Activation of Mammalian Target of Rapamycin (mTOR)by Insulin Is Associated with Stimulation of 4EBP1 Bindingto Dimeric mTOR Complex 1*
Received for publication, April 13, 2006, and in revised form, May 30, 2006 Published, JBC Papers in Press, June 23, 2006, DOI 10.1074/jbc.M603566200
Lifu Wang‡, Christopher J. Rhodes§, and John C. Lawrence, Jr.‡1
From the ‡Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908 and the §Pacific Northwest ResearchInstitute and Department of Pharmacology, University of Washington, Seattle, Washington 98122
Insulin stimulates protein synthesis by promoting phospho-rylation of the eIF4E-binding protein, 4EBP1. This effect israpamycin-sensitive and mediated by mammalian target ofrapamycin (mTOR) complex 1 (mTORC1), a signaling complexcontaining mTOR, raptor, and mLST8. Here we demonstratethat insulin produces a stable increase in the kinase activity ofmTORC1 in 3T3-L1 adipocytes. The response was associatedwith amarked increase in 4EBP1 binding to raptor inmTORC1,and it was abolished by disrupting the TOR signaling motif in4EBP1. The stimulatory effects of insulin on both 4EBP1 kinaseactivity and binding occurred rapidly and at physiological con-centrations of insulin, and both effects required an intactmTORC1. Results of experiments involving size exclusion chro-matography and coimmunoprecipitationof epitope-tagged sub-units provide evidence that themajor insulin-responsive form isdimeric mTORC1, a structure containing two heterotrimers ofmTOR, raptor, and mLST8.
As the major anabolic hormone in mammals, insulin stimu-lates protein synthesis in a wide variety of cell types. Thisresponse is mediated in part by mTOR,2 a phosphatidylinositol3-kinase-related protein kinase that controls the phosphoryla-tion of multiple factors involved in the control of cell growthand proliferation (1, 2). mTOR functions in two signaling com-plexes, mTORC1 andmTORC2 (1, 3). Both complexes containmTORandmLST8 (also known asG�L), a protein homologousto � subunits of heterotrimeric G proteins (4, 5). One definingfeature of the complexes is the third subunit, either raptor inmTORC1 (5–7) or rictor (also known as mAVO3) in mTORC2(8, 9).
4EBP1 (also known as PHAS-I) is an important target ofmTOR signaling. 4EBP1 binds eIF4E, the mRNA cap-bindingprotein, and it represses cap-dependent translation by compet-itively blocking the binding of eIF4G to eIF4E (2, 10). ActivatingmTORwith insulin stimulates the phosphorylation of 4EBP1 infour sites (11, 12), including Thr-36 and Thr-45, the two sitespreferred by mTOR in vitro (13, 14), causing 4EBP1 to dissoci-ate fromeIF4E. This allows eIF4E to engage eIF4G, a scaffoldingprotein that binds eIF3 and eIF4A (10, 15). eIF3 is a complexinitiation factor that binds the small ribosomal subunit and sev-eral key initiation factors, and eIF4A is a helicase that unwindsmRNA to facilitate binding and/or scanning by the 40 S riboso-mal subunit (10, 15). Thus, the phosphorylation of 4EBP1 leadsto the recruitment of the small ribosomal subunit and impor-tant initiation factors to the 5�-end of the message to begin theprocesses of scanning and selection of the start codon.The finding that the effects of insulin and insulin-like growth
factor 1 on 4EBP1 were attenuated by rapamycin provided thefirst evidence that mTOR controlled 4EBP1 (16, 17). Becauserapamycin inhibits mTORC1 but not mTORC2 (8, 9), the sen-sitivity to rapamycin also implicates mTORC1. The functionsof the mTORC1 subunits are not fully understood. mLST8,which consists almost entirely of seven WD40 repeats, bindsnear the catalytic domain of mTOR and is required for the fullactivity of the mTOR kinase (4). Raptor possesses a uniqueNH2-terminal region followed by threeHEATmotifs and sevenWD40 repeats that are believed to mediate protein-proteininteractions (7). Raptor binds themTOR substrates, 4EBP1 andS6K1, and it has been suggested that raptor might function topresent substrates to mTOR for phosphorylation (6). 4EBP1can be readily phosphorylated in vitro by mTORC1 (6) but notbymTORC2, which lacks raptor (9). The substrate interactionswith raptor are mediated by TOR signaling (TOS) motifs (18–22). In 4EBP1 this motif is formed by the COOH-terminal fiveamino acids (FEMDI) (21). Disrupting the TOSmotif by a Phe3Ala point mutation markedly decreases phosphorylation of theprotein in response to activation by mTOR signaling in cells(21) and by mTORC1 in vitro (20, 22).Incubating cells with insulin (23), serum (13), or certain
growth factors (24, 25) has been reported to increase the pro-tein kinase activity ofmTOR.However, such changes inmTORactivity have not been detected in other studies, and the con-clusion that insulin produces a stable increase in the kinaseactivity of mTOR is controversial. Previous studies of insulinaction on mTOR activity in vitro have not discriminated
* This work was supported by National Institutes of Health Grants DK52753and DK28312. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
1 To whom correspondence should be addressed: Dept. of Pharmacology,University of Virginia Health System, P. O. Box 800735, 1300 Jefferson ParkAve., Charlottesville, VA 22908. Tel.: 434-924-1584; Fax: 434-982-3878;E-mail: [email protected].
2 The abbreviations used are: mTOR, mammalian target of rapamycin; C-RapAb, antibody to the COOH-terminal region of raptor; eIF3, eIF4E, and eIF4G,eukaryotic initiation factors 3, 4E, and 4G, respectively; FKBP12, FK506-binding protein of Mr � 12,000; GST, glutathione S-transferase; mTAb2,mTOR antibody 2; mTORC1 and mTORC2, mTOR complex 1 and 2; TOS,TOR signaling; HA, hemagglutinin; ERK, extracellular signal-regulatedkinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfon-ic acid.
between the twomTOR signaling complexes, and in some casesthe conditions used to extract mTOR would have disruptedmTORC1. Because mTORC1mediates the effects of insulin onthe phosphorylation of 4EBP1 in cells, we conducted experi-ments to measure mTORC1 activity and the interaction ofmTORC1 with 4EBP1.
EXPERIMENTAL PROCEDURES
Adipocyte Culture and Extract Preparation—3T3-L1 fibro-blasts were grown in Dulbecco’s modified Eagle’s medium con-taining 10% newborn calf serum. Fibroblasts were converted toadipocytes by using differentiation medium as described previ-ously (17). Cells were cultured in Dulbecco’s modified Eagle’smedium containing 10% fetal bovine serum for 10–12 daysafter adding the differentiation medium. For experiments, theculture medium was replaced with a solution containing 145mM NaCl, 5.4 mM KCl, 1.4 mM CaCl2, 1.4 mM MgSO4, 25 mMNaHCO3, 5 mM glucose, 5 mg/ml bovine serum albumin, 0.2mM sodium phosphate, and 10 mM HEPES, pH 7.4. The cellswere incubated at 37 °C without or with a maximally effectiveconcentration of insulin (0.6 �M) and/or other additions. Toterminate the incubation the adipocytes were rinsed once withchilled phosphate-buffered saline (145 mM NaCl, 5.4 mM KCl,and 10 mM sodium phosphate, pH 7.4) and then homogenized(0.8 ml of buffer/10-cm-diameter dish) in a glass tissue grinderwith a Teflon pestle driven at 1,000 rpm. HomogenizationBuffer was composed of Buffer A supplemented with 1 mMphenylmethylsulfonyl fluoride, 10 �g/ml leupeptin, 10 �g/mlaprotinin, 10�g/ml pepstatin, and 0.5�Mmicrocystin. BufferAcontained 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM dithi-othreitol, 0.1% Tween 20 (unless otherwise indicated), 10 mMsodium phosphate, and 50 mM �-glycerophosphate, pH 7.4.Homogenates were centrifuged at 12,000 � g for 10 min, andthe supernatants were retained for analyses.Antibodies—Antibodies to the COOH-terminal region of
4EBP1 (26) and the phosphospecific antibodies to the Thr-36and Thr-45 sites (12) have been described previously (12, 26).The 4EBP1 antibodies bind wild type 4EBP1 and F113A equallywell (22), and the phosphospecific antibodies bind 4EBP1 phos-phorylated in either Thr-36 or Thr-45 (12) as the amino acidsequences surrounding these sites are almost identical. ThemTOR antibodies, mTAb1 and mTAb2, were described previ-ously (27). Antibodies (designated N-Rap Ab) to the region inraptor (amino acids 36–53) originally targeted by Kim et al. (7)were generated as described previously (22). mTAb2 andN-Rap Ab were used to detect mTOR and raptor, respectively,by immunoblotting. mLST8 antibodies were described previ-ously (28) as were phosphospecific antibodies to the Ser-2448site in mTOR (29).In pilot experiments we attempted to immunoprecipitate
mTORC1 by using N-Rap Ab. Although raptor was readilydetected in immunoprecipitates obtained with this antibody,neither mTOR nor mLST8 were found (results not presented).Thus, N-Rap Ab either promoted dissociation of mTOR andraptor or was unable to bind raptor associated with mTOR. Togenerate raptor antibodies that could be used to immunopre-cipitate mTORC1, a peptide having an NH2-terminal Cys fol-lowed by 12 amino acids (YISVYSVEKRVR) corresponding to
the COOH-terminal region of raptor was coupled to keyholelimpet hemocyanin, and the conjugate was used to immunizerabbits. The resulting raptor antibodies (C-Rap Ab) were puri-fied using a column containing an affinity resin prepared bycoupling the peptide to Sulfolink beads (Pierce). Rictor anti-bodies were generated in a similarmanner except that a peptide(CRHSPDTAEGQLKEDRE) based on amino acids 263–278 inmouse rictor was used.Monoclonal antibody 12CA5, which recognizes the HA
epitope tag, was purified from hybridoma culture medium.Phosphospecific antibodies to the Ser-473 site inAkt2, theThr-389 site in S6K1, and the activating sites in the ERK1 and ERK2isoforms of mitogen-activated protein kinase were from CellSignaling Technology Inc.Purification of Recombinant Proteins—His-tagged forms of
wild type 4EBP1 and a 4EBP1 protein havingAla at position 113(F113A) were expressed in bacteria and purified as describedpreviously (22). To assess purity and to confirm protein con-centrations, samples were subjected to SDS-PAGE and thenstained with Coomassie Blue (22). Complexes of eIF4E boundto His-tagged forms of either 4EBP1 or 4EBP2 were purified asdescribed previously (30). Glutathione S-transferase (GST)-FKBP12 was prepared as described previously (31).Immunoprecipitation of mTORC1—Two strategies were
used to recover mTORC1. In the first, the raptor subunit of theendogenous complex was targeted by using C-Rap Ab. In theother, an HA antibody was used to capture mTORC1 contain-ing an epitope-tagged raptor that had been overexpressed in theadipocytes. Several antibodies to mTOR, raptor, and mLST8proved unsuitable either because they activated mTOR orbecause they failed to immunoprecipitate mTORC1 (resultsnot shown). Adipocyte extract samples (800�l) were incubatedwith C-RapAb (2�g) bound to protein A-agarose beads (15�l)or with 12CA5 (2 �g) bound to protein G-agarose beads (15 �l)at 4 °C for 12 h with constant mixing. As a control for specific-ity, rabbit or mouse nonimmune IgG was substituted for theC-Rap Ab or 12CA5, respectively. The beads were then washedonce with 1 ml of Buffer A, once with 1 ml of Buffer A plus 0.5mM NaCl, and then twice with 1 ml of Buffer A.Expression of HA-raptor in 3T3-L1 Adipocytes by Adenoviral
Mediated Gene Transfer—Virus for expressing HA-raptor wasprepared using the system developed by He et al. (32). BrieflycDNA encoding HA-tagged raptor was excised with KpnI andNotI from the pBluescript construct described previously (22)and inserted between the KpnI and NotI sites in the shuttlevector, pAdTrack. The resulting plasmid was cotransformedwith the adenoviral backbone plasmid, pAdEasy-1, into BJ5183bacteria. Recombined plasmid was selected and transfectedinto human embryonic kidney 293 cells to generate virus, whichwas amplified and thenpurified byCsCl gradient centrifugationto create a high titer viral stock. 3T3-L1 adipocytes wereinfected essentially as described by Kasuga and co-workers(33). The efficiency of infection judged by expression ofgreen fluorescent protein, which is also encoded by the HA-raptor virus, was �50%. Virus encoding �-galactosidase wasused as a control.ExpressionConstructs formTOR, Raptor, andmLST8—Most of
22, 28). To generate a construct for expressingMyc-raptor, cDNAencoding raptor was excised from an HA-raptor-pcDNA3 vector(22) by using EcoRI and inserted into the EcoRI site inpCMVTag3B (Stratagene).The construct for expressinguntaggedmLST8 was prepared by excising mLST8 cDNA from the HA-mLST8 vector (28) and then inserting the mLST8 fragment intopcDNA3.To generate vector for expressing FLAG-mTOR, a frag-ment corresponding to bp 1–632 of themTORcoding regionwasamplified by PCR with AU1-mTOR pcDNA3 template and thefollowing primers: 5�-GGCGGATCCCACCATGCTTGGGA-CAGGCCCTG-3� and 5�-GGCAGTCGACTCTAGAGCCACA-GCTCCTTCACGGATG-3�. The fragment was digested withBamHIandSalI, and theproductwas insertedbetween theBamHIand SalI sites in pCMVTag2A (Stratagene) to generateNT-pCMVTag2A. To complete construction of the FLAG-mTOR expression construct (mTOR-pCMVTag2A), EcoRI andXbaI were used to excise a fragment from AU1-mTOR pcDNA3that was inserted between the EcoRI (which cuts in the mTORcoding region in NT-pCMVTag2A) and XbaI sites in theNT-pCMVTag2A construct. The region of the FLAG-mTORcDNA generated by PCR was sequenced and found to be free oferrors.Immune Complex Assay of mTORC1 Activity—Immune
complex beads were rinsed with 1 ml of Buffer B (50 mM NaCl,0.1 mM EGTA, 1 mM dithiothreitol, 0.5 �M microcystin LR, 10mM HEPES, and 50 mM �-glycerophosphate, pH 7.4) and sus-pended in 60�l of Buffer B. After removing a sample for immu-noblotting mTOR and raptor, the kinase reactions were initi-ated by adding to 20 �l of the suspension 5 �l of Buffer Asupplemented with 0.5 mM [�-32P]ATP (PerkinElmer Life Sci-ences, 1,000 mCi/mmol), 50 mM MnCl2, and 1 �g of the His-tagged form of either wild type 4EBP1 or F113A. Unless other-wise stated, reactions were terminated after 30 min at 30 °C byadding SDS sample buffer. The relative amounts of 32P incor-porated into the 4EBP1 proteins were determined by phospho-rimaging following SDS-PAGE. Measurements under theseconditions reflect the initial rate of phosphorylation as less than5% of the available substrates were phosphorylated, and thereactions proceed linearly for 60 min (see later, Fig. 2b).4EBP1 Binding to C-Rap Ab Immune Complexes—Immune
complexes from 400 �l of extract (7.5 �l of beads) were sus-pended in Buffer A (500 �l) containing 50 ng of the His-taggedformof either 4EBP1 or F113A. After incubating at 21 °C for 1 hwith constant mixing, the beads were washed four times with 1ml of Buffer A, twice with Buffer A plus 0.5 M NaCl, and thenonce with a solution containing 1 mM EDTA, 1 mM EDTA,and 50 mM Tris-HCl, pH 7.4. The relative amounts of the4EBP1 proteins retained by the beads were determined byimmunoblotting.Binding of Raptor to 4EBP1 Affinity Resins—His-tagged
4EBP1 or F113A proteins (1 mg) were coupled to CNBr-acti-vated Sepharose (86 mg) in 0.5 M NaCl and 0.1 M NaHCO3 (pH8.3). After 12 h at 4 °C, the resins were washed exhaustively asdirected by the supplier (Amersham Biosciences). The beadswere then suspended in 0.5 ml of buffer (0.5 M NaCl and 50mMTris-HCl, pH7.4) and stored at 4 °Cprior to use. For binding, analiquot (15�l) of the beads was added to 800�l of extract. Afterincubating at 4 °C for 12 hwith constantmixing, the beadswere
washed as described above for 4EBP1 binding to C-Rap Abimmune complexes. The relative amounts of raptor andmTORretained by the beads were determined by immunoblotting.Electrophoretic Analyses—SDS-PAGE and immunoblotting
were conducted as described previously (12). Binding of pri-mary antibodies was monitored by using the appropriate alka-line phosphatase-conjugated secondary antibodies, whichweredetected by using CDP-Star reagent (PerkinElmer Life Sci-ences). Relative signal intensities of bands in immunoblotsweredetermined by scanning laser densitometry of x-ray films or byusing a Fujifilm LAS 3000 LCD camera system.Gel Filtration—Adipocytes were incubated without or with
insulin, rinsed three times with Buffer A (minus detergents anddithiothreitol) that had been chilled on ice, and scraped fromthe dishes (six 10-cm-diameter dishes per treatment). Toenhance resolution by the column, the volume of extract wasminimized by homogenizing cells in 500 �l of HomogenizationBuffer. The homogenates were centrifuged at 12,000 � g for 10min. The supernatants were retained and passed through a0.45-�m filter. Extract samples (350 �l) were applied to aSuperose 6HR10/30 column (AmershamBiosciences) that hadbeen equilibrated in Buffer A. The flow rate was maintained at0.2 ml/min, and 1-ml fractions were collected.Other Materials—Recombinant human insulin (Novolin R)
was from Novo Nordisk. Rapamycin, LY294002, and U0126were from Calbiochem-Novabiochem. Epidermal growth fac-tor was from Upstate Biologicals. Fibroblast growth factor-1was provided by Dr. David Ornitz (Washington University).Farnesylthiosalicylic acid was provided by Dr. Wayne Bardin(Thyreos, New York, NY). Tween 20 was from Fischer. CHAPSwas from Roche Applied Science. Caffeine, insulin-like growthfactor 1, Nonidet P-40, Triton X-100, and wortmannin werefrom Sigma.
RESULTS
Stable Activation of mTORC1—mTORC1 was immunopre-cipitated from extracts of 3T3-L1 adipocytes by using an anti-body (C-Rap Ab) to the COOH-terminal region of raptor (Fig.1A). Raptor, mTOR, and mLST8 were detected in the immunecomplexes, confirming that intact mTORC1 was recovered(Fig. 1B). To measure mTORC1 activity, immune complexeswere incubated with [�-32P]ATP and 4EBP1. Treating adipo-cytes with insulin increased 4EBP1 phosphorylation bymTORC1 (Fig. 1B). No activity was detected in complexes iso-lated with nonimmune IgG. The 3-fold increase in mTORC1activity produced by insulin (Fig. 1B) is comparable to theincrease in phosphorylation of endogenous 4EBP1 when 32P-labeled 3T3-L1 adipocytes are incubated with the hormone(17). mTORC1 isolated with C-Rap Ab was not able to phos-phorylate F113A (Fig. 1C), a 4EBP1 protein having a pointmutation that disrupts the TOS motif (22).In the present experiments, 3T3-L1 adipocytes were incu-
bated in buffer lacking amino acids to isolate the effects due toinsulin from thosemediated by amino acids, which also activatemTOR signaling (34). Activating mTOR signaling with insulinhas been shown previously to increase 4EBP1 phosphorylationin 3T3-L1 adipocytes incubated in buffer without added aminoacids (11, 17). The incubation buffer was supplemented with
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albumin to bind fatty acids released from the adipocytes. To besure that the albumin was not replacing a requirement foramino acids, control experiments were conducted in bufferlacking albumin. Removing the albumin did not attenuate theeffects of insulin onmTOR activity.3 Thus, the insulin responseis not occurring secondarily to an increase in amino acidtransport.To confirm findingswith theC-RapAb,HA-raptorwas over-
expressed in 3T3-L1 adipocytes by using an adenovirus vector.HA antibodies were then used to isolate mTORC1 (Fig. 1D).HA-raptor, mTOR, and mLST8 were immunoprecipitated,indicating that the epitope-tagged raptor was incorporated intomTORC1 (Fig. 1E). Insulin did not change the amount ofmTOR ormLST8 that coimmunoprecipitated with HA-raptor,but it clearly increased the 4EBP1 kinase activity (Fig. 1E).mTORC1 subunits and kinase activity were not detected inimmune complexes isolated with HA antibodies from cellsinfected with a control adenovirus.The effects of insulin on mTORC2 were investigated by iso-
lating this complex by using antibodies to rictor (Fig. 2A). Insu-lin decreased the electrophoretic mobility of rictor, suggestingthat phosphorylation of rictor was increased by the hormone.Less mTOR was recovered with the rictor antibodies than withtheC-RapAb, consistentwith the interpretation thatmTORC2is less abundant than mTORC1 in 3T3-L1 adipocytes. Little if
any 32Pwas introduced into 4EBP1 bymTORC2 in the immunecomplexes isolated with the rictor antibodies. Even after cor-recting for theamountofmTORcatalytic subunitpresent,phos-phorylation of 4EBP1 by mTORC2 was negligible comparedwith that by mTORC1 (Fig. 2B). The incorporation of 32P into4EBP1 by C-Rap Ab immune complexes increased linearly forat least 1 h (Fig. 2B). Thus, 32P incorporationmeasured after 30min, as in Fig. 1B, reflects the initial rate of phosphorylation.To investigate further the 4EBP1 kinase activity in C-Rap Ab
complexes and to confirm that the phosphorylation detectedwas mediated by mTOR, the complexes were incubated withadditions that have been shown previously to inhibit mTOR.Incubating C-Rap immune complexes with either rapamycin(not shown) or FKBP12 alone was without effect on the phos-phorylation of 4EBP1 (Fig. 2C); however, the combination ofrapamycin plus FKBP12 attenuated phosphorylation of 4EBP1(Fig. 2C). The inhibition of mTOR by rapamycin is unusual inthat to inhibit, rapamycin must first bind to an intracellularreceptor, FKBP12 (35). When complexed with FKBP12, rapa-mycin binds with high affinity to a domain, designated the FRB,which is located upstream of the kinase domain in mTOR.Thus, the requirement for both rapamycin and FKBP12 to sup-press 4EBP1 kinase activity supports the conclusion that theinsulin-stimulated increase in activity is due to mTORC1.Rapamycin is not an active site inhibitor of mTOR, and as
noted in other studies (13, 14), FKBP12-rapamycin did not fullyinhibit the mTOR kinase. In contrast, LY294002, which is3 L. Wang and J. C. Lawrence, Jr., unpublished observations.
FIGURE 1. Insulin activates mTORC1. 3T3-L1 adipocytes were incubated with insulin for 30 min. A, immune complex kinases assays were performed with[�-32P]ATP and 4EBP1 (BP1) proteins as substrates after conducting immunoprecipitations using C-Rap antibodies (Ab) coupled to protein A (PA)-agarose.Nonimmune IgG (NI) was used as a control. B, after SDS-PAGE a phosphorimage of 32P-labeled 4EBP1 and immunoblots of mTOR, raptor, mLST8, andphospho-Thr-36/45 (P-T36/45) were prepared. The effect of insulin (I) on 32P incorporation into 4EBP1 (corrected for mTOR recovery) is expressed as the -foldincrease relative to control (C) (mean � S.E. from 10 experiments). C, mTORC1 kinase assays were conducted using 4EBP1 and F113A as substrates. A CoomassieBlue-stained gel is presented to show the proteins (1 �g of each loaded) that were used as substrate. D, HA-raptor was overexpressed in 3T3-L1 adipocytes byusing an adenoviral vector before mTORC1 was isolated with HA antibodies bound to protein G (PG)-agarose. E, immunoblots of mTOR, raptor, and mLST8recovered with HA antibodies from cells infected with HA-raptor virus (HA-Rap) or with a control virus (�-galactosidase (�-Gal)). Phosphorylation of 4EBP1 inimmune complex kinase assays was assessed by 32P incorporation and immunoblotting with phosphospecific antibodies. The -fold increase in 32P incorpora-tion into 4EBP1 (corrected for recovery of mTOR) produced by insulin is presented (mean � S.E. from three experiments). IP, immunoprecipitate.
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believed to bind in the active site of the kinase, essentially abol-ished activity (Fig. 2C). The kinase activity in the C-Rapimmune complexes was also inhibited by two other inhibitorsof mTOR, caffeine (36) and farnesylthiosalicylate (28) (Fig. 2C).These results provide additional evidence that the kinase activ-ity in C-Rap Ab immunoprecipitates was due to mTORC1.It has been proposed that to promote dissociation of eIF4E
from 4EBP1, mTOR must phosphorylate the 4EBP1-eIF4Ecomplex. Therefore, we compared the phosphorylation of free4EBP1 to that of a complex of recombinant 4EBP1-eIF4E thathad been purified by using m7GTP affinity chromatography.The recombinant proteinswere stainedwithCoomassie Blue toensure that equal amounts of 4EBP1 were added as substrate
(Fig. 2D). The phosphorylation of both the free and the eIF4E-bound forms of 4EBP1 by mTORC1 was increased in responseto insulin. Indeed there was virtually no difference in phospho-rylation of the two forms (Fig. 2D). The 4EBP isoform, 4EBP2,also has a TOS motif, and its phosphorylation in cells is inhib-ited by rapamycin (30). Recombinant 4EBP2 was phosphoryla-ted by mTORC1 (Fig. 2D) but somewhat more slowly than4EBP1.Enhancement of TOS Motif-dependent Binding of 4EBP1 to
mTORC1 by Insulin—Several potential mechanisms may beenvisioned through which insulin could activate mTOR. Forexample, the hormone might act to increase the intrinsic activ-ity of the mTOR kinase. Alternatively kinase activity ofmTORC1 could be enhanced as a result of an increase in sub-strate binding to the complex. To investigate the latter possibil-ity, C-Rap Ab immune complexes were incubated with purified4EBP1 (Fig. 3A). 4EBP1 that bound to raptor was detected byimmunoblotting after washing complexes to remove theunbound protein (Fig. 3B). Insulin increased 4EBP1 binding by�5-fold (Fig. 3C). Little if any endogenous 4EBP1 was found inthe C-RapAb immune complexes (Fig. 3B), an observation thatagrees with previous findings of Hara et al. (6). Thus, the resultswere not complicated by differences in the occupancy of bind-ing sites in raptor with endogenous 4EBP1. Phosphorylation of4EBP1has been shown to inhibit binding of 4EBP1 to raptor (6).Therefore, it seems likely that the failure to recover endogenous4EBP1 in C-Rap immune complexes is due to the fact that evenin control cells 4EBP1 is phosphorylated to some extent (11).Disrupting the TOSmotif dramatically decreased binding of
4EBP1 to the immune complexes (Fig. 3B), and binding ofF113A tomTORC1was not increased by insulin (Fig. 3C). Coo-massie Blue staining confirmed that the same amounts of thetwo recombinant proteins were added to the reaction (forexample, see Fig. 1C). Moreover the 4EBP1 antibodies usedhave been shown to recognize 4EBP1 and F113A equally well(22). Hence the failure to detect F113Abinding to theC-RapAbimmune complexes was due to the effect of the mutation oninhibiting the interaction with raptor and not to inability of the4EBP1 antibody to recognize F113A.As another approach to investigate binding, recombinant
4EBP1 proteins were coupled to agarose beads, which werethen used as an affinity resin to bind mTORC1 (Fig. 3D). Adi-pocyte extracts were incubated with the beads, which werewashed before preparing mTOR and raptor immunoblots (Fig.3E). Insulin markedly increased the amounts of both mTORand raptor retained by the beads. Binding of raptor was inhib-ited by supplementing extracts with 4EBP1 but not by addingF113A (Fig. 3F ). Moreover neither mTOR nor raptor wasretained by F113A beads (Fig. 3E), indicating that the recoveryof mTORC1 required a functional TOS motif. Because equalamounts of 4EBP1 and F113A were coupled to the beads, wewere confident that the failure of the F113A beads to captureraptor was not due to the absence of the mutant protein on thebeads. Nevertheless we conducted experiments to confirmindependently that the F113A protein was present and that theintegrity of the protein had not been destroyed by the couplingprocess. Because the TOSmotif does not participate in bindingof 4EBP1 to eIF4E (10), functionality of the F113A beads could
FIGURE 2. Time course of phosphorylation, effects of inhibitors, and phos-phorylation of 4EBP-eIF4E complexes by mTORC1. 3T3-L1 adipocyteswere incubated without or with insulin for 30 min before extracts were pre-pared. A, mTORC1 and mTORC2 were immunoprecipitated by using antibod-ies to raptor (C-Rap Ab) and rictor, respectively. The relative amounts of mTOR,raptor, and rictor associated with the two mTOR complexes were determinedby immunoblotting. The shift in mobility of rictor caused by insulin is indica-tive of covalent modification of the protein. B, mTORC1 and mTORC2 frominsulin-treated cells were incubated with [�-32P]ATP and 4EBP1 for either 30or 60 min. The kinase reactions were stopped by adding SDS sample buffer,and aliquots were subjected to SDS-PAGE. The relative amounts of 32P incor-porated into 4EBP1 were determined by phosphorimaging. The results pre-sented were corrected for the recovery of mTOR, which was estimated fromimmunoblots, and are mean values (�S.E.) from three experiments. C,washed C-Rap Ab immune complexes were incubated for 30 min in kinasereaction mixtures containing no additions (None) or the following: 10 �M
LY294002, 10 �M GST-FKBP12 (FKBP12), 10 �M GST-FKBP12 plus 10 �M rapa-mycin (Rapa), 1 mM caffeine, or 20 �M farnesylthiosalicylate (FTS). A phospho-rimage of 32P-labeled 4EBP1 is presented. D, phosphorylation of free andeIF4E-bound forms of recombinant 4EBP1 or 4EBP2 by C-Rap immune com-plexes or by complexes recovered with nonimmune (NI) IgG was measuredafter Coomassie Blue staining to normalize the 4EBP concentrations in thedifferent preparations. IP, immunoprecipitate; BP-4E, 4EBP1-eIF4E; BP2-4E,4EBP2-elF4E.
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be judged by their ability to bind toeIF4E. Relatively little eIF4E fromcontrol cells was retained. However,eIF4E was readily detected on both4EBP1 beads and F113A beads thathad been incubated with extractsfrom insulin-treated cells (Fig. 3E).The increase in response to insulinis due to the effect of the hormoneon promoting dissociation of theendogenous 4EBP1-eIF4E complex,an action that increases the amountof eIF4E available to bind the resin(37). Thus, both 4EBP1 and F113Abound to the beads were competentto bind eIF4E.Time Course and Concentration
Dependence of the Insulin Responsesand Effects of Growth Factors andInhibitors—With a maximally effec-tive concentration of insulin, in-creases in both binding and activitywere clearly evident after only 2 min(Fig. 4A), and themaximumeffectsonboth were observed after 20 min ofincubation with insulin (Fig. 4B). Tocompare the timecourseofmTORC1activation with the phosphorylationof known effectors of insulin, immu-noblots were prepared with phos-phospecific antibodies to sites in Akt,mTOR, S6K1, and 4EBP1 (Fig. 4A).Insulin markedly increased the phos-phorylation of Ser-473 in Akt. This
response reachedamaximumafter only 2minasdid thephospho-rylation of mTOR in Ser-2448, which is phosphorylated inresponse to an increase in Akt activity. Insulin also markedlyincreased the phosphorylation of sites in 4EBP1 and S6K1. Thetime courses of the stimulatory effects of insulin on 4EBP1 kinaseactivity and raptor binding to 4EBP1 were very similar (Fig. 4B).
The phosphorylation of 4EBP1 and S6K1 (Fig. 4C) inresponse to insulin treatment reached a maximum slightlybefore mTOR kinase activity reached a maximum. This doesnot mean that the activation of mTORC1 observed in Fig. 4B istoo slow to account for phosphorylation of S6K1 and 4EBP1because the time required to reach a new level of phosphoryla-tion following activation of a protein kinase in a cell depends onthe rate of dephosphorylation the protein.Significant effects of insulin on both binding and activity
were observed at a concentration of 6 nM (Fig. 5A), which is inthe physiological range of insulin concentrations. Insulin-like growth factor 1 was as equally effective as insulin inincreasing mTORC1 binding to 4EBP1 (Fig. 5B), mTORC1kinase activity (Fig. 5C), and the activation of Akt and S6K1(Fig. 5D). In contrast, epidermal growth factor, which was atleast as effective as insulin in activating mitogen-activatedprotein kinase, as assessed by the phosphorylation of ERK1/2
FIGURE 3. Activation of mTORC1 by insulin is associated with increased 4EBP1 binding to raptor. 3T3-L1adipocytes were incubated without and with insulin. A, mTORC1 was immunoprecipitated using C-Rap Abbound to protein A-agarose and incubated with recombinant 4EBP1 proteins. B, immunoblots of mTOR, raptor,and 4EBP1 proteins bound after washing immune complexes that had been incubated without or with 4EBP1(wild type (WT)) or F113A. C, relative amounts of 4EBP1 proteins bound, expressed as a percent maximum(means � S.E. from three experiments) are presented. D, mTORC1 was recovered from adipocyte extracts byusing 4EBP1 coupled to agarose beads. E, immunoblots of mTOR, raptor, and eIF4E bound to 4EBP1 beads or toF113A beads. F, extracts from insulin-treated cells were supplemented with recombinant 4EBP1 or F113Abefore incubation with 4EBP1 beads. The amounts of raptor and eIF4E bound were determined by immuno-blotting. CON, control; INS, insulin; PA, protein A; BP1, 4EBP1.
FIGURE 4. Time courses of responses to insulin. 3T3-L1 adipocytes wereincubated for increasing times with insulin. A, mTORC1 was isolated fromextracts by using C-Rap Ab. Recovery of mTOR and raptor in the C-Rap Abcomplexes was monitored by immunoblotting. Kinase activity wasassessed by phosphorimaging after incubating samples of the complexeswith 4EBP1 and [�-32P]ATP. Other samples were incubated with 4EBP1before the beads were washed, and the amount of 4EBP1 bound wasdetermined by immunoblotting. Extract samples were also subjected toSDS-PAGE, and immunoblots were prepared with phosphospecific anti-bodies to sites in 4EBP1, Akt, mTOR, and S6K1. B, mTORC1 kinase activityand 4EBP1 binding expressed as percentages of the respective maximumresponses (means � S.E. of three experiments). C, 4EBP1 and S6K1 phos-phorylation in cells determined from immunoblots with the phospho-Thr-36/45 (P-T36/45) antibodies and the phospho-Thr-389 (P-T389) antibodies,respectively. IP, immunoprecipitate; P-, phospho-.
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(Fig. 5D), did not increase 4EBP1 binding to mTORC1 (Fig.5B) or the kinase activity of the complex (Fig. 5C).To investigate further the pathways leading to the stimula-
tory effects of insulin on mTORC1 activity and 4EBP1 binding,adipocytes were incubated with inhibitors of several signalingpathways. The effects of insulin on both activity and bindingwere abolished by treating adipocytes with wortmannin at aconcentration (100 nM) sufficient to blockAkt activation (Fig. 5,E and F) but too low to inhibit mTOR directly (38). Inhibitingactivation ofmitogen-activated protein kinase withU0126 (Fig.5F) did not attenuate the effect of insulin on the kinase activityor 4EBP1 binding function of mTORC1 (Fig. 5E). Rapamycindid not block 4EBP1 binding tomTORC1.However, rapamycintreatment did reduce mTORC1 kinase activity (Fig. 5E), sug-gesting that the high affinity complex formed when rapamycin
and endogenous FKBP12 bindmTORpersisted following homoge-nization of the cells and immuno-precipitation of mTORC1.Stimulation of Kinase Activity
and Substrate Binding by InsulinRequire IntactmTORC1—Todeter-mine whether the effects of insulinon 4EBP1 binding to raptor wereretained after disrupting mTORC1,extracts were supplemented withTriton X-100 or Nonidet P-40,which dissociate mTOR and raptor(6, 7). These detergents had rela-tively little effect on the recovery ofraptor from insulin-treated cells(Fig. 6A); however, both detergentsabolished the effect of insulin bymarkedly increasing the amount ofraptor retained from controlextracts. The nonionic detergentsalso abolished insulin-stimulated4EBP1 kinase activity (Fig. 6B),although this was not surprising inview of previous studies showingthat nonionic detergents abolish the4EBP1 kinase of mTOR (6). Bothinsulin-stimulated kinase activity(Fig. 6B) and the hormonal effect on4EBP1 binding (Fig. 6A) were pre-served in the presence of Tween 20and CHAPS, which do not causedissociation of mTORC1.Dimeric mTORC1 Is Insulin-
responsive—To define better thenature of the insulin-responsivemTOR signaling complex, extractproteins were size-fractionated byusing a Superose 6 HR 10/30 column(Fig. 7). Immunoblotting fractionsfrom the column with antibodies tomTOR (Fig. 7A) revealed two majorpeaks, centered at fractions 9 and 13
(Fig. 7D). Based on the elution position of standards, we estimatedtheMr of the complexes in the peaks, designatedhighMr peak andlow Mr peak to be 2,000,000 and 840,000, respectively. Threemajor peaks of raptor were detected (Fig. 7B), two in the samepositions as the mTOR peaks and a third centered on fraction 17(Mr � 350,000) (Fig. 8E) where little, if any, mTOR was found.mLST8wasdetected inpeaks located in thepositionof thehighMrand low Mr peaks of mTOR and raptor (Fig. 7C); a third peak ofmLST8 eluted in later fractions where no mTOR or raptor wasdetected (Fig. 7F). Thus, this third peak represents mLST8 notassociated withmTOR complexes.Insulin did not change the elution pattern of mTOR, except
that it consistently caused a small increase in the amount of theprotein found in fraction 11, which was located in the valleybetween the high and lowMr peaks (Fig. 7D). Raptor in fraction
FIGURE 5. Relative effects of insulin, growth factors, and inhibitors on mTORC1. A, 3T3-L1 adipocytes wereincubated for 30 min with the following concentrations of insulin: 0, 0.06, 6, and 600 nM. 4EBP1 binding andkinase activity were assessed in C-Rap immune complexes. The results represent the respective maximumchanges due to insulin and are mean values � S.E. of three experiments. B–D, adipocytes were incubated for 15min without additions (None) or with insulin, insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF),or fibroblast growth factor (FGF) at a concentration of 5 nM. Extracts were incubated with 4EBP1 beads. B, afterwashing the beads to remove unbound proteins, samples were subjected to SDS-PAGE, and immunoblots ofmTOR, raptor, and eIF4E were prepared. C, mTORC1 was immunoprecipitated using C-Rap Ab. The amounts ofmTOR and raptor recovered were determined by immunoblotting. mTORC1 activity was measured using4EBP1 as substrate. A phosphorimage of the 32P-labeled 4EBP1 product and an immunoblot prepared usingphospho-Thr-36/45 (P-T36/45) antibodies are shown. D, extract samples were subjected to SDS-PAGE beforeimmunoblots were prepared using phosphospecific antibodies to sites in Akt, S6K1, and ERK1/2. Results (B–D)are representative of three experiments. E and F, 3T3-L1 adipocytes were incubated without additions (None)or with one of the following: 20 nM rapamycin (Rapa), 100 nM wortmannin (Wm), or 10 �M U0126. After 30 min,insulin was added as indicated, and the incubations were continued for 30 min before extracts were prepared.mTORC1 was immunoprecipitated by using C-Rap Ab. E, mTOR kinase activity and 4EBP1 binding to mTORC1were measured. F, to confirm the effectiveness of the inhibitors, samples of extracts were subjected to SDS-PAGE, and immunoblots were prepared with phosphospecific antibodies to sites in Akt, mTOR, S6K1, andERK1/2. IP, immunoprecipitate; P-, phospho-.
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11 was not changed by insulin, but the amount of raptor in thehigh Mr peak was increased somewhat by the hormone. Theamount of raptor in the lowMr peakwas not changed by insulin,and the amount of mLST8 was not significantly altered by thehormone in either the high or the lowMr peaks (Fig. 7, E and F ).
Insulin increased the 4EBP1 kinase activity measured inC-Rap immune complexes isolated from both peaks of mTOR,although the bulk of the kinase activity was found in the lowMrpeak (Fig. 7I). Insulin also increased the amount of bothmTORand raptor retained by 4EBP1 beads in both the high and lowMrpeaks (Fig. 7, G and H). In contrast, insulin was without effecton the amount of raptor captured from the peak at fraction 17(Fig. 7H). Because nomTORwas detected in this peak, the lackof effect of insulin on raptor binding to 4EBP1 in this fractionprovides additional evidence that insulin action on 4EBP1bind-ing to raptor depends on the association of raptor and mTOR.The predictedMr of anmTOR (Mr � 289,000)-raptor (Mr �
149,000)-mLST8 (Mr � 36,000) heterotrimer is �474,000,which is considerably lower than that of the species in the lowMr peak. Assuming an equal stoichiometry of subunits, a com-plex containing two such heterotrimers has a predicted Mrclosest to the complexes in the low Mr peak. However, because
FIGURE 6. Effects of detergents on the stimulatory effects of insulin onkinase activity and 4EBP1 binding. A, adipocyte extracts were preparedwithout detergents. The following detergents (0.2% final concentration)were added to samples of the extracts: Tween 20 (TW20), Triton X-100(X100), Nonidet P-40 (NP40), and CHAPS (CHAP). Extracts were incubatedwith 4EBP1 beads, which were then washed with buffers containing thesame detergents before the amounts of mTOR and raptor retained by thebeads were determined by immunoblotting. Relative amounts of raptorbound (expressed as percent maximum) were determined. Mean values(�S.E.) from three experiments are presented. B, mTORC1 was isolatedfrom adipocyte extracts in the presence of 0.1% Tween 20 by using C-RapAb. Detergents (0.2% final concentration) were added to the washed com-plexes, which were then incubated with 4EBP1 and [�-32P]ATP. 32P-La-beled 4EBP1 was detected by phosphorimaging. The relative amounts of32P incorporated were determined. Mean values (�1⁄2 the range) fromduplicate experiments are presented. CON, control; INS, insulin.
FIGURE 7. Separation of mTOR and raptor complexes by gel filtration chromatography. 3T3-L1 adipocytes were incubated without (CON) or withinsulin (INS) for 30 min before extracts were prepared and fractionated by using a Superose 6 column as described under “Experimental Procedures.”Samples (100 �l) of each fraction were subjected to SDS-PAGE before mTOR (A), raptor (B), and mLST8 (C) immunoblots were prepared. The arrows onthe mLST8 blots identify the bands corresponding to mLST8, which is resolved from a species of slightly higher electrophoretic mobility that cross-reactswith the mLST8 antibodies. The relative amounts of mTOR (D), raptor (E), and mLST8 (F) in each fraction were determined by band intensities of theimmunoblots. Results are expressed as percentages of the respective maximum peak fraction in each experiment and are means � S.E. of fourexperiments. Samples (900 �l) of fractions were incubated with 4EBP1 beads, which were washed before the amounts of mTOR (G) or raptor (H) retainedby the beads were determined. I, kinase activities in C-Rap immune complexes isolated from 900-�l samples of the fractions were measured andexpressed relative to the peak activity. Results are relative to the respective peak values and are means � S.E. of three experiments. Mr of mTOR andraptor complexes was estimated from the elution positions of blue dextran 2000 (Mr � 2,000,000), thyroglobulin (Mr � 669,000), and ferritin (Mr �440,000).
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the subunits ofmTORC1may associatewith other proteins, thecomposition of the complex cannot be inferred by size fraction-ation experiments alone. To address this issue, we coexpressedAU1-mTOR, FLAG-mTOR, HA-raptor, Myc-raptor, and anon-tagged form of mLST8 in human embryonic kidney 293cells.The overexpressed epitope-tagged forms of mTOR and rap-
tor fractionated in a manner very similar to that of the endog-enous proteins from 3T3-L1 adipocytes (Fig. 8, A and B), indi-cating that the complexes formed after overexpression wererepresentative of the native complexes. The two peaks ofepitope-tagged mTOR proteins were again designated the highand low Mr peaks (Fig. 8C). Three peaks of tagged raptor weredetected, two in exactly the same position as the high and lowMr peaks and a third associated with fractions that did not con-tain AU1- or FLAG-tagged mTOR (Fig. 8D). To examine theinteraction between mTOR and raptor proteins in these col-umn fractions, immunoprecipitations were conducted usingantibodies to one of the unique epitope tags (Fig. 8, E and F).Immunoblotting with antibody to the appropriate tags enabledus to determine whether any two of the tagged overexpressedproteins associated. For example, after isolating complexeswith AU1 antibodies, immunoblotting revealed that FLAG-mTOR, HA-raptor, and Myc-raptor coimmunoprecipitatedwith the AU1-mTOR in the low Mr peak (Fig. 8E). Similarlyimmunoblotting samples of complexes immunoprecipitatedwith HA antibodies indicated that Myc-raptor as well as bothepitope-tagged forms of mTOR coimmunoprecipitated withHA-raptor (Fig. 8F). The associations between epitope-tagged
forms of mTOR and raptor together with the estimated sizes ofthe complexes from the experiments in Fig. 7 are consistentwith the view that the low Mr peak contains dimeric mTORC1(two mTOR-raptor-mLST8 heterotrimers).The estimated size of epitope-tagged raptor not associated
with mTOR (Mr � 350,000) is similar to that (Mr � 300,000)predicted of a raptor dimer. The finding thatMyc-raptor coim-munoprecipitated with HA-raptor (Fig. 8F ) confirms that rap-tor dimers existed in this peak, although we cannot exclude thepossibility that other smaller proteins are also associated withthe raptor dimers. The proteins from the high Mr peak immu-noprecipitated less efficiently than those from the low Mr peak(Fig. 8, E and F ). This complicated the analyses, although wewere able to detect signals indicative of coimmunoprecipita-tion. Thus, it seems likely that multimers of the mTORC1 sub-units are present in the highMr peak, although additional stud-ies will be needed to define the nature of these complexes.
DISCUSSION
Two key findings of the present study are that insulin pro-motes a stable increase in the kinase activity of dimericmTORC1 and that the increase in kinase activity is associatedwith a marked increase in the binding of substrate to raptor.The stimulatory effects of insulin on mTOR activity and bind-ing occurred rapidly and at physiological concentrations of thehormone. Both effects of insulinwere dependent upon theTOSmotif in the substrate, 4EBP1, and it was essential to preserveintact mTORC1 to detect the effect of insulin on kinase activityand 4EBP1 binding.Our studies provide the first evidence of dimeric mTORC1,
although there is recent evidence of complexes containingmul-tiple TOR proteins in yeast and flies. Zhang et al. (39) used abiochemical approach, as well as a genetic strategy involvingintragenic complementation, to demonstrate that Drosophilamelanogaster cells contain functional complexes harboringmultiple TOR proteins. Wullschleger et al. (40) presented evi-dence of a dimeric TORC2 in Saccharomyces cerevisiae. Theseinvestigators also showed that mTOR proteins harboring dif-ferent epitope tags could be coimmunoprecipitated, indicatingthat complexes containing multiple mTOR proteins exist inmammalian cells; however, they did not determine the numberof mTOR proteins found in the coimmunoprecipitating com-plexes or whether the epitope-tagged proteins were inmTORC1 ormTORC2 (40). Although the present experimentsdid not directly address which subunits mediate dimerizationof mTORC1, the results would be consistent with a model sim-ilar to that proposed by Wullschleger et al. (40) in which thestructure is maintained by interactions between the HEAT andFAT domains of a pair of TOR proteins. However, our resultssuggest that interactions between raptor proteins might alsocontribute to the stability of the higher order complex as raptordimers not associatedwithmTORwere detected. In addition todimeric mTORC1, an even larger complex was evident fromthe high Mr peak, which eluted in the position of the Mr2,000,000 marker. The nature of this larger complex is poorlydefined, and it may represent mTORC1 associated with otherproteins, such as the eIF3 complex, which was shown recentlyto bind mTOR (41, 42).
FIGURE 8. Coimmunoprecipitation of epitope-tagged mTORC1 subunitsafter gel filtration chromatography. AU1-mTOR, FLAG-mTOR, HA-raptor,Myc-Raptor, and untagged mLST8 were coexpressed in human embryonickidney 293 cells. The cell extract was fractionated using the Superose 6 col-umn, and immunoblots were prepared to detect epitope-tagged mTOR (A) orraptor (B) proteins in the different fractions. The relative amounts of taggedmTOR (C) or raptor (D) proteins relative to the respective maximum peakfraction were determined. Immunoprecipitations with antibodies to AU1 orHA were conducted, and immunoblots were prepared to identify proteinsthat coimmunoprecipitated with AU1-mTOR (E) or HA-raptor (F). IP,immunoprecipitate.
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We have found that kinase-dead mTOR (43) as well as �rdmTOR, a protein rendered constitutively active by deletion of aputative regulatory domain (24), associatewith raptor and elutein the low Mr peak,3 indicating that formation of the dimericmTORC1 does not depend on mTOR activity. Insulin did notsignificantly change the amount of dimeric mTORC1, indicat-ing that the hormonal control is not due to gross alterations inthe multimeric state of mTOR. Themechanism through whichinsulin activates mTORC1 is still undefined, and at this point,any of the subunits in the complex must be considered as can-didates for mediating the insulin response.There have been significant advances in defining the
upstream elements in the mTOR signaling pathway. The stim-ulation of 4EBP1 phosphorylation by insulin depends on acti-vation ofAkt (23, 24, 44). The rapidity of the phosphorylation ofSer-473 in response to insulin would be consistent with posi-tioning Akt upstream of mTOR (Fig. 4). Insulin also rapidlyincreasedphosphorylationofmTORinSer-2448,which isphos-phorylated in response to Akt activation (2). Recent evidenceindicates that this site is phosphorylated by S6K1 (45, 46),which is downstreamofmTOR.However, rapamycin abolishedS6K1 activation by insulin, but it did not did not abolish Ser-2448 phosphorylation in either primary (23) or 3T3-L1 adipo-cytes (Fig. 5F ). Consequently S6K1 cannot be the only kinasephosphorylating Ser-2448 in adipocytes. In any event, it isunlikely that phosphorylation of Ser-2448 is the primary signalfor activating mTORC1 as Sekulic et al. (24) found that mTORharboring an Ala-2448 mutation, which ablates the Ser-2448phosphorylation site, supported the activation ofmTOR signal-ing by insulin in human embryonic kidney 293 cells.Another link between Akt and mTOR involves the GTP-
binding protein Rheb and the tuberous sclerosis proteins TSC1and TSC2 (1, 2). These two proteins form a complex that sup-presses mTOR activity by functioning as a GTPase-activatingprotein to decrease the active, GTP-bound form of Rheb. TSC2is phosphorylated by Akt, which has been proposed to inhibitthe GTPase-activating function of TSC2 (47, 48), although thispoint remains hypothetical. GST-Rheb-GTP binding to HA-mTOR in cells was associated with an increase in mTOR activ-ity measured in vitro with S6K1-(355–525) (49), a fragmentlacking the TOS motif necessary for binding to raptor. More-over Rheb-GTP did not increase the binding of raptor to 4EBP1(49). Therefore, it would be premature to conclude that Rhebmediates the TOS-dependent effects of insulin on kinase activ-ity and 4EBP1 binding to mTORC1 described in the presentstudy.Although the mechanism is unclear, it is reasonable to sus-
pect that the effect of insulin on increasing binding of 4EBP1 tomTORC1 contributes to the increase in mTOR activity. Theparadigm in which kinase activity is increased as a result ofincreased substrate binding to an associated noncatalytic pro-tein represents an unusual mechanism for protein kinase acti-vation. For such a kinase system to operate efficiently, phospho-rylation of the substrate must decrease its affinity for raptor.Otherwise