RapidTurnoverofthemTORComplex1(mTORC1)Repressor ... · 2008-01-25 · (PRAS)40.Incontrast,mTORC2containsG L,rapamycin-in-sensitivecompanion of mTOR (rictor) ... histidinol (Sigma-Aldrich)

Rapid Turnover of the mTOR Complex 1 (mTORC1) RepressorREDD1 and Activation of mTORC1 Signaling followingInhibition of Protein Synthesis*

Received for publication, August 9, 2007, and in revised form, November 14, 2007 Published, JBC Papers in Press, December 10, 2007, DOI 10.1074/jbc.M706643200

Scot R. Kimball‡1,2, A. N. Dang Do‡1, Lydia Kutzler‡, Douglas R. Cavener§, and Leonard S. Jefferson‡

From the ‡Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine,Hershey, Pennsylvania 17033 and the §Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

mTORC1 is a complex of proteins that includes the mamma-lian target of rapamycin (mTOR) and several regulatory pro-teins. It is activated by a variety of hormones (e.g. insulin) andnutrients (e.g. amino acids) that act to stimulate cell growth andproliferation and repressed by hormones (e.g. glucocorticoids)that act to reduce cell growth. Curiously, mTORC1 signaling isreported to be rapidly (e.g. within 1–2 h) activated by inhibitorsof protein synthesis that act on either mRNA translation elon-gation or gene transcription. However, the basis for themTORC1 activation has not been satisfactorily delineated. Inthe present study,mTORC1 signalingwas found to be activated inresponse to inhibition of either the initiation or elongation phasesof mRNA translation. Changes in mTORC1 signaling wereinversely proportional to alterations in the expression of themTORC1 repressor, REDD1, but not the expression of TRB3 orTSC2.Moreover the cycloheximide-induced increase inmTORC1signaling was significantly attenuated in cells lacking REDD1,showing thatREDD1plays an integral role in the response. Finally,the half-life of REDD1 was estimated to be 5 min or less. Overall,the results are consistent with a model in which inhibition of pro-tein synthesis leads to a loss of REDD1 protein because of its rapiddegradation, and in part reduced REDD1 expression subsequentlyleads to de-repression ofmTORC1 activity.

The mammalian target of rapamycin (mTOR),3 a Ser/Thrprotein kinase, is an important regulator of cell growth (1).mTOR exists in two distinct signaling complexes referred to asmTOR complex (mTORC)1 and mTORC2 (2). mTORC1 con-tains G-protein �-subunit-like protein (G�L), the regulatoryassociated protein of mTOR (raptor), the Ras homologenriched in brain (Rheb), and proline-rich Akt substrate(PRAS)40. In contrast, mTORC2 contains G�L, rapamycin-in-sensitive companion of mTOR (rictor), and hSIN1. Repression

of mTORC1 signaling using the selective inhibitor rapamycinnot only leads to a reduction in the size of cells in culture (3) butalso prevents cardiac hypertrophy associated with pressureoverload (4–6), resistance exercise-induced skeletal musclehypertrophy (7–9), and regrowth of the liver after partial hep-atectomy (10). In contrast, constitutive activation of mTORC1can lead to uncontrolled cell growth and cancer (11). mTORC1signaling is activated in response to growth-promoting hor-mones such as insulin (12), IGF-1 (13), or EGF (14). The signal-ing pathways through which these hormones act to increasemTOR signaling (e.g. the phosphatidylinositide (PI) 3-kinaseand extracellular-regulated protein kinase (ERK) pathways)converge on a GTPase activator protein referred to as tuberoussclerosis complex (TSC)2 (Tuberin) (15). TSC2, in a complexwith TSC1 (Hamartin), promotes the GTPase activity of the rashomolog enriched in brain (Rheb). Rheb binds directly tomTOR, and when present as a Rheb�GTP complex, activatesmTOR. Conversely, the binding of Rheb�GDP to mTOR isinhibitory. By activating the GTPase activity of Rheb, TSC2causes a redistribution of Rheb from the stimulatory Rheb�GTPcomplex into the inhibitory GDP-bound form. mTORC1 sig-naling is also activated by nutrients, particularly amino acids(16). Amino acids may also act through Rheb to activatemTORC1 (17–19); however, the mechanism through whichthey do so appears to be unrelated to TSC2 (19, 20). In contrastto the activating effect of insulin/IGF-1 and amino acids, cata-bolic hormones such as glucocorticoids (21–23) and pro-in-flammatory cytokines (24) and conditions that reduce the ATP:AMP ratio (25) repress mTORC1 activity. For example,glucocorticoids act rapidly (i.e. within 4 h) to upregulate theexpression of the mTORC1 repressor, regulated in develop-ment and DNA damage responses (REDD1) (23). IncreasedREDD1 expression promotes the assembly of the activeTSC1�TSC2 complex, leading to decreasedmTORC1 signaling.Reports in the literature suggest that mTORC1 signaling is

up-regulated following the inhibition of protein synthesis (26–31); however, a satisfactory explanation for this observation hasnot been forthcoming in regard to the regulators described inthe preceding paragraph. In most cases (26–30), inhibitors ofthe elongation phase of mRNA translation have been used torepress protein synthesis, and one report (31) suggests thataccumulation of intracellular amino acids under these condi-tions might be responsible for the observed activation ofmTORC1 signaling. Another possibility is that the activationof mTORC1 is mediated specifically through the inhibition of

* This study was supported in part by Grant DK-13499 from the National Insti-tutes of Health and a grant from the Pennsylvania Department of Healthusing Tobacco Settlement Funds. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Cellular and

Molecular Physiology, H166, The Pennsylvania State University College ofMedicine, 500 University Drive, Hershey, PA 17033. Tel.: 717-531-8970; Fax:717-531-7667; E-mail: [email protected].

3 The abbreviations used are: mTOR, mammalian target of rapamycin; MEF,mouse embryo fibroblast; Rheb, Ras homolog enriched in brain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 6, pp. 3465–3475, February 8, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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elongation, perhaps in amanner analogous to the generation ofthe signaling molecule ppGpp in bacteria (32). To date, therehave been no reports to indicate whether or not inhibitors ofthe initiation phase of mRNA translation might produce a sim-ilar activation of mTORC1 signaling. Another condition underwhich activation ofmTORC1 signaling occurs is following inhi-bition of gene transcription with actinomycin D treatment (30,33). In this case, the activation of mTORC1 signaling occursprior to detectable inhibition of global rates of protein synthe-sis, so it is unlikely that the effect is due to an accumulation ofintracellular amino acids. A potential explanation for theobserved activation of mTORC1 signaling under all of theseconditions is that inhibitors of protein synthesis acting eitheron the elongation or initiation phases of mRNA translation, orgene transcription, cause the rapid loss of a specific protein thatacts to repress mTORC1. Such a protein would have to turnoverrapidly because activation of mTORC1 signaling is observed

within 1–2 h of inhibiting the elonga-tion phase of mRNA translation (30).In the present study,mTORC1 sig-

naling was shown to be activatedwhen eithermRNA translation initia-tion or elongation was inhibited.Moreover, activation of mTORC1signaling was proportional to theextent of inhibition of protein synthe-sis. A search for a potential upstreamrepressor of mTORC1 whose turn-over was rapidly increased upon inhi-bition of mRNA translation revealedthat REDD1, but not TSC2 or TRB3,expression was directly proportionalto changes in protein synthesis andinversely proportional to alterationsin mTORC1 signaling. In addition,the half-life of REDD1 was esti-mated to be �5 min. Together withthe observation that induction ofmTORC1 signaling by cyclohexi-mide is dramatically attenuated inREDD1�/� mouse embryo fibro-blasts (MEFs), the results presentedherein are consistent with a modelin which inhibitors of protein syn-thesis acting on either the initiationor elongation phase ofmRNA trans-lation, or gene transcription, cause arapid reduction in REDD1 proteinexpression, resulting in activation ofmTORC1 activity.

EXPERIMENTAL PROCEDURES

Cell Culture—Wild type(GCN2�/�) and GCN2�/� MEFs(kindly provided by Drs. David Ronand Heather Harding, NY UniversitySchool ofMedicine)weremaintainedat 37 °C in high glucose Dulbecco’s

modifiedEagle’smedium (Invitrogen) containing 10% fetal bovineserum (Atlas Biologicals) and 1%penicillin-streptomycin (Invitro-gen). Wild type (REDD1�/�) and REDD1�/� MEFs (kindly pro-vided by Dr. Leif Ellisen, Harvard Medical School) were similarlymaintained. In all experiments, cells were �50–70% confluent.On the day of the study, cells were randomly divided into fivegroups. One group of cells (control) was placed in serum-freemedium. Two other groups of cells were placed in serum-freemedium containing either 1 �M cycloheximide (A. G. Scientific,Inc.) or 5 mM histidinol (Sigma-Aldrich). The final two groups ofcells were placed in serum-freemedium lackingHis, and in one ofthose groups, the medium also contained 5 mM histidinol. Cellswere returned to the incubator for 2 h prior to harvest unless oth-erwise indicated.Measurement ofGlobal Rates of Protein Synthesis—Global rates of

protein synthesiswere estimated by the incorporation of [35S]methi-onine and [35S]cysteine into protein as described previously (34).

FIGURE 1. Regulation of protein synthesis and eIF2� phosphorylation in wild type and GCN2�/� MEFs bycycloheximide, His deprivation, and/or histidinol treatment. Wild type (A and C) and GCN2�/� (B and D)MEFs were treated with 1 �M cycloheximide or 5 mM histidinol or were deprived of His in the presence orabsence of 5 mM histidinol for 2 h prior to harvest. Global rates of protein synthesis were measured as theincorporation of [35S]methionine and [35S]cysteine into protein as described under “Experimental Procedures.”The results are presented as cpm of [35S]methionine and [35S]cysteine incorporated into protein correctedfor the amount of protein in cell homogenates and represent the mean � S.E. of nine dishes of cells percondition. The phosphorylation state of eIF2� in wild type (C) and GCN2�/� (D) MEFs was assessed by Westernblot analysis using an antibody specific for eIF2� phosphorylated on Ser51 as described under “ExperimentalProcedures.” Values for phosphorylated eIF2� were corrected for the total amount of the protein present in thesample. Representative blots are shown as insets to C and D. Lane 1, control cells; lane 2, cells treated withcycloheximide; lane 3, cells treated with 5 mM histidinol; lane 4, cells deprived of His; lane 5, cells deprived of Hisand treated with 5 mM histidinol. The results represent the mean � S.E. of three dishes of cells per condition.Means not sharing a superscript are significantly different (p � 0.05).

Protein Synthesis Inhibition Leads to Rapid REDD1 Turnover

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Western Blot Analysis—Cells were harvested by scraping inSDS sample buffer (0.0625 M Tris-HCl, pH 6.8; 12.5% v/v glyc-erol; 1.25% SDS; 1.25% v/v �-mercaptoethanol; 0.1% bromphe-

nol blue), boiled for 5min, and equalvolumes of each sample were sub-jected to Western blot analysis asdescribed previously (35). Briefly,samples were resolved by SDS-poly-acrylamide gel electrophoresis(SDS-PAGE), transferred onto a0.45-�m polyvinylidene fluoridemembrane (Pall Life Sciences), andthe membrane was blocked with 5%nonfat dry milk. Membranes werethen incubated overnight at 4 °Cwith one of the following antibodies:anti-phospho-Ser51 (BIOSOURCEInternational, Invitrogen) or totaleIF2�, anti-4E-BP1 (Bethyl Labora-tory), anti-S6K1 (Bethyl), anti-TSC2 (Cell Signaling) anti-TRB3(EMD Chemicals, Inc.), or anti-REDD1 (ProteinTech Group Inc.).Membranes were then incubated insecondary antibody (Bethyl) atroom temperature for one hour andblotswere developedwith enhancedchemiluminescence (ECL) or ECLPlus reagents (Amersham Bio-sciences). Images were capturedusing a GeneGnome HR Bioimager(SynGene) and quantitated usingGeneTools software (SynGene).Sucrose Density Gradients—For

polysome analysis, cells werescraped in buffer (50 mM HEPES,pH 7.4; 75 mM KCl; 5 mM MgCl2;250 mM sucrose; 1:10 volume 10%Triton X-100, and 13% sodiumdeoxycholate; 0.1 mg/ml cyclohexi-mide), and the suspension wasrocked for 10 min at 4 °C to lyse thecells. The homogenate was centri-fuged at 3,000 � g for 15 min at 4 °Ccentrifugation, and the supernatantwas subjected to sucrose densitygradient centrifugation as describedpreviously (36). Briefly, discontinu-ous 9-step 20% (10 mM HEPES, pH7.4; 250 mM KCl; 5 mM MgCl2; 0.5mM EDTA; 20% w/w sucrose) to47% (10mMHEPES, pH 7.4; 250mMKCl; 5 mM MgCl2; 0.5 mM EDTA;47% w/w sucrose) sucrose densitygradients were formed as previouslydescribed (36), except that the KClconcentration was changed to 75mM. An equal amount of protein

was loaded onto each gradient and centrifuged at 288,200� g at4 °C for 1 h 50 min. Gradients were fractionated using an ISCOgradient pump (Teledyne Isco, Inc.) while the absorbance at

FIGURE 2. Analysis of polysomal aggregation by sucrose density gradient centrifugation. Wild type (A, C,E, G, and I) and GCN2�/� (B, D, F, H, and J) cells were treated with cycloheximide (C and D) or histidinol (E and F)or were deprived of His with (I and J) or without (G and H) histidinol treatment as described in the legend to Fig.1. Cell supernatants were subjected to sucrose density gradient centrifugation as described under “Experimen-tal Procedures.” The black arrow in each panel denotes the location of 80 S monomers. The ratio of the riboso-mal content of the non-polysomal to the polysomal fraction (NP/P) is shown as an inset to each panel. Thedashed vertical line in each panel denotes the separation between the two fractions. The results are represent-ative of two studies that were individually analyzed per condition.




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254 nm was continuously recorded. The ratio of ribosomespresent in the nonpolysomal fraction to those present in poly-somes was calculated from the area under the curve for eachfraction.RNA Isolation and Quantitative Real-time PCR—RNA

was extracted from wild type and GCN2�/� MEFs usingTRIzol reagent according to the manufacturer’s protocol(Invitrogen, Carlsbad, CA). RNA (1 �g) was reverse-tran-scribed and subjected to quantitative real-time PCR asdescribed previously (23), with the exception that primerscorresponding to the mouse REDD1 and GAPDH mRNAswere used for amplification. The primers used were as fol-lows: GAPDH forward primer, 5�-GTTGTCTCCTGCGAC-TTCA-3�; reverse primer, 5�-TGCTGTAGCCGTATTCA-TTG-3�; REDD1 forward primer, 5�-TGGTGCCCACCTT-TCAGTTG-3�; reverse primer, 5�-GTCAGGGACTGGCT-

GTAACC-3�. mRNA expressionlevels were normalized to GAPDHmRNA expression.Measurement of Intracellular

AminoAcidContent—Formeasure-ment of intracellular amino acidcontent, cells were washed twice inice-cold phosphate-buffered saline,harvested in 3% perchloric acid, andcentrifuged at 1,000 � g for 3 min.The supernatant was neutralizedwith 1 M K2HPO4, mixed, and cen-trifuged at 1,000 � g for 3 min. Thesupernatant was subjected to aminoacid analysis by HPLC (37) in theGeneral Clinical Research Center atThe Pennsylvania State UniversityCollege of Medicine.Statistical Analysis—Statistical

analyses were performed by Stu-dent’s t test or one-way analysis ofvariance with the correspondingpost-test using the InStat softwareprogram (GraphPad) as noted in thefigure legends.

RESULTS

In the present study, wild typeMEFs and MEFs lacking GCN2(GCN2�/�) were employed asexperimental model systems to dis-tinguish mRNA translation initia-tion- versus elongation-inducedchanges in mTORC1 signaling fol-lowing selective inhibition of eitherprocess. Changes in mTORC1 sig-naling that occurred in response toinhibition of translation elongationby cycloheximide in MEFs werecompared with changes associatedwith inhibition of initiation in cellsdeprived of His or in cells treated

with the histidinyl-tRNA synthetase inhibitor, histidinol, toinduce His-tRNA deacylation (38). Deprivation of essentialamino acids such as His leads to activation of the protein kinaseGCN2 by deacylated tRNA (39). GCN2 subsequently phospho-rylates the �-subunit of eIF2 (eIF2�) on Ser51, resulting in inhi-bition of initiation relative to elongation. Initially, the effective-ness of cycloheximide andHis deprivation in inhibiting proteinsynthesis was assessed by measuring changes in [35S]methi-onine and [35S]cysteine incorporation into protein.As shown inFig. 1A, global rates of protein synthesis were reduced to �15%of the control value in wild type MEFs treated with cyclohexi-mide and to about 30% of the control value in wild type MEFsdeprived of His. Addition of histidinol to His-containingmedium was significantly less effective in repressing proteinsynthesis compared withHis deprivation. However, the combi-nation ofHis deprivation and histidinol treatment reduced pro-

FIGURE 3. Effect of inhibition of translation initiation or elongation on mTOR signaling in wild type andGCN2�/� MEFs. Wild type (A and C) and GCN2�/� (B and D) MEFs were treated with cycloheximide or histidinolor were deprived of His with or without histidinol treatment as described in the legend to Fig. 1. Phosphoryla-tion of 4E-BP1 (A and B) and S6K1 (C and D) was assessed by changes in migration during SDS-polyacrylamidegel electrophoresis as described under “Experimental Procedures.” Phosphorylation of 4E-BP1 was calculatedas the proportion of the protein present in the hyperphosphorylated �-form and phosphorylation of S6K1 wascalculated as the proportion of the protein present in the hyperphosphorylated �, �, and � forms. Represent-ative blots are shown as insets to the panels. Lane 1, control cells; lane 2, cells treated with cycloheximide; lane3, cells treated with 5 mM histidinol; lane 4, cells deprived of His; lane 5, cells deprived of His and treated with 5mM histidinol. The results represent the mean � S.E. of 8 –9 dishes of cells per condition. Means not sharing asuperscript are significantly different (p � 0.05).




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tein synthesis to�10%of the control value. InGCN2�/�MEFs,the inhibition of protein synthesis caused by cycloheximidewassimilar in magnitude to the decrease observed in wild typeMEFs (Fig. 1B). As reported previously using perfused liversfromGCN2�/� mice (40), the inhibition of protein synthesis inGCN2�/� MEFs caused by His deprivation and/or histidinoltreatment was also similar in magnitude to wildtype MEFs. Toconfirm that the GCN2�/� MEFs lacked functional GCN2, thephosphorylation state of eIF2� was measured by Western blotanalysis using an antibody that specifically recognizes the pro-tein when Ser51 is phosphorylated. As shown in Fig. 1C, His

deprivation, treatment with histidi-nol, and His deprivation combinedwith histidinol treatment all led to asignificant increase in eIF2� phos-phorylation on Ser51 comparedwithwild type control MEFs or wild typeMEFs treated with cycloheximide.In contrast, eIF2� phosphorylationon Ser51 was not increased inGCN2�/� MEFs in response to anyof the treatments.A possible explanation for the

decrease in protein synthesis thatoccurred in GCN2�/� MEFs duringHis deprivation and/or histidinoltreatment is that in the absence ofGCN2, accumulation of deacylatedtRNA resulting from deprivation ofan essential amino acid leads to aninhibition of translation elongationrather than initiation. To assess thispossibility, polysome aggregationwas measured in wildtype andGCN2�/� MEFs using sucrose den-sity gradient centrifugation. In thistype of analysis, inhibition of initia-tion relative to elongation leads todisaggregation of polysomeswith anincrease in the proportion of ribo-somes present as 80 S monomers(41). In contrast, inhibition of elon-gation relative to initiation has theopposite effect, i.e. ribosomes accu-mulate in polysomes. As expected,cycloheximide treatment resultedin a decrease in the number of ribo-somes present as 80 S monomersconcomitant with accumulation ofribosomes in polysomes in bothwild type and GCN2�/� MEFs (Fig.2), indicative of an inhibition ofelongation. In wildtype MEFs, Hisdeprivation, histidinol treatment, ora combination of His deprivationand histidinol treatment all caused adecrease in the number of ribo-somes present in the polysomal

fraction of the gradient with a corresponding increase in ribo-somes present as 80 S monomers, a result consistent with inhi-bition of initiation. In contrast, in GCN2�/� MEFs, His depri-vation and/or histidinol treatment resulted in an inhibition ofelongation as indicated by a shift of ribosomes from 80 Smono-mers into polysomes.As an index of mTORC1 signaling, changes in phosphoryla-

tion of two proteins that are direct substrates of mTORC1,4E-BP1, and S6K1 (42), were measured as altered migrationduring SDS-polyacrylamide gel electrophoresis. The electro-phoretic migration of both proteins is inversely proportional to

FIGURE 4. Inhibition of mRNA translation results in reduced expression of REDD1, but not TSC2 or TRB3.Wild type (A, C, and E) and GCN2�/� (B, D, and F) cells were treated with cycloheximide or histidinol or weredeprived of His with or without histidinol treatment as described in the legend to Fig. 1. The expression of TSC2(A and B), TRB3 (C and D), and REDD1 (E and F) was measured by Western blot analysis as described under“Experimental Procedures.” Representative blots are shown as insets to the panels. Lane 1, control cells; lane 2,cells treated with cycloheximide; lane 3, cells treated with 5 mM histidinol; lane 4, cells deprived of His; lane 5,cells deprived of His and treated with 5 mM histidinol. The results are presented as mean and S.E. of 7 (TSC2), 6(TRB3), or 9 (REDD1) dishes of cells. Means not sharing a superscript are significantly different, p � 0.05.




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their phosphorylation state, with the most highly phosphoryla-ted forms of the proteins exhibiting the slowest migration dur-ing electrophoresis. As shown in Fig. 3, cycloheximide- andHisdeprivation-induced changes in phosphorylation of both pro-teins were inversely proportional to alterations in protein syn-thesis. Thus, all four treatments led to increased phosphoryla-tion of both 4E-BP1 and S6K1. However, the magnitude of theincrease was greater in cells treated with cycloheximide ortreated with histidinol in medium lacking His compared withcells either deprived of His or treated with histidinol alone.

Moreover, the pattern of responsein GCN2�/� MEFs (Fig. 3, B andD)was identical to that in wild typecells (Fig. 3, A and C). Interestingly,phosphorylation of both 4E-BP1and S6K1 in untreated wild typeMEFs tended to be higher than inuntreated GCN2�/� MEFs, a pointdiscussed further below.A previous report (26) suggested

that activation of mTORC1 signal-ing that occurred in response toinhibition of translation elongationby agents such as cycloheximidewasdue to accumulation of intracellularconcentrations of amino acids. Todetermine whether or not such achange would explain the activationof mTORC1 signaling observed inthe present study, the intracellularcontent of His and the branched-chain amino acids was measured incontrol cells and cells deprived ofhistidine. The content of thebranched-chain amino acids wasmeasured because in many cellsthey are as potent as a completemixture of amino acids in activatingmTORC1 (43). It was found that theintracellular His content wasreduced to 43% of the control valuein His-deprived wild typeMEFs andthe intracellular contents of Leu, Ile,and Val in His-deprived wild typeMEFs was 105, 93, and 91% of thecontrol values, respectively (n � 3experiments; within each experi-ment three dishes of cells were inde-pendently analyzed). Similarly, inHis-deprived GCN2�/� MEFs, theintracellular contents of Leu, Ile,and Val were 116, 114, and 111% ofthe control value, respectively.Thus, an increase in the intracellu-lar branched-chain amino acids isunlikely to explain activation ofmTORC1 signaling associated withHis deprivation in MEFs.

An alternative explanation for the activation of mTORC1signaling observed in the present study is that inhibition of pro-tein synthesis resulted in increased turnover of a mTORC1repressor. TSC2 is arguably the best known mTORC1 repres-sor. However, as shown in Fig. 4,A and B, TSC2 expression wasunchanged in either wild type or GCN2�/� MEFs, respectively,in response to cycloheximide treatment orHis deprivationwithor without histidinol treatment. TRB3 binds to and inhibitsAkt, an upstream activator of mTORC1 signaling (44, 45), andthus acts as a mTORC1 repressor. However, similar to TSC2,

α βγδ

FIGURE 5. Time course of cycloheximide-induced changes in REDD1 expression and mTORC1 signaling.Cycloheximide was added to the culture medium of wild type (A, C, and E) and GCN2�/� (B, D, and F) MEFs andat the times indicated in the figure, the cells were quickly harvested as described under “Experimental Proce-dures.” The expression of REDD1 (A and B) was measured as described in the legend to Fig. 4, and 4E-BP1 (C andD) and S6K1 (E and F) phosphorylation was assessed as described in the legend to Fig. 3. Representative blotsare shown as insets to the panels. The time of exposure to cycloheximide in min is depicted below each blot. Theresults represent the mean � S.E. of six dishes of cells per condition. Means not sharing a superscript aresignificantly different, p � 0.05.




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no consistent change in TRB3 expression was observed ineither wild type (Fig. 4C) or GCN2�/� MEFs (Fig. 4D) underany of the conditions used in the present study. REDD1 isinduced in response to a variety of cell stresses (46) and actsupstream of TSC2 to repress mTORC1 (47, 48). In the presentstudy, the pattern of REDD1 expression in both wild type (Fig.4E) andGCN2�/� (Fig. 4F)MEFsmirrored exactly the changesin protein synthesis in response to cycloheximide treatmentandHis deprivationwith orwithout histidinol treatment. Thus,cycloheximide treatment and a combination of His deprivationand histidinol treatment caused a larger reduction in REDD1expression compared with either histidinol treatment or Hisdeprivation alone.Todeterminewhether or not decreasedREDD1expression is

critical for the activation of mTORC1 signaling under the con-ditions used in the present study, we considered exogenousexpression of the protein prior to cycloheximide treatment orHis deprivation. However, the success of such an approachwould require that expression of REDD1 be maintained duringthe treatment period. Therefore, prior to initiating expressionstudies, an estimate of REDD1 protein half-life was obtained. Inthese studies, cycloheximide was added to wild type orGCN2�/� MEFs, and the cells were harvested at various timesand analyzed for REDD1 expression and mTORC1 signaling.As shown in Fig. 5, A and B, REDD1 expression was dramati-cally reduced within 5 min of cycloheximide addition to eitherwild type or GCN2�/� MEFs, respectively, suggesting that thehalf-life of the protein was less than 2 min. The activation ofmTORC1 signaling, as assessed by phosphorylation of4E-BP1 (Fig. 5, C and D) and S6K1 (Fig. 5, E and F), wasslightly delayed compared with the decrease in REDD1expression, but was nonetheless rapidly induced after cyclo-heximide addition. Interestingly, the activation of mTORC1signaling was delayed to a greater extent in GCN2�/� MEFscompared with wild type cells, a finding in agreement withthe greater REDD1 expression observed in GCN2�/� MEFscompared with wild type cells.

To assess whether the observedchanges in REDD1 content wereassociated with alterations inREDD1 mRNA, RNA was isolatedfromwild type andGCN2�/� MEFsat various times after addition ofcycloheximide to the cell culturemedium. As shown in Fig. 6A,REDD1 mRNA content did notchange during a one hour incuba-tion with cycloheximide, a time atwhichmTORC1 signaling wasmax-imally increased.Moreover, REDD1mRNA content did not decrease inGCN2�/�MEFs incubated for up to1 h with cycloheximide, but insteadwas significantly increased (Fig. 6B).Interestingly, REDD1 mRNAexpression was greater in GCN2�/�

MEFs compared with wild typecells, a finding that may explain the

higher REDD1 protein expression in those cells (Fig. 5, A andB). Overall, the rapid fall in REDD1 protein content is unlikelyto be due to changes in REDD1 mRNA content.Based on its apparently short half-life, we anticipated that

REDD1 would need to be expressed at very high levels relativeto the native protein to effectively repress mTORC1 signalingduring inhibition of protein synthesis. Indeed, exogenouslyexpressed REDD1 was rapidly degraded in MEFs (data notshown). Therefore, an alternative approach to assessing the roleof changes in REDD1 expression in increasing TORC1 signal-ing under conditions of repressed mRNA translation was uti-lized. In this approach, wild type or REDD1�/� MEFs weretreated with cycloheximide, and the effect on mTORC1 signal-ing was assessed as changes in phosphorylation of 4E-BP1 andS6K1. The time point chosen for these studies was based on thefinding that cycloheximide-induced changes in REDD1 expres-sion aremaximal by 15min (Fig. 5,A andB). As shown in Fig. 7,A and C, signaling through mTORC1 was significantlyincreased within 15 min of exposure to cycloheximide in wildtype MEFs, concomitant with decreased REDD1 expression(Fig. 7E). However, the increase in mTORC1 signaling inducedby cycloheximide was dramatically reduced in MEFs lackingREDD1 compared with wild type MEFs. For example, the rela-tive increase in 4E-BP1 phosphorylation in REDD1�/� MEFs15 min after addition of cycloheximide was only 26% of theincrease in wild type MEFs. As one would predict, inREDD1�/� MEFs, basal mTORC1 signaling was elevated com-pared with wild type MEFs, as assessed by a small, but signifi-cant, increase in phosphorylation of both 4E-BP1 and S6K1.

DISCUSSION

The results of the present study confirm earlier reportsshowing that inhibition of the elongation phase of mRNAtranslation results in activation of mTORC1 signaling (26–31).The results extend the earlier reports to show that inhibition ofthe initiation phase of mRNA translation similarly results inactivation of mTORC1. Thus, inhibition of mRNA translation

FIGURE 6. Time course of cycloheximide-induced changes in REDD1 mRNA expression. Cycloheximidewas added to the culture medium of wild type (A) and REDD1�/� (B) MEFs and at the times indicated in thefigure, the cells were quickly harvested as described under “Experimental Procedures.” RNA was isolated fromcells and analyzed by quantitative real-time PCR as described under “Experimental Procedures.” REDD1 mRNAcontent was normalized for the expression of GAPDH mRNA. The results represent the mean � S.E. of six dishesof cells per condition. Means not sharing a superscript are significantly different, p � 0.05.




