.

Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (ß2-AR) in living cells, revealing the differential regulation of these receptors' dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.

Phospholipase Signaling in Breast Cancer.

Breast cancer progression results from subversion of multiple intra- or intercellular signaling pathways in normal mammary tissues and their microenvironment, which have an impact on cell differentiation, proliferation, migration, and angiogenesis. Phospholipases (PLC, PLD and PLA) are essential mediators of intra- and intercellular signaling. They hydrolyze phospholipids, which are major components of cell membrane that can generate many bioactive lipid mediators, such as diacylglycerol, phosphatidic acid, lysophosphatidic acid, and arachidonic acid. Enzymatic processing of phospholipids by phospholipases converts these molecules into lipid mediators that regulate multiple cellular processes, which in turn can promote breast cancer progression. Thus, dysregulation of phospholipases contributes to a number of human diseases, including cancer. This review describes how phospholipases regulate multiple cancer-associated cellular processes, and the interplay among different phospholipases in breast cancer. A thorough understanding of the breast cancer-associated signaling networks of phospholipases is necessary to determine whether these enzymes are potential targets for innovative therapeutic strategies.

Proteomic analysis of the palmitate-induced myotube secretome reveals involvement of the annexin A1-formyl peptide receptor 2 (FPR2) pathway in insulin resistance.

Elevated levels of the free fatty acid palmitate are found in the plasma of obese patients and induce insulin resistance. Skeletal muscle secretes myokines as extracellular signaling mediators in response to pathophysiological conditions. Here, we identified and characterized the skeletal muscle secretome in response to palmitate-induced insulin resistance. Using a quantitative proteomic approach, we identified 36 secretory proteins modulated by palmitate-induced insulin resistance. Bioinformatics analysis revealed that palmitate-induced insulin resistance induced cellular stress and modulated secretory events. We found that the decrease in the level of annexin A1, a secretory protein, depended on palmitate, and that annexin A1 and its receptor, formyl peptide receptor 2 agonist, played a protective role in the palmitate-induced insulin resistance of L6 myotubes through PKC-θ modulation. In mice fed with a high-fat diet, treatment with the formyl peptide receptor 2 agonist improved systemic insulin sensitivity. Thus, we identified myokine candidates modulated by palmitate-induced insulin resistance and found that the annexin A1- formyl peptide receptor 2 pathway mediated the insulin resistance of skeletal muscle, as well as systemic insulin sensitivity.

Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation.

Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.

Potential pancreatic lipase inhibitory activity of phenolic constituents from the root bark of Morus alba L.

Detailed phytochemical investigation from the root bark of Morus alba resulted in the isolation of eleven new compounds, including seven 2-arylbenzofuran derivatives (morusalfurans A-G), three flavonoids (morusalnols A-C), and one geranylated stilbene (morusibene A), as well as 22 known compounds. The structures of the identified compounds were elucidated based on a comprehensive analysis of spectroscopic data and Mosher's method. Compounds 2, 3, 6-8, 11, 23, 24, and 29 showed potent inhibition of PL in comparison with the positive control treatment (orlistat, IC50=0.012µM), with IC50 values ranging from 0.09 to 0.92µM.

Obesity resistance and increased energy expenditure by white adipose tissue browning in Oga(+/-) mice.

AIMS/HYPOTHESIS: O-GlcNAcylation plays a role as a metabolic sensor regulating cellular signalling, transcription and metabolism. Transcription factors and signalling pathways related to metabolism are modulated by N-acetyl-glucosamine (O-GlcNAc) modification. Aberrant regulation of O-GlcNAcylation is closely linked to insulin resistance, type 2 diabetes and obesity. Current evidence shows that increased O-GlcNAcylation negatively regulates insulin signalling, which is associated with insulin resistance and type 2 diabetes. Here, we aimed to evaluate the effects of Oga (also known as Mgea5) haploinsufficiency, which causes hyper-O-GlcNAcylation, on metabolism. METHODS: We examined whether Oga(+/-) mice developed insulin resistance. Metabolic variables were determined including body weight, glucose and insulin tolerance, metabolic rate and thermogenesis. RESULTS: Oga deficiency does not affect insulin signalling even at hyper-O-GlcNAc levels. Oga(+/-) mice are lean with reduced fat mass and improved glucose tolerance. Furthermore, Oga(+/-) mice resist high-fat diet-induced obesity with ameliorated hepatic steatosis and improved glucose metabolism. Oga haploinsufficiency potentiates energy expenditure through the enhancement of brown adipocyte differentiation from the stromal vascular fraction of subcutaneous white adipose tissue (WAT). CONCLUSIONS/INTERPRETATION: Our observations suggest that O-GlcNAcase (OGA) is essential for energy metabolism via regulation of the thermogenic WAT program.

Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

Endothelial deletion of phospholipase D2 reduces hypoxic response and pathological angiogenesis.

OBJECTIVE: Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1α and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo. APPROACH AND RESULTS: Pld2 knockout ECs exhibited decreased hypoxia-induced cellular responses in survival, migration, and thus vessel sprouting. Analysis of hypoxia-induced gene expression revealed that PLD2 deficiency disrupted the upregulation of hypoxia-inducible factor-1α target genes, including VEGF, PFKFB3, HMOX-1, and NTRK2. Consistent with this, PLD2 contributed to hypoxia-induced hypoxia-inducible factor-1α expression at the translational level. The roles of PLD2 in hypoxia-induced in vivo pathological angiogenesis were assessed using oxygen-induced retinopathy and tumor implantation models in endothelial-specific Pld2 knockout mice. Pld2 endothelial-specific knockout retinae showed decreased neovascular tuft formation, despite a larger avascular region. Tumor growth and tumor blood vessel formation were also reduced in Pld2 endothelial-specific knockout mice. CONCLUSIONS: Our findings demonstrate a novel role for endothelial PLD2 in the survival and migration of ECs under hypoxia via the expression of hypoxia-inducible factor-1α and in pathological retinal angiogenesis and tumor angiogenesis in vivo.

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise.

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.

Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single-Molecule Diffusivity in the Membrane of Living Cells.

We present a single-molecule diffusional-mobility-shift assay (smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells. We found that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and ß-adrenergic receptors, as determined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.

Proteomic analysis of hypoxia-induced U373MG glioma secretome reveals novel hypoxia-dependent migration factors.

High-grade gliomas are one of the most common brain tumors and notorious for poor prognosis due to their malignant nature. Gliomas have an extensive area of hypoxia, which is critical for glioma progression by inducing aggressiveness and activating the angiogenesis process in the tumor microenvironment. To resolve the factors responsible for the highly malignant nature of gliomas, we comprehensively profiled the U373MG glioma cell secretome-exosome and soluble fraction under hypoxic and normoxic conditions. A total of 239 proteins were identified from the exosome and soluble fractions. Vascular endothelial growth factor, stanniocalcin 1 (STC1) and stanniocalcin 2, and insulin-like growth factor binding protein 3 and 6, enriched in the soluble fraction, and lysyl oxidase homolog 2 enriched in the exosomal fraction were identified as upregulated proteins by hypoxia based on a label-free quantitative analysis. STCs and insulin-like growth factor binding proteins, which were identified as secretory proteins under hypoxic conditions, were highly correlated with glioma grade in human patients by microarray analysis. An in vitro scratch wound assay revealed that STC1 and 2 have important functions in the induction of cell migration in a hypoxia-dependent manner, suggesting that they are hypoxia-dependent migration factors.

CXCL12 secreted from adipose tissue recruits macrophages and induces insulin resistance in mice.

AIMS/HYPOTHESIS: Obesity-induced inflammation is initiated by the recruitment of macrophages into adipose tissue. The recruited macrophages, called adipose tissue macrophages, secrete several proinflammatory cytokines that cause low-grade systemic inflammation and insulin resistance. The aim of this study was to find macrophage-recruiting factors that are thought to provide a crucial connection between obesity and insulin resistance. METHODS: We used chemotaxis assay, reverse phase HPLC and tandem MS analysis to find chemotactic factors from adipocytes. The expression of chemokines and macrophage markers was evaluated by quantitative RT-PCR, immunohistochemistry and FACS analysis. RESULTS: We report our finding that the chemokine (C-X-C motif) ligand 12 (CXCL12, also known as stromal cell-derived factor 1), identified from 3T3-L1 adipocyte conditioned medium, induces monocyte migration via its receptor chemokine (C-X-C motif) receptor 4 (CXCR4). Diet-induced obese mice demonstrated a robust increase of CXCL12 expression in white adipose tissue (WAT). Treatment of obese mice with a CXCR4 antagonist reduced macrophage accumulation and production of proinflammatory cytokines in WAT, and improved systemic insulin sensitivity. CONCLUSIONS/INTERPRETATION: In this study we found that CXCL12 is an adipocyte-derived chemotactic factor that recruits macrophages, and that it is a required factor for the establishment of obesity-induced adipose tissue inflammation and systemic insulin resistance.

Emodin regulates glucose utilization by activating AMP-activated protein kinase.

AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentration than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and [(14)C]glucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca(2+)/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of type 2 diabetic models.

Airway activation of formyl peptide receptors inhibits Th1 and Th17 cell responses via inhibition of mediator release from immune and inflammatory cells and maturation of dendritic cells.

Formyl peptide receptors (FPRs) are chemoattractant receptors that mediate inflammatory cell responses to infection. Recent evidence indicates that noneosinophilic asthma phenotypes can be developed by both Th1 and Th17 cell responses when exposed to LPS-containing allergens. In this study, we evaluated the effects of airway activation of FPRs by their synthetic agonist, Trp-Lys-Tyr-Met-Val-D-Met (W-peptide), on the development of Th1 and Th17 cell responses in a noneosinophilic asthma mouse model. A noneosinophilic asthma mouse model was generated by intranasal sensitization with 10 µg of LPS plus 75 µg of OVA on days 0, 1, 2, and 7. Mice were then challenged with 50 µg of OVA alone on days 14, 15, 21, and 22. W-peptide was administered during the sensitization period, and immune and inflammatory responses were evaluated after OVA challenge. Lung inflammation after OVA challenge was partly abolished by airway activation of FPRs during sensitization. Maturation of dendritic cells (DCs) and migration of DCs from the lung to lung-draining lymph nodes were inhibited by FPR activation. In addition, airway activation of FPRs inhibited allergen-specific T cell proliferation in the lymph nodes. Production of IL-12 and IL-6 (Th1- and Th17-polarizing cytokines) from lung DCs was decreased by airway activation of FPRs. This effect resulted in the inhibition of allergen-specific Th1 and Th17 cell responses. Airway activation of FPRs during sensitization effectively prevents the development of Th1 and Th17 cell responses induced by LPS-containing allergens via multiple mechanisms, such as inhibition of DC maturation and migration and the production of Th1- and Th7-polarizing cytokines.

Periostin-binding DNA aptamer inhibits breast cancer growth and metastasis.

Periostin is an extracellular matrix (ECM) protein that is overexpressed in a variety of human cancers, and its functions appear to be linked to tumor growth, metastasis, and angiogenesis. Recent clinical evidence suggests that aberrant periostin expression is correlated with poor outcome in patients with breast cancer. To identify novel tools to regulate the functional role of periostin, we generated benzyl-d(U)TP-modified DNA aptamers that were directed against human periostin (PNDAs) and characterized their functional roles in breast cancer progression. PNDA-3 selectively bound to the FAS-1 domain of periostin with nanomolar affinity and disrupted the interaction between periostin and its cell surface receptors, αvß3 and αvß5 integrins. PNDA-3 markedly antagonized the periostin-induced adhesion, migration, and invasion of breast cancer cells and blocked the activation of various components of the αvß3 and αvß5 integrin signal transduction pathways. In a 4T1 orthotopic mouse model, PNDA-3 administration significantly reduced primary tumor growth and distant metastasis. Thus, our results demonstrated that periostin-integrin signaling regulates breast cancer progression at multiple levels in tumor cells and the tumor microenvironment. DNA aptamers targeting periostin may potentially be used to inhibit breast cancer progression.

Phosphoinositides differentially regulate protrudin localization through the FYVE domain.

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

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.