The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism

The adaptor protein p66Shc unexpectedly inhibits glucose metabolism, which could be a useful therapeutic target for diabetes or cancer. Negatively Adapting Metabolism Deficiency of the adaptor protein p66Shc, which inhibits signaling through receptor tyrosine kinases, improves the glucose tolerance of mice that are a genetic model of obesity. Soliman et al. found that in both transformed and nontransformed cells, p66Shc silencing enhanced glucose metabolism, increased the abundance of intermediates in various biosynthetic pathways, and increased cell size. The change in metabolism required the mTOR pathway, which couples energy, nutrient, and growth factor (including insulin) signals to processes that mediate cellular growth and proliferation. Thus, p66Shc suppresses mTOR signaling, and manipulating the abundance of p66Shc or interfering with p66Shc function could be used to treat diseases characterized by disruptions in this pathway, including diabetes and cancer. Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control the way cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoform of the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistance to diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucose metabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derived carbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. This change in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibition of mTOR with rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike the other isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucose uptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism.

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abstract: A search for substrates of a growth-promoting kinase revealed a regulatory feedback loop involved in tumor suppression. The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

title: Phosphoproteomic Analysis Identifies Grb10 as an mTORC1 Substrate That Negatively Regulates Insulin Signaling
abstract: A search for substrates of a growth-promoting kinase revealed a regulatory feedback loop involved in tumor suppression. The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor–bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal–regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

title: Peripheral Disruption of the Grb10 Gene Enhances Insulin Signaling and Sensitivity In Vivo
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title: Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation
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title: A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction
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title: The p66shc adaptor protein controls oxidative stress response and life span in mammals
abstract: Gene mutations in invertebrates have been identified that extend life span and enhance resistance to environmental stresses such as ultraviolet light or reactive oxygen species. In mammals, the mechanisms that regulate stress response are poorly understood and no genes are known to increase individual life span. Here we report that targeted mutation of the mouse p66shc gene induces stress resistance and prolongs life span. p66shc is a splice variant of p52shc/p46shc (ref. 2), a cytoplasmic signal transducer involved in the transmission of mitogenic signals from activated receptors to Ras. We show that: (1) p66shc is serine phosphorylated upon treatment with hydrogen peroxide (H2O2) or irradiation with ultraviolet light; (2) ablation of p66shc enhances cellular resistance to apoptosis induced by H2O2 or ultraviolet light; (3) a serine-phosphorylation defective mutant of p66shc cannot restore the normal stress response in p66shc-/- cells; (4) the p53 and p21 stress response is impaired in p66shc-/- cells; (5) p66shc-/- mice have increased resistance to paraquat and a 30% increase in life span. We propose that p66shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals.

title: Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance
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title: The Shc locus regulates insulin signaling and adiposity in mammals
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title: Genetic deletion of p66(Shc) adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress.
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title: Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA.
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title: Complex N-Glycan Number and Degree of Branching Cooperate to Regulate Cell Proliferation and Differentiation
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title: The hexosamine biosynthetic pathway couples growth factor-induced glutamine uptake to glucose metabolism.
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title: Electron Transfer between Cytochrome c and p66Shc Generates Reactive Oxygen Species that Trigger Mitochondrial Apoptosis
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title: Reactive oxygen species have a causal role in multiple forms of insulin resistance
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title: Activation of a metabolic gene regulatory network downstream of mTOR complex 1.
abstract: Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.

title: Stimulation of de Novo Pyrimidine Synthesis by Growth Signaling Through mTOR and S6K1
abstract: Coordinating Metabolism Growth factors help to coordinate metabolism with growth in part by stimulating the activity of the protein kinase mTORC1 (mechanistic target of rapamycin complex 1). Ben-Sahra et al. (p. 1323, published online 21 February) and Robitaille et al. (p. 1320, published online 21 February) independently identified a key target of mTORC1—carbamolyl-phosphate synthase 2, or CAD, the rate-limiting enzyme for de novo synthesis of pyrimidines. Metabolomic profiling and phosphoproteomic analyses of normal cells and cells lacking signaling by mTORC1 converged on CAD as a key point at which growth-promoting signals also ramp up production of nucleic acids. In addition to its role in stimulating protein and lipid synthesis, the kinase mammalian target of rapamycin stimulates nucleotide biosynthesis. Cellular growth signals stimulate anabolic processes. The mechanistic target of rapamycin complex 1 (mTORC1) is a protein kinase that senses growth signals to regulate anabolic growth and proliferation. Activation of mTORC1 led to the acute stimulation of metabolic flux through the de novo pyrimidine synthesis pathway. mTORC1 signaling posttranslationally regulated this metabolic pathway via its downstream target ribosomal protein S6 kinase 1 (S6K1), which directly phosphorylates S1859 on CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase), the enzyme that catalyzes the first three steps of de novo pyrimidine synthesis. Growth signaling through mTORC1 thus stimulates the production of new nucleotides to accommodate an increase in RNA and DNA synthesis needed for ribosome biogenesis and anabolic growth.

title: Quantitative Phosphoproteomics Reveal mTORC1 Activates de Novo Pyrimidine Synthesis
abstract: Coordinating Metabolism Growth factors help to coordinate metabolism with growth in part by stimulating the activity of the protein kinase mTORC1 (mechanistic target of rapamycin complex 1). Ben-Sahra et al. (p. 1323, published online 21 February) and Robitaille et al. (p. 1320, published online 21 February) independently identified a key target of mTORC1—carbamolyl-phosphate synthase 2, or CAD, the rate-limiting enzyme for de novo synthesis of pyrimidines. Metabolomic profiling and phosphoproteomic analyses of normal cells and cells lacking signaling by mTORC1 converged on CAD as a key point at which growth-promoting signals also ramp up production of nucleic acids. In addition to its role in stimulating protein and lipid synthesis, the kinase mammalian target of rapamycin stimulates nucleotide biosynthesis. 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.

