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(313), ra17. [DOI: 10.1126/scisignal.2004785] 7Science Signaling(18 February 2014) Pasculescu, Yong Zheng, David M. Sabatini, James W. Dennis and Tony PawsonBirsoy, Judy Pawling, Maria E. Frigolet, Huogen Lu, I. George Fantus, Adrian Mohamed A. Soliman, Anas M. Abdel Rahman, Dudley W. Lamming, KivançMetabolism
The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic`
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R E S E A R C H A R T I C L E
M E T A B O L I S M
The Adaptor Protein p66Shc InhibitsmTOR-Dependent Anabolic MetabolismMohamed A. Soliman,1,2,3* Anas M. Abdel Rahman,1† Dudley W. Lamming,4 Kivanç Birsoy,4
Judy Pawling,1 Maria E. Frigolet,1,5 Huogen Lu,1,5 I. George Fantus,1,5 Adrian Pasculescu,1
Yong Zheng,1 David M. Sabatini,4,6,7,8,9 James W. Dennis,1,2* Tony Pawson1,2‡
(Published 18 February 2014 | Revised 4 March 2014)Dow
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Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control theway cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoformof the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistanceto diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucosemetabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derivedcarbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. Thischange in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibitionof mTORwith rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike theother isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucoseuptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism.
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INTRODUCTION
A common mechanism through which activated receptor tyrosine kinases(RTKs) control intracellular pathways involves recruitment of Src homology2 (SH2)–containing proteins with regulatory or adaptor functions (1). Forexample, the Grb10 adaptor is an inhibitor of insulin signaling that is sta-bilized by mammalian target of rapamycin (mTOR)–mediated phosphoryl-ation and suppresses insulin sensitivity in vivo (2–5). Loss of inhibitors canresult in dysregulation of growth factor signaling, promoting the rewiring ofmetabolic pathways in amanner that supports rapid growth and cell survival.A major change that occurs in such proliferating cells is enhanced glucoseuptake and catabolism, accompanied by increased lactate production, a phe-nomenon referred to as the “Warburg effect” (6, 7). This rewiring providesproliferating cells with biosynthetic precursors from increased glucose-derived carbon intermediates that are essential for increasing cell biomass.
The gene for the Shc1 adaptor protein encodes three isoforms in mam-mals: p46, p52, and p66. These proteins share a modular arrangement of aphosphotyrosine binding (PTB) domain, a collagen homology 1 (CH1) region,and an SH2 domain (Fig. 1A). p66Shc and p52Shc or p46Shc (p52/p46Shc)are encoded by two transcripts that differ in the use of alternative 5′ codingexons, whereas p46Shc and p52Shc originate fromdifferent translation startsites in the same mRNA, such that p46Shc is an N-terminally truncatedform of p52Shc (8). p52/p46Shc isoforms are scaffolds that associate withactivated RTKs and amplify signaling to the Ras-ERK (extracellular signal–
1Lunenfeld-TanenbaumResearch Institute,MountSinaiHospital, Toronto,OntarioM5G 1X5, Canada. 2Department of Molecular Genetics, University of Toronto,Toronto, Ontario M5S 1A8, Canada. 3Department of Biochemistry, Faculty ofPharmacy, Cairo University, Cairo 11562, Egypt. 4Whitehead Institute for Bio-medicalResearch,NineCambridgeCenter,Cambridge,MA02142,USA. 5Bantingand Best Diabetes Centre, Toronto General Research Institute, Faculty of Medicine,University of Toronto, Toronto, Ontario M5G 1L6, Canada. 6Department of Biology,Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA. 7DavidH.Koch Institute for IntegrativeCancerResearchatMIT, 77MassachusettsAvenue,Cambridge, MA 02139, USA. 8Broad Institute, Seven Cambridge Center, Cam-bridge, MA 02142, USA. 9Howard Hughes Medical Institute, MIT, Cambridge,MA 02139, USA.*Corresponding author. E-mail: [email protected] (M.A.S.); [email protected] (J.W.D.)†Present address: School of Pharmacy, Yarmouk University, Irbid 21163, Jordan.‡Deceased.
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regulated kinase)mitogen-activated protein kinase and phosphatidylinositol3′-kinase (PI3K)–Akt pathways. The p66Shc isoform emerged with verte-brates and is characterized by an N-terminal collagen homology 2 (CH2)extension (9). p66Shc has been reported to promote oxidative stress andproapoptotic signaling in HeLa cells and murine embryonic fibroblasts(MEFs) (9–11). Unlike p52/p46, the abundance of p66Shc is substantiallydecreased in ErbB2-overexpressing breast cancer cell lines, suggesting thatp66Shc may function as an antagonist of p52/p46Shc, possibly acting as atumor suppressor (12). Inactivation of p66Shc in mice improves glucose toler-ance and insulin sensitivity (13, 14) and confers resistance to hyperglycemia-induced endothelial dysfunction (15). However, under nutrient stress conditions,mice lacking p66Shc are short-lived (16). These studies suggest that p66Shcmay suppress metabolism by dampening growth factor signaling.
Using targeted mass spectrometry (MS)–based metabolomics, we haveanalyzed the effects of p66Shc onmetabolic pathways.Our data suggest thatsilencing of p66Shc improves glucose uptake and redirects glucose carbontoward anabolic metabolism and increased cell size. In addition, we showthat the effects of p66Shc aremediated, in part, bymTOR complexes 1 and2 (mTORC1 and mTORC2, respectively). Our work reveals a role forp66Shc as an inhibitor of growth factor signaling and cell metabolism.
RESULTS
Loss of p66Shc enhances glycolytic metabolismTo elucidate the involvement of p66Shc in cellularmetabolism,weperformeda targeted metabolomic analysis using liquid chromatography–tandemMS (LC-MS/MS) in multiple reaction monitoring (MRM) mode. We mea-sured ~250 metabolites in positive- or negative-mode runs (table S1) andvalidated metabolite spectral patterns using standards. To assess the roleof p66Shc in cancer cell metabolism, we initially used HeLa cells that werestably transfectedwith either a short hairpin RNA (shRNA) that specificallytargets the isoform p66Shc or a control hairpin (Fig. 1B) (17). When weanalyzed the profiles of metabolites extracted from the two cell types, wenoted that loss of p66Shc resulted in increased abundance of intermediatesof glucose metabolism (Fig. 1C and fig. S1). Specifically, p66Shc deficien-cy was accompanied by significant increases in glucose-6-phosphate (G6P)and downstream glycolytic intermediates including fructose-6-phosphate
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(F6P), fructose-1,6-bisphosphate (F1,6BP), phosphoenolpyruvate (PEP),and pyruvate (Fig. 1D). p66Shc-deficient HeLa cells producedmore lactatethan did control cells, consistent with the Warburg effect. The metabolicshift also redirects glucose-derived citrate from the tricarboxylic acid cycleinto lipid synthesis to generate biomass (18). Consistent with this effect,p66Shc-deficient cells had higher citrate concentrations (Fig. 1D). Thesedata suggest that depletion of p66Shc is sufficient to enhance anabolic me-tabolism in HeLa cells.
Loss of p66Shc promotes glucose metabolism throughthe pentose phosphate and hexosaminebiosynthesis pathwaysIn addition to their role in glycolysis, G6P and F6P are precursors of thehexosamine biosynthesis and pentose phosphate pathways, which are es-sential anabolic pathways in proliferating cells. The hexosamine bio-synthesis pathway provides uridine diphosphate N-acetylglucosamine(UDP-GlcNAc) as a substrate for O-GlcNAcylation of cytosolic proteinsand N- and O-glycosylation of proteins produced in the secretory pathway.The pentose phosphate pathway provides ribose for nucleic acid synthesisandNADPH [reduced form of nicotinamide adenine dinucleotide phosphate(NADP+)] to maintain the reductive environment of the cell. In HeLa cells
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lacking p66Shc, we observed increases inthe abundance of N-acetylglucosamine-6-phosphate (GlcNAcP) and UDP-GlcNAc(nearly four- and twofold, respectively), themajor products of the hexosamine biosyn-thesis pathway (Fig. 1E). A two- to three-fold increase in UDP-GlcNAc enhancesN-glycosylation of growth factor recep-tors, thereby promoting positive feedbackto signaling (19, 20). The concentrations ofribose-5-phosphate (R5P) and xylulose-5-phosphate (X5P) were also increased, whichmay involve the oxidative pentose phosphatepathway, which contributes to redox balance(Fig. 1F).
Restoring p66Shc expression inp66Shc-deficient MEFs inhibitsglycolytic metabolismTo test whether p66Shc was necessary forenhanced glycolytic metabolism, we stablyinfected immortalized p66Shc knockout(KO) MEFs with either green fluorescentprotein (GFP) or 3xFLAG-p66Shc retroviralvectors (hereinafter referred to as KO andp66+ MEFs, respectively) (Fig. 2A). Princi-pal components analysiswasused to evaluatereplicate consistency, and revealed a notabledifference in the overall metabolic profiles ofthe p66Shc KO and p66+ cells (fig. S2).Consistentwith thep66Shc-mediated effectsin HeLa cells, F6P andG6Pwere among themetaboliteswith themost significant changes,and the abundance of glycolytic intermedi-ateswas generally lower in p66+MEFs com-pared to theKOcells (Table1).Theabundanceof glycolytic intermediates changed to a sim-ilar extent in MEFs as in HeLa cells; we ob-served a nearly threefold decrease in G6P
concentrations and a concomitant decrease in downstream three-carbonglycolytic metabolites including PEP and lactate in p66+ cells (Fig. 2B).
The lower concentration of citrate in p66+ cells was accompanied by aconcomitant decrease in the amounts of the fatty acid synthesis precur-sors acetyl–coenzymeA (CoA) andmalonyl-CoA (Fig. 2C).Malonyl-CoAis a high-energy and committed intermediate in fatty acid biosynthesisand is thus exclusively an anabolic metabolite. These results suggest thatp66Shc inhibits de novo lipid biosynthesis. In addition, the abundance ofintermediates in the hexosamine biosynthesis and pentose phosphatepathways was decreased in p66+ MEFs (Fig. 2, D and E). This profile con-firms that p66Shc deficiency in nontransformed cells enhanced glycolyticflux at the expense of decreasing oxidative mitochondrial metabolism.Indeed, p66Shc-deficient MEFs displayed lower oxygen consumption(Fig. 2F) but improved energy utilization [adenosine 5′-monophosphate(AMP)/adenosine 5′-triphosphate (ATP)] (Fig. 2G).
p66Shc expression inhibits amino acid biosynthesis andpyrimidine metabolismThe difference in glucose uptake between p66Shc-deficient and p66Shc-competent MEFs was ~33% at most. However, the difference in the abun-dance of glycolytic metabolites ranged between 1.5- and 3-fold. These results
Fig. 1. p66Shc deficiency enhances glycolytic metabolism and causes a Warburg shift. (A) Diagram of the
three isoformsofmammalianShc1. (B) Abundanceof the threeShc1 isoforms inHeLacells stably expressingshRNAs targeting GFP or p66Shc. n = 3 biological replicates. (C) Summary of the changes in intracellularmetabolite amounts associated with p66Shc knockdown in HeLa cells analyzed by LC-MS/MS. (D to F) Foldchange of glycolytic (D), hexosamine biosynthesis pathway (E), and pentose phosphate pathway (F) inter-mediates in p66Shc-deficient and p66Shc-competent HeLa cells as measured by LC-MS/MS. Error barsrepresent SD of three biological replicates (*P < 0.05, **P < 0.01, ***P < 0.001).February 2014 Vol 7 Issue 313 ra17 2
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suggested a contribution from other nutrients such as amino acids thatreversibly exchange with glycolytic intermediates. We detected higheramounts of nonessential amino acids, including alanine, serine, and as-partate, in p66Shc-deficient MEFs (Fig. 3A). To determine whether theeffect of p66Shc signaling on the steady-state abundance of nonessentialamino acids reflects regulation of metabolic flux through de novo syn-thesis, we measured the relative flux with a pulse of stable isotope–labeled
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15N-glutamine. Increased incorporation of labeled nitrogen into non-essential amino acids was detected in p66Shc KO MEFs (Fig. 3B). In-creased flux of nitrogen from extracellular glutamine into nonessentialamino acids is consistent with the reprogramming that favors anabolism.
Pyrimidine derivatives [dCTP (2′-deoxycytidine 5′-triphosphate) andUTP (uridine 5′-triphosphate)] were among the top 10 metabolites whoseabundancewas significantly decreased in p66+MEFs (Table 1). Pyrimidine
Fig. 2. Rescue of p66Shc expression in p66Shc KO MEFs (p66+ cells) hexosamine biosynthesis pathway intermediates (E) in p66Shc-deficient
inhibits glycolytic metabolism in favor of oxidative metabolism. (A) Abun-dance of the three Shc1 isoforms in p66Shc KO MEFs stably infected withGFP (KO) or FLAG-p66Shc (p66+). n = 3 biological replicates. (B to E)p66Shc expression inhibits glycolytic metabolism. Data represent foldchange of glycolytic intermediates (B), acetyl-CoA (ACoA) and malonyl-CoA (MCoA) (C), pentose phosphate pathway intermediates (D), and(white) and p66Shc-competent (black)MEFs asmeasured by LC-MS/MS.(F) p66Shc deficiency inhibits oxygen consumption rate as measuredby an XF-24 flux analyzer. (G) Relative AMP/ATP ratio in p66Shc KOand p66+ MEFs by LC-MS/MS. In all experiments, error bars repre-sent SD of at least three biological replicates (*P < 0.05, **P < 0.01,***P < 0.001).
Table 1. The 10 metabolites that showed the most statistically significant changes of p66+ over KO MEFs. n = 3 biological replicates.FDR, false discovery rate.
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FDR
Dihydroxyacetone phosphate (DHAP)
0.23 2.44 × 10−5 0.000244 Fructose-6-phosphate (F6P) 0.29 7.64 × 10−5 0.000270333 Glucose-6-phosphate (G6P) 0.36 8.11 × 10−5 0.000270333 Fructose-1,6-bisphosphate (F1,6BP) 0.29 0.000207 0.000452 Malonyl–coenzyme A (MCoA) 0.30 0.000226 0.000452 Erythrose-4-phosphate (E4P) 0.26 0.000598 0.000915714 NADH/NAD+ 0.34 0.000641 0.000915714 Acetyl coenzyme A (ACoA) 0.29 0.001544 0.00193 2′-Deoxycytidine 5′-triphosphate (dCTP) 0.41 0.00265 0.002944444 Uridine 5′-triphosphate (UTP) 0.45 0.003853 0.00385313 ra17 3
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nucleotides are required as high-energy donors for phospholipid and glyco-conjugate biosynthesis. p66+ MEFs showed lower amounts of the pyrimi-dine synthesis intermediates orotate and dihydroorotate as compared toKOMEFs (Fig. 3C). These results confirm that p66Shc inhibits anabolicmetabolic pathways, and demonstrate a prominent role for p66Shc in
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reprogramming cell metabolism toward glucose catabolism and oxida-tive respiration.
p66Shc regulates redox homeostasisBy associating with cytochrome c, p66Shc promotes the generation of re-active oxygen species (ROS) (10, 21). Consistent with these findings, weobserved a higher ratio of reduced to oxidized glutathione (GSH/GSSG)in p66Shc KO MEFs compared to p66+ MEFs (Fig. 3D). The ratio ofNADH/NAD+ also reflects the redox state of the cell (22), and a nearlythreefold increase in NADH/NAD+ indicates a more reducing environmentin p66Shc-deficient cells (Fig. 3E). NADH is produced in the second half ofglycolysis during the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–catalyzed step, and this may contribute to the altered NADH/NAD+ ratio.The higher GSH/GSSG ratio in KO MEFs is consistent with the reportedlower amounts of ROS in p66Shc KO MEFs (21). A previous report hasshown that ROS contributes to insulin resistance and decreases glucosemetabolism (23).
p66Shc is necessary and sufficient to alter glucoseuptake and metabolismTo directly test whether p66Shc alters glucose uptake, we measured 3H-labeled 2-deoxyglucose (2-DG) uptake at different time points. The rateof 2-DG uptake in p66Shc-deficient HeLa cells and p66Shc KO MEFswas greater than that in p66Shc-expressing cells (Fig. 4, A and B). Con-sistent with the enhanced glucose consumption and higher glycolytic me-tabolism in p66Shc-deficient cells, we observed increased concentrationsof extracellular lactate in themedia ofHeLa cells andMEFs lacking p66Shc(Fig. 4, C andD). However,Western blots showed no apparent difference inthe abundance of glucose transporter 1 (GLUT1) between p66Shc-competent and p66Shc-deficient cells (fig. S3).
To monitor the effect of p66Shc on glucose catabolism, we examinedthe metabolites of 13C-labeled glucose in control and p66Shc-deficientHeLa cells. The metabolism of [1,2-13C2]glucose generates M0, M1, andM2 mass isotopomers corresponding to ion fragments that contain zero,one, or two labeled carbons, respectively (table S2). The rapid transfer of cellsfrom unlabeledmedium to identicalmedium containing [1,2-13C2]glucoseensures that metabolism is minimally disturbed (24). Consistent withenhanced glycolytic metabolism in p66Shc-depleted cells, the amounts ofthe M2-labeled form of G6P and downstream intermediates including M2F6P andM2 pyruvatewere increased in p66Shc-depletedHeLa cells (Fig. 4,F and G). p66Shc silencing enhanced the amount of labeled glucose-derivedcitrate and acetyl-CoA nearly twofold, consistent with the redirection ofglucose-derived carbons toward lipid biosynthesis. Conversely, reexpres-sing p66Shc in p66ShcKOMEFs inhibited the increase in the abundance of[1,2-13C2]–labeled glycolytic metabolites and downstream anabolic inter-mediates, including acetyl-CoA and citrate, in p66Shc-deficient cells (Fig. 4H).Additionally, p66Shc inhibited the de novo synthesis of amino acids (fig. S4).Collectively, our data suggest that p66Shc is necessary and sufficient to re-program glucose utilization into anabolic pathways.
Lack of p66Shc enhances glycolytic flux andanabolic metabolismTo study the dynamics of incorporation of isotope-labeled glucose intodownstream anabolic metabolites, we performed kinetics flux profilingin p66Shc KO and p66+ MEFs, using [1,2-13C2]glucose, by measuringthe relative amounts of labeled glycolytic intermediates over six time points(0, 1, 2.5, 5, 10, and 15min). Consistentwith the steady-state data,we foundthat p66Shc inhibited labeled glucose flux intoglycolytic intermediates suchas pyruvate in p66+ MEFs (Fig. 5A). In addition, the amounts of labeledcitrate, UDP-GlcNAc, and R5P were lower in cells expressing p66Shc
Fig. 3. p66Shc expression inhibits the biosynthesis of amino acids and
pyrimidines and regulates redox homeostasis. (A) Fold change of steady-state alanine, serine, and aspartate, measured by LC-MS/MS. (B) Foldchange of intracellular amounts of 15N-labeled alanine, serine, and aspar-tate in p66Shc KO and p66+ cells grown in media containing 15N-glutaminefor 2 hours. (C) Fold change of de novo pyrimidine synthesis intermediates,N-carbamoyl aspartate, orotate, and uridine 5′-monophosphate (UMP).(D and E) Relative GSH/GSSG ratio (D) and reduced/oxidized NAD+(NADH/NAD+) (E) in p66Shc KO and p66+ MEFs. In all experiments, er-ror bars represent SD of at least three biological replicates (*P < 0.05,***P < 0.001).
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(Fig. 5, B to D). UDP-GlcNAc is a high-energy donor required for protein glyco-sylation and an example of an anabolicmetabolite in which the GlcNAc portionis not catabolized back to glucose (20).Overall, these results suggest that increasedglycolytic flux in p66Shc-deficient cellsaccounts for the observed increase in me-tabolites at steady state, consistent with arole for p66Shc as an inhibitor of glucosecatabolism.
p66Shc inhibits growth factorsignaling to the metabolicsensor mTOR
Several aspects of the metabolic profile of p66Shc KO MEFs resemblethose of cells with chronic mTOR activation; these include enhancedamounts of glycolytic and pentose phosphate pathway intermediates(G6P, F6P, and R5P) (25), increased amounts of de novo pyrimidine me-tabolites (26, 27), and increased lipid biosynthesis precursors (acetyl-CoA and malonyl-CoA) (25). To monitor mTOR signaling, we examinedphosphorylation of Thr389 in the ribosomal protein S6 kinase (S6K1), asubstrate of mTORC1, and of Thr473 in Akt, which is a direct target ofmTORC2 (28). After serum stimulation, p66Shc-deficient HeLa cellsdisplayed markedly increased phosphorylation of both mTORC1 andwww
mTORC2 targets compared to control cells, despite having equal abun-dance of the p52Shc and p46Shc isoforms (Fig. 6A). Similar results wereobtained with insulin stimulation (Fig. 6B). Pretreatment of cells withthemTOR inhibitor Torin1 (29) abolished the phosphorylation of mTORtargets in both cell types, confirming the specificity of the signal (Fig. 6,A andB).We also found that amino acid activation of themTORC1 path-way was enhanced in p66Shc-deficient HeLa cells (fig. S5A). In p66+
MEFs, p66Shc inhibited the activation of mTOR targets after stimulationwith insulin-like growth factor 1 (IGF-1) and insulin, but notwith epidermalgrowth factor (EGF) (Fig. 6C). This observation is consistent with previous
Fig. 4. p66Shc is necessary and sufficient to alter glucose uptake and gly-colytic metabolism. (A and B) Tracing of 3H-labeled 2-DG uptake over
with [1,2-13C2]glucose as a tracer. Blue circles indicate 13C-labeled car-bons. (F and G) Fold change of 13C-labeled glycolytic (F) and hexosamine
time in p66Shc-competent and p66Shc-deficient HeLa cells stably ex-pressing shRNAs targeting GFP (A) or p66Shc and p66Shc KO and p66+
MEFs (B). (C and D) Fold change of extracellular lactate in media fromp66Shc-competent and p66Shc-deficient HeLa cells (C) and MEFs (D).(E) Schematic diagram of 13C labeling patterns of metabolic products
biosynthesis pathway (G) intermediates in p66Shc-competent (white) andp66Shc-deficient (black) HeLa cells. (H) 13C-labeled glycolytic intermedi-ates in p66Shc KO (white) and p66+ (black) MEFs. Error bars representSD of at least three independently prepared samples (*P < 0.05, **P <0.01, ***P < 0.001).
Fig. 5. Glucose flux in p66Shc KO and p66+ MEFs. (A to D) Relative amounts of 13C-labeled pyruvate(A), citrate (B), UDP-GlcNAc (C) and R5P (D) after incubation of cells with [1,2-13C2]glucose for theindicated times. Data are represented as the ratio of 13C-labeled metabolites to an internal standard(D7-glucose). Error bars represent SD of at least three independently prepared samples (*P < 0.05).
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reports showing that p66Shc has little effect on EGF signaling (9). The phos-phorylation of mTORC1 andmTORC2 targets was decreased in p66+MEFsafter serum (Fig. 6D) and amino acid stimulation (fig. S5B). In contrast tothe inhibitory effects of p66Shc expression onmTOR signaling inKOMEFs,mTOR activation was sustained in KO MEFs stably overexpressing thep52Shc isoform (Fig. 6E). This confirms earlier reports that the p66Shcand p52Shc isoforms have opposing effects attributable to the uniqueN-terminal CH2 region of p66Shc (9, 30).
Loss of p66Shc causes an increase in cell sizemTOR functions as a central regulator of cell growth (31). The inhibitoryeffect of p66Shc on mTOR activation therefore predicted that p66Shc defi-ciencywould lead to a larger cell size. Indeed, stable reexpression of p66Shcin p66Shc KO MEFs caused a decrease in cell size as measured by FACS(fluorescence-activated cell sorting) forward scatter analysis (Fig. 6F).These findings were confirmed using Coulter counter measurement (fig.S6A). Conversely, HeLa cells depleted of p66Shc displayed an increasein median cell size, consistent with redirection of glucose-derived carbontoward biomass synthesis (fig. S6B). Together, these results suggest thatp66Shc acts as a negative regulator of the nutrient-sensing mTOR signalingpathway, leading to inhibition of cell growth and anabolic metabolism.
The effects of p66Shc on glycolytic metabolism aremediated through mTORTo determine which mTOR complex contributes to the metabolic pheno-type observed in p66Shc null cells, we treated p66Shc KO and p66+ MEFs
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with rapamycin for 16 hours, a time frame that inhibits both mTORC1 andmTORC2 enzymes (Fig. 7A). In line with reports linking mTORC1 andmTORC2 to glycolytic metabolism (25, 32, 33), we found that inhibitionof both mTOR complexes partly reversed the metabolic phenotype ofp66Shc KO MEFs, notably diminishing the increase in the hexosaminebiosynthesis pathway (UDP-GlcNAc) and the pentose phosphate pathway(R5P) (Fig. 7B). A similar trend was observed in de novo pyrimidinesynthesis (Fig. 7C). Furthermore, inhibition of Akt, a kinase that isupstream of mTORC1 and downstream of mTORC2 (28), significantlydecreased the amounts of glycolytic metabolites in p66Shc-deficientMEFs (fig. S7).
p66Shc alters gene expressionTo understand how p66Shc signals to mTORC1 and mTORC2, we at-tempted to find unique p66Shc binding partners by MS. However, allproteins that bound to p66Shc in our experiments have also been re-ported to bind to p52Shc after serum stimulation (table S4) (34). Thesteady-state condition of p66Shc KO and p66+ MEFs is likely to includedifferences in gene expression. To address this question,we performedRNAsequencing (RNA-seq) analysis comparing the expression profiles ofp66Shc KO and p66+ MEFs. Our data showed significant differences in theexpression of about 400 genes but not for genes encoding glycolytic enzymes,including hexokinase and phosphofructokinase, or those encoding glucosetransporters (table S3). This suggests that changes in steady-state abundanceof p66Shc regulate metabolism largely through signaling and post-translational modification.
Fig. 6. p66Shc inhibits mTOR activation and cell size. (A) Effect of p66Shc onmTORC1-S6K1 and mTORC2-Akt activation. HeLa cells stably transfectedwith indicated shRNAs were serum-starved, treated with either dimethylsulfoxide or Torin for 1 hour, and then stimulated with serum (10 min). Celllysates were analyzed for the indicated proteins and phosphorylationstates by immunoblotting. n = 3 biological replicates. (B) Experimentwas done as in (A), but cells were stimulated with 100 nM insulin (10 min).n = 3 biological replicates. (C) Effect of reconstitution of p66Shc ex-pression in p66Shc KO MEFs on mTOR activation in response to EGF,
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insulin, and IGF-1 stimulation. Serum-starved cells were stimulated with100 nM EGF, IGF, or insulin (10 min). n = 3 biological replicates. (D)Experiment was done as in (C), but cells were stimulated with serum.n = 3 biological replicates. (E) Stable expression of retroviral FLAG-p66Shc, but not FLAG-p52Shc, in p66Shc null cells inhibits the mTORpathway. Experiment was done as in (C). n = 3 biological replicates. (F)Cell size distribution of KO and p66+ MEFs using flow cytometry forwardscattering (right). The average cell size of KO and p66+ MEFs from threeindependent experiments is represented (left). ***P < 0.001.
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DISCUSSION
Our results provide evidence that p66Shc expression is associated withdampened signaling response to insulin stimulation and reduced glycolytic
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metabolism. Hyperglycemia has been pre-viously shown to induce p66Shc expres-sion (35). Our data suggest that p66Shcmediates feedback inhibition of growthsignaling and glucose metabolism incells (Fig. 7D). For metabolites that areintermediates in both catabolic and ana-bolic directions, the delivery of 13C-labeledglucose reveals competing delivery fromcold sources including storage products,autophagy, and uptake of other nutrients.For central metabolites such as pyruvate,citrate, R5P, and UDP-GlcNAc, our fluxanalysis showed faster 13C incorporationand an increase to a higher steady state inp66Shc-deficient cells, indicating a largercontribution from13C-glucose than the non-radioactive sources in cells lacking p66Shc.
Furthermore, p66Shc deficiency inmiceimproves glucose tolerance and insulinsensitivity (13, 14). p66Shc silencing inboth transformed and nontransformed im-mortalized cells improves glucose uptakeand increases lactate production, metabo-lite abundance for fatty acid biosynthesis,the hexosamine pathway, and the pentosephosphate pathway, and amino acids, con-tributing to increased cell size. This meta-bolic shift depends in part on the mTORpathway because rapamycin treatment re-
versed the glycolytic shift caused by p66Shc loss (Fig. 4D). These effectsare particularly striking because the p52Shc isoform is identical to p66Shcin all but the N-terminal CH2 region, and has the opposing function of pro-moting the activation of the Ras-ERKand PI3K-Akt pathways. p66Shc acts
FoRtoinwp3M1apwiac5litthssad**
ig. 7. mTOR mediates the effects of p66Shcn glycolytic and pyrimidine metabolism. (A)apamycin treatment for 16 hours is sufficientinhibit both mTORC1 and mTORC2 signal-g in p66Shc KO and p66+ MEFs. Cell lysatesere analyzed for the indicated proteins andhosphorylation states by immunoblotting. n =biological replicates. (B) p66Shc KO and p66+
EFswere treatedwith vehicle or rapamycin for6 hours, and the amounts of glycolytic, hexos-mine biosynthesis pathway, and pentosehosphate pathway metabolic intermediatesere quantified by LC-MS/MS [analysis of var-nce (ANOVA) analysis, n = 3 biological repli-ates]. (C) Fold change of UMP and cytidine′-monophosphate (CMP) pyrimidinemetabo-es. Cells were treated as in (B). (D) Model fore role of p66Shc in inhibiting growth factorignaling to glycolytic metabolism and bio-ynthetic pathways through inhibition of mTORctivation. Error bars represent SDof three in-ependently prepared samples (*P < 0.05,P < 0.01, ***P < 0.001).
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as an inhibitor of mTORC1 and mTORC2 signaling, pathways that sup-port protein synthesis associated with increased cell size. mTOR inhibitionreduced anabolic metabolites, partially restoring the p66Shc-competentphenotype, suggesting that mTOR-independent pathways also contributeto the p66Shc-deficient phenotype. p66Shc expression has been reportedto be inversely correlated to tumorigenesis (12), which is consistent withour finding that p66Shc loss enhances mTOR signaling and anabolic metab-olism. It is possible that p66Shc competes with p52Shc for binding partnersin a dynamic manner that disrupts downstream signaling to mTOR. Timecourses for receptor recruitment of signaling complexes may reveal themolecular basis for p66Shc action on signaling and anabolic metabolism.
RNA-seq analysis showed no differences in the expression of genes en-coding metabolic enzymes and glucose transporters between p66Shc-competent and p66Shc-deficient cells. Transcripts showing a change in abun-dance were clustered in the category of secretory proteins, and additionalstudies are required to assess their contribution to the p66Shc phenotype.Rapid forms of regulation such as allostery and posttranslational modifica-tions to metabolic enzymes and transporters may account for much of theobserved p66Shc-dependent metabolic reprogramming. However, on a lon-ger time scale, the stable cell lines used in our experiments have adapted top66Shc expression or loss, and the attendant changes in signaling and me-tabolism fluxmay be reinforced by some of the changes in gene expression.
In summary, our results indicate that the Shc1 locus not only regulates mito-genic signaling but also modifies anabolic metabolism in an mTOR-dependentmanner. Our data point to the prospect that monitoring or modulating p66Shcabundance could be important in the management of pathological conditionsin which metabolic signaling is dysregulated, such as cancer and diabetes.
MATERIALS AND METHODS
Reagentswere obtained from the following sources: antibodies to phospho-Thr389
S6K1, phosho-Thr308 Akt, phospho-Ser473 Akt, Akt, and S6K1 from CellSignalingTechnology;mouseand rabbitShc1antibodies fromBDTransductionLaboratories; FLAG and tubulin antibodies, amino acids, insulin, IGF-1, andEGF from Sigma-Aldrich; FuGENE 6 and Complete Protease Cocktail fromRoche; rapamycin from LC Laboratories; Dulbecco’s modified Eagle’s medium(DMEM) fromGibco; inactivated fetal calf serum (IFS) fromHyClone; aminoacid–free RPMI from US Biological; bicinchoninic acid assay reagent andproteinG–Sepharose fromPierce; andAkt Inhibitor IV fromEMD4Biosciences.
Cell lines and tissue cultureHeLa cells andMEFswere cultured inDMEMwith 10% IFS and penicillin/streptomycin. p66Shc KO MEFs were provided by P. G. Pelicci (IFOM).3xFLAG-p66Shc–expressing MEFs were generated by retroviral infectionof gateway-compatible pMX vector with murine p66Shc complementaryDNA (cDNA). Stably infected cells were maintained under puromycin(1 µg/ml) selection. For stable p66Shc knockdown in HeLa cells, we usedthe following hairpin sequence: sense, 5′-cggaatgagtctctgtcatcgct(tt)-3′;antisense, 5′-agcgatgacagagactcattccg(tt)-3′.
Amino acid stimulationAlmost confluent cultures in six-well culture plates were rinsed once withamino acid–free RPMI, incubated in amino acid–free RPMI for 50min, andstimulated with leucine (52 mg/ml) for 10 min. For amino acid starvation,cells in 10-cm culture dishes or coated glass coverslips were rinsedwith andincubated in amino acid–free RPMI for 50 min and stimulated with a 10×amino acidmixture for 10min as indicated in the figure legends. After stim-ulation, the final concentration of amino acids in the mediawas the same asthat in RPMI. The 10× amino acid mixture was prepared from individualamino acid powders.
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Cell lysis and Western blotsCells were rinsed once with ice-cold phosphate-buffered saline (PBS) andlysed in ice-cold lysis buffer [40mMHepes (pH 7.4), 2mMEDTA, 10mMpyrophosphate, 10 mM glycerophosphate, 0.5% NP-40, and one tablet ofEDTA-free protease inhibitors (Roche) per 10 ml]. The soluble fractions ofcell lysateswere isolated by centrifugation at 13,000 rpm for 10min by cen-trifugation in a microfuge.
Proteins were denatured by the addition of 2× sample buffer and boilingfor 5 min, resolved by 8% SDS–polyacrylamide gel electrophoresis, andanalyzed by immunoblotting for the indicated proteins in the figures.
Metabolomic profilingThe relative abundances of metabolites were determined in HeLa cells andMEFs using the following protocol (36): Cells were seeded in six-wellplates in replicates. After 24 hours, the media were removed; the cells werewashed two times with warm PBS and were placed on dry ice. The metab-olites were immediately extracted by adding 1 ml of extraction solution(40% acetonitrile, 40% methanol, and 20% water) containing internalstandards [D7-glucose and 13C915N-tyrosine (500 and 300 µg/ml, respec-tively)], and then the cells were scraped and collected in 1.5-ml vials. Themixture was shaken for 1 hour at 4°C and 1400 rpm in a Thermomixer(Eppendorf). The samples were spun down at 14,000 rpm for 10 min at4°C (Eppendorf), and supernatants were transferred into fresh tubes andevaporated to dryness in a CentreVap concentrator at 40°C (Labconco).The dry extract samples were stored at −80°C until LC/MS analysis. The drymetabolite extracts were reconstituted in 100 µl of water. For positive- andnegative-mode analysis, the sample was injected twice through a reversed-phase column (Inertsil ODS-3, 4.6-mm internal diameter, 150-mm length, and3-µmparticle size;DionexCorporation). Inpositive-modeanalysis, the mobile-phase gradient ramped from 5 to 90% of acetonitrile in 16 min; after 1 minat 90%, the composition returned to 5% acetonitrile in 0.1% acetic acidin 2 min. In negative mode, the acetonitrile composition ramped from 5 to90% in 10 min; after 1 min at 90%, the gradient returned to 5% acetonitrilein buffer A (0.1% tributylamine, 0.03% acetic acid, 15% methanol). Thetotal run time in both modes was 20 min, the samples were stored at 4°C,and the injection volume was 10 µl. An automated washing procedure wasdeveloped before and after each sample to avoid any sample carryover.
The eluted metabolites were analyzed at the optimum polarity in MRMmode on an electrospray ionization triple-quadrupole mass spectrometer(ABSciex4000Qtrap). The MS data acquisition time for each run was20 min, and the dwell time for each MRM channel was 10 ms. CommonMS parameters were the same as the tuning conditions described above,except GS1 and GS2 were 50 psi; CURwas 20 psi; CADwas 3 and 7 forpositive and negative modes, respectively; and source temperature (TEM)was 400°C. Signal was normalized to internal standard and cell number.MetaboAnalystwasused toanalyze thedata (37).TheLC-MS/MSsystemdoesnot resolve hexose and hexosamine isomers including glucose/galactose andGlcNAc/GalNAc (N-acetylgalactosamine). To monitor trends in metabolicpathways, we referred to these isomers in their glucose (Glc) forms.
StatisticsUnpaired two-tailed Student’s t tests were performed for comparing twogroups. For multiple comparison analysis, one-way ANOVA followed byBonferroni’s post hoc test was used. All experiments represent at least threebiological replicates.
2-Deoxy-D-[3H]glucose uptake assayMEFs and HeLa cells grown in six-well plates were rinsed with Hepes-buffered saline [140 mMNaCl, 20 mMHepes, 5 mMKCl, 2.5 mMMgSO4,1 mMCaCl2 (pH 7.4)]. 2-DG uptakewas performed for 3, 5, and 10min in
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Hepes-buffered saline containing 2mMunlabeled 2-DG and 3H-labeled 2-DG(0.2 mCi/ml) at room temperature. The reaction was terminated by washingthree times in ice-cold 0.9% NaCl (w/v). To quantify the radioactivity incor-porated, cells were lysed with 0.05 N NaOH, and lysates were counted withscintillation fluid in ab-counter.Nonspecificuptakewasdetermined in thepres-ence of cytochalasin B (10 mM) during the assay. The results are expressed aspicomoles of 2-DG transported per milligram of protein per minute.
Oxygen consumption rate measurementCells were seeded at 50,000 cells per well in XF-24 cell culture plates. Theday after the seeding, cell culture medium was replaced with unbufferedRPMI, and oxygen consumption was measured using an XF-24 flux ana-lyzer as described in (38).
Isotope labeling and kinetic profilingMedia of cell cultures in six-well dishes were replaced with unlabeled freshDMEM 2 hours before exposure to labeled glucose. The media were thenreplaced with DMEM containing 25 mM [1,2-13C2]glucose or
15N-labeledglutamine for the times indicated in the figures legends (Cambridge IsotopeLaboratories). Metabolites were extracted and analyzed as mentioned in the“Metabolomic profiling” section using differentMRM transitions that weredeveloped for [1,2-13C2]glucose as detailed in table S1.
MS analysis of the p66Shc protein interactionsThe p66Shc protein interaction network was determined following a protocoldescribed in (34). p66+MEFs were washed three times with ice-cold PBS andlysed in NP-40 lysis buffer [50 mM Hepes-NaOH (pH 8), 150 mM NaCl,1 mM EGTA, 0.5% NP-40, 100 mMNaF, 2.5 mMMgCl2, 10 mMNa4P2O7,1mMdithiothreitol, 10%glycerol] supplementedwith protease andphospha-tase inhibitors [50 mM b-glycerophosphate, aprotinin (10 µg/ml), leupeptin(10 µg/ml), 1 mM Na3VO4, 100 nM calyculin A, 1 mM phenylmethylsulfonylfluoride].The total cell lysateswere centrifugedat 10,000 rpmfor 15min topelletnuclei and insoluble material. Nuclear-free lysates were precleared by 1-hourincubation with protein A–Sepharose and normalized for total protein con-centration using the Bio-Rad protein assay. 3xFLAG-p66Shc was immuno-precipitated by incubating lysates with 5 µl (bed volume) of anti-FLAGM2antibody–conjugated agarose for 4 hours at 4°C.
RNA sequencingcDNA library preparation was done using the Illumina TruSeq RNA Sam-ple Prep Kit v2 (cat. no. RS-122-2001). Briefly, 1 µg of high-quality totalRNAwas isolated from p66Shc KO and p66+ MEFs to generate the cDNAlibrary according to the manufacturer’s protocol. The generated bar-codedcDNA library had an average fragment size of 350 to 400 base pairs. Thisbar-coded library was quality-checked with the Agilent Bioanalyzer andquantified by quantitative polymerase chain reaction (qPCR) with KAPASYBR FAST Universal 2x qPCR Master Mix (Kapa Biosystems, cat.no. KK4602). The quality-checked libraries were then loaded on a flowcell for cluster generation using Illumina cBot andTruSeq PEClusterKit v3(cat. no. PE-401-3001). Sequencing was done on HiSeq2000 with TruSeqSBS Kit v3 (paired-end, 200 cycles, cat. no. FC401-3001). The real-timebase call (.bcl) files were converted to FASTQ files using CASAVA 1.8.2(on CentOS 6.0 data storage and computation Linux servers).
SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/7/313/ra17/DC1Fig. S1. A volcano plot for the metabolomic analysis of HeLa cells stably expressingshRNA targeting GFP or p66Shc.Fig. S2. Unsupervised principal components analysis for targeted metabolomic analysis ofp66Shc KO and p66+ MEFs.
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Fig. S3. The abundance of GLUT1 in p66Shc-competent and p66Shc-deficient HeLa cellsand MEFs is similar.Fig. S4. p66Shc inhibits de novo synthesis of nonessential amino acids.Fig. S5. Effect of amino acid deprivation on serum-mediated activation of mTORC1 inp66Shc-deficient cells.Fig. S6. Effect of p66Shc expression on cell size.Fig. S7. Effect of Akt inhibition on the amounts of glycolytic metabolites in p66Shc-competent and p66Shc-deficient MEFs.Table S1. LC-MS/MS transitions for the metabolites measured in this study (Excel file).Table S2. LC-MS/MS transitions for [1,2-13C2]glucose intermediates.Table S3. List of genes showing differential regulation upon p66Shc expression.Table S4. List of identified p66Shc-interacting proteins.
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Acknowledgments: This work is dedicated to the memory of the late Dr. Tony Pawsonwho passed away during the revision process.We are indebted to P. G. Pelicci andG. Cortopassifor providing p66Shc null and control MEFs and to K. Chan and C. Y. Ho for performing theRNA-seq experiments. We thank M. Stacey and K. Riabowol for critical reading of the man-uscript, as well as J. Gish and J. Woodgett for their encouragement and insight. Funding:This research was supported by grants from Genome Canada through the Ontario GenomicsInstitute, and funding from the Ontario Research Fund Global Leadership Round in Genomics& Life Sciences (ORF-GL2) and the Canadian Cancer Society to T.P. and J.W.D. J.W.D. isalso supported by grants from Canadian Institutes of Health Research (MOP-62975), theSydney C. Cooper Program, and the Canada Research Chairs Program. I.G.F. is supportedby the Canadian Institute of Health Research (MOP-49409). D.M.S. is supported by grantsfrom the NIH and Department of Defense and awards from the Keck and LAM Foundation.A.M.A.R. was supported by MITACS-accelerate. M.E.F. is supported by the National Scienceand Technology Council, CONACYT, Mexico. D.W.L. is supported by the Ruth KirschsteinNational Research Service and NIH K99 Awards. D.M.S. is an investigator of the HowardHughes Medical Institute. M.A.S. is supported by a Vanier Canada Graduate Scholarshipand an Ontario Graduate Scholarship. Author contributions: M.A.S. initiated the project,devised and conducted experiments, interpreted results, and wrote the manuscript. A.M.A.R.and J.P. performed the metabolomics experiments and analysis. A.P. performed the statisticalanalysis. D.W.L. assisted with project design. K.B. performed the oxygen consumption rateexperiment. Y.Z. conducted and interpreted the proteomics analysis. M.E.F. and H.L. con-ducted the 2DG experiment. I.G.F. and D.M.S. assisted with experiment design and re-sources. J.W.D. and T.P. supervised the project, edited the manuscript, provided fundingresources, and directed the project. Competing interests: The authors declare that theyhave no competing interests. Data and materials availability: The metabolomics and MSdata have been deposited at ftp://PASS00385:[email protected]/.
Submitted 3 October 2013Accepted 23 January 2014Final Publication 18 February 201410.1126/scisignal.2004785Citation: M. A. Soliman, A.M. Abdel Rahman, D.W. Lamming, K. Birsoy, J. Pawling, M. E. Frigolet,H. Lu, I. G. Fantus, A. Pasculescu, Y. Zheng, D. M. Sabatini, J. W. Dennis, T. Pawson, Theadaptor protein p66Shc inhibits mTOR-dependent anabolic metabolism. Sci. Signal. 7,ra17 (2014).
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