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per se, not decreased elongation, leads to activation ofmTORC1 signaling. How does inhibition of translation activatemTORC1? Previously, it was suggested that the activation

of mTORC1 signaling was due toaccumulation of intracellular aminoacids that could occur as a result oftheir reduced utilization for proteinsynthesis in conjunction withunchanged cellular uptake andappearance from protein degrada-tion (26). The results of the presentstudy showing that mTORC1 sig-naling was activated in MEFsdeprived ofHis without a significantchange in the intracellular contentof any of the branched-chain aminoacids suggests that activation ofmTORC1 can occur without adetectable increase in intracellularamino acids.An alternative explanation for the

activation of mTORC1 signalingthat occurs upon inhibition ofmRNA translation is that synthesisof a short-lived protein that acts as arepressor of mTORC1 is reduced.The most proximal repressor ofmTORC1 that has been identifiedthus far is TSC2. In cells in whichTSC2 expression is repressed, theproportion of Rheb present in thestimulatory GTP complex is consti-tutively elevated, and mTORC1 sig-naling is activated (49, 50). More-over, in such cells, mTORC1signaling is resistant to amino aciddeprivation (18, 51). However, inthe present study, TSC2 expressionin MEFs was unaffected by eithercycloheximide treatment or Hisdeprivation with or without treat-ment with histidinol. Another pro-tein that acts to repress mTORC1signaling is TRB3. TRB3 binds toand prevents activation of anupstream modulator of mTORC1function, Akt (44). By inhibitingAkt, TRB3 represses insulin-in-duced activation of mTORC1 (45).Interestingly, the half-life of TRB3 isestimated to be less than 2 h (52),making it a potential candidate formTORC1 regulation under condi-tions of reduced protein synthesis.However, similar to TSC2, therewas no detectable change in TRB3expression in the present study.REDD1 was originally identified

as a gene that is transcriptionally up-regulated in response tohypoxia (53–56), and subsequently shown to be regulated byother cellular stresses such as exposure to arsenite (57), dexa-

FIGURE 7. Activation of mTORC1 signaling by cycloheximide is attenuated in REDD1�/� compared withwild type MEFs. Cycloheximide was added to the culture medium of wild type (A, C, and E) and REDD1�/�

(B, D, and F) MEFs and 15 min later, the cells were quickly harvested as described under “Experimental Proce-dures.” The expression of 4E-BP1 phosphorylation (A and B), S6K1 phosphorylation (C and D), and REDD1expression (E and F) was assessed as described in the legend to Fig. 5. Representative blots are shown as insetsto the panels. The time of exposure to cycloheximide in min is depicted below each blot. The results representthe mean � S.E. of six dishes of cells per condition. Means not sharing a superscript are significantly different,p � 0.05. †, p � 0.01 versus REDD1�/� cells at time � 0.




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methasone (23, 58), or agents that cause DNA damage (59).More recent studies have shown that REDD1 acts downstreamof Akt (47, 54) and upstream of TSC2 (48, 53, 60) to repressmTORC1. In the present study, REDD1 expression was dra-matically decreased in both wild type and GCN2�/� MEFstreated with cycloheximide as well as in response to His depri-vation. Thus,mTORC1 signalingwas indirectly proportional toREDD1 content (Fig. 8), strongly suggesting that decreasedREDD1 expression was causative in the observed activation ofmTORC1.It is noteworthy that both REDD1 protein (Fig. 4, E and F)

and mRNA (Fig. 6, A and B) were more highly expressed inGCN2�/� compared with wild type MEFs. The basis for theincreased REDD1 expression is unknown, but the finding thatbasal phosphorylation of both 4E-BP1 and S6K1 was lower inGCN2�/� compared with wild type MEFs provides furthersupport for the idea that up-regulated REDD1 expression leadsto further repression of mTORC1 signaling.Although not examined herein, it is likely that reduced

REDD1 expression also explains the observation made in pre-vious studies that inhibition of gene transcription results inmTORC1 activation. For example,mTORC1 signaling is signif-icantly activatedwithin 1–2hof exposure to actinomycinD (30,33). The half-life of the REDD1 mRNA is relatively short, i.e. afew hours (58). Thus, inhibition of gene transcription wouldlead to a rapid reduction in REDD1mRNA expression and sub-sequently to decreased REDD1 protein, thereby activatingmTORC1 signaling.An obvious question is why has not the link between inhibi-

tion of protein synthesis and activation of mTORC1 signalingbeen more widely observed? For example, in L6 myoblasts, Hisdeprivation had no obvious effect on mTORC1 signaling (61).The answer to this question is unknown, butmay in part involvethe magnitude of the inhibition of protein synthesis engen-dered by activation of GCN2 in different cell types. For exam-ple, in many mammalian cells and tissues, activation of GCN2

in response to amino acid depriva-tion reduces protein synthesis toonly about 70% of the control value(e.g. 62). In contrast, at the concen-tration used in the present and paststudies, cycloheximide leads to areduction in global rates of proteinsynthesis to �15% of the controlvalue. Thus, the relatively smallchanges in protein synthesis thatoccur in response to activation ofGCN2 in some cells may allowREDD1 mRNA translation to pro-ceed at a rate sufficient to sustainexpression of the protein at levelsnecessary to maintain mTORC1regulation. An alternative explana-tion for the dearth of studies report-ing activation ofmTORC1 signalingin response to amino acid depriva-tion is that in many cases cells weredeprived of Leu, either alone or in

combinationwith other amino acids. Leucine is thought to acti-vate mTORC1 through a signaling pathway independent ofTSC2 (19, 20). In the present study, we found that Leu depriva-tion resulted in reduced mTORC1 signaling in MEFs, eventhough REDD1 expression was lower in Leu-deprived com-pared with control cells (data not shown). Thus, the negativeinfluence of Leu deprivation on mTORC1 signaling appears tobe dominant to the positive input provided by reduced REDD1.Therefore, it is likely that in many studies, the negative regula-tion of mTORC1 caused by Leu deprivation masks the up-reg-ulation of mTORC1 signaling that would otherwise be inducedby reduced REDD1 expression.Acute inhibition of mRNA translation likely leads to

decreased expression of a number of proteins that have shorthalf-lives (e.g. a few minutes). Thus, although the changes inmTORC1 signaling that manifest in cells in which mRNAtranslation is inhibited are inversely proportional to changes inREDD1 expression (Fig. 8), such a correlation does not provethat REDD1 is directly involved in the effect. Therefore, in thepresent study, cycloheximide was used to inhibit mRNA trans-lation in MEFs lacking REDD1. In those cells, the increase inmTORC1 signaling was significantly reduced compared withcontrol MEFs, suggesting that, in large part, the increase inmTORC1 signaling was due to changes in REDD1 expression.However, the effect was not completely absent in REDD1�/�

MEFs, and therefore another protein(s) is likely to be involved.A likely candidate for the unknown regulatory protein isREDD2 (RTP801l). Like REDD1, REDD2 represses signalingthrough mTORC1 (47). Moreover, similar to REDD1, REDD2mRNA expression is induced by hypoxia (54, 63). Unfortu-nately, we are not aware of a source of anti-REDD2 antibodies,and examination of REDD2 mRNA content is unlikely to beinformative, based on the assumption that, similar to REDD1expression, rapid changes in REDD2 protein expression wouldprobably be a result of repressed REDD2 protein expressionrather than to changes in REDD2 mRNA content.

FIGURE 8. REDD1 expression is inversely proportional to mTORC1 signaling. Wild type and GCN2�/� cellswere treated with cycloheximide or histidinol or were deprived of His with or without histidinol treatment asdescribed in the legend to Fig. 1. Values for REDD1 expression are from Fig. 4 and values for 4E-BP1 and S6K1phosphorylation are from Fig. 3. Note that the same samples were analyzed for all three proteins, so that adirect comparison could be made. The results represent the mean � S.E. of 8 –9 dishes of cells per condition.The results of a least squares analysis are presented in the figure.




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In summary, the results of the present study reveal the novelfinding that inhibitors of protein synthesis acting either ontranslation initiation or elongation cause a rapid increase inREDD1 turnover, leading to activated signaling throughmTORC1. Moreover, the finding that mTORC1 signaling isinversely proportional to changes in REDD1 expression suggestthat even small changes in REDD1 expression will have a sig-nificant effect on mTORC1 signaling. Finally, the results revealthat REDD1 has an exceptionally short half-life, making it apossible target for regulation under a variety of conditions thatalter mTORC1 signaling. Future studies will be required toidentify the other protein(s) that are involved in the increase inmTORC1 signaling that occur in response to inhibition ofmRNA translation.

Acknowledgments—We thank Rick Horetsky, Holly Lacko, and Sha-ron Rannels for technical assistance, andDrs. David Ron andHeatherHarding (NYUniversity School ofMedicine) andDr. Leif Ellisen (Har-vard Medical School) for kindly supplying the GCN2�/� andGCN2�/�MEFs andREDD1�/� andREDD1�/�MEFs, respectively.

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JeffersonScot R. Kimball, A. N. Dang Do, Lydia Kutzler, Douglas R. Cavener and Leonard S.

Activation of mTORC1 Signaling following Inhibition of Protein SynthesisRapid Turnover of the mTOR Complex 1 (mTORC1) Repressor REDD1 and

doi: 10.1074/jbc.M706643200 originally published online December 10, 20072008, 283:3465-3475.J. Biol. Chem.

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RapidTurnoverofthemTORComplex1(mTORC1)Repressor ... · 2008-01-25 · (PRAS)40.Incontrast,mTORC2containsG L,rapamycin-in-sensitivecompanion of mTOR (rictor) ... histidinol (Sigma-Aldrich)