title: mTOR: from growth signal integration to cancer, diabetes and ageing
abstract: In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.

title: TOR Signaling in Growth and Metabolism
abstract: The target of rapamycin (TOR) is a conserved Ser/Thr kinase that regulates cell growth and metabolism in response to environmental cues. Here, highlighting contributions from studies in model organisms, we review mammalian TOR complexes and the signaling branches they mediate. TOR is part of two distinct multiprotein complexes, TOR complex 1 (TORC1), which is sensitive to rapamycin, and TORC2, which is not. The physiological consequences of mammalian TORC1 dysregulation suggest that inhibitors of mammalian TOR may be useful in the treatment of cancer, cardiovascular disease, autoimmunity, and metabolic disorders.

title: Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c.
abstract: Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.

title: Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2)
abstract: The mechanistic target of rapamycin (mTOR) exists in two complexes that regulate diverse cellular processes. mTOR complex 1 (mTORC1), the canonical target of rapamycin, has been well studied, whereas the physiological role of mTORC2 remains relatively uncharacterized. In mice in which the mTORC2 component Rictor is deleted in liver [Rictor‐knockout (RKO) mice], we used genomic and phosphoproteomic analyses to characterize the role of hepatic mTORC2 in vivo. Overnight food withdrawal followed by refeeding was used to activate mTOR signaling. Rapamycin was administered before refeeding to specify mTORC2‐mediated events. Hepatic mTORC2 regulated a complex gene expression and post‐translational network that affects intermediary metabolism, ribosomal biogenesis, and proteasomal biogenesis. Nearly all changes in genes related to intermediary metabolic regulation were replicated in cultured fetal hepatocytes, indicating a cell‐autonomous effect of mTORC2 signaling. Phosphoproteomic profiling identified mTORC2‐related signaling to 144 proteins, among which were metabolic enzymes and regulators. A reduction of p38 MAPK signaling in the RKO mice represents a link between our phosphoproteomic and gene expression results. We conclude that hepatic mTORC2 exerts a broad spectrum of biological effects under physiological conditions. Our findings provide a context for the development of targeted therapies to modulate mTORC2 signaling.—Lamming, D. W., Demirkan, G., Boylan, J. M., Mihaylova, M. M., Peng, T., Ferreira, J., Neretti, N., Salomon, A., Sabatini, D. M., Gruppuso, P. A. Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2).  (2014). www.fasebj.org

title: Temporal regulation of EGF signaling networks by the scaffold protein Shc1
abstract: Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation.

title: Hyperglycemia-induced p66shc inhibits insulin-like growth factor I-dependent cell survival via impairment of Src kinase-mediated phosphoinositide-3 kinase/AKT activation in vascular smooth muscle cells.
abstract: Hyperglycemia has been shown to induce the p66shc expression leading to increased reactive oxygen species (ROS) generation and apoptosis. In the present study, we demonstrated that hyperglycemia induced p66shc expression in vascular smooth muscle cells. This induction was associated with an increase in apoptosis as assessed by the increase of capspase-3 enzymatic activity, cleaved caspase-3 protein, and the number of dead cells. The ability of IGF-I to inhibit apoptosis was also attenuated. Further studies showed that hyperglycemia-induced p66shc inhibited IGF-I-stimulated phosphoinositide (PI)-3 kinase and AKT activation. Mechanistic studies showed that knockdown of p66shc enhanced IGF-I-stimulated SHPS-1/p85, p85/SHP-2, and p85/Grb2 association, all of which are required for PI-3 kinase/AKT activation. These responses were attenuated by overexpression of p66shc. IGF-I-stimulated p85 and AKT recruitment to the cell membrane fraction was altered in the same manner. Disruption of p66shc-Src interaction using either a blocking peptide or by expressing a p66shc mutant that did not bind to Src rescued IGF-I-stimulated PI-3 kinase/AKT activation as well as IGF-I-dependent cell survival. Although the highest absolute level of ROS was detected in p66shc-overexpressing cells, the relative increase in ROS induced by hyperglycemia was independent of p66shc expression. Taken together, our data suggest that the increase in p66shc that occurs in response to hyperglycemia is functioning to inhibit IGF-I-stimulated signaling and that the incremental increase in SMC sensitivity to IGF-I stimulation that occurs in response to p66shc induction of ROS is not sufficient to overcome the inhibitory effect of p66shc on Src kinase activation.

title: Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst
abstract: MetaboAnalyst is an integrated web-based platform for comprehensive analysis of quantitative metabolomic data. It is designed to be used by biologists (with little or no background in statistics) to perform a variety of complex metabolomic data analysis tasks. These include data processing, data normalization, statistical analysis and high-level functional interpretation. This protocol provides a step-wise description on how to format and upload data to MetaboAnalyst, how to process and normalize data, how to identify significant features and patterns through univariate and multivariate statistical methods and, finally, how to use metabolite set enrichment analysis and metabolic pathway analysis to help elucidate possible biological mechanisms. The complete protocol can be executed in ∼45 min.

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abstract: There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anticancer therapy are under way. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, SLC16A1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Furthermore, forced MCT1 expression in 3-BrPA–resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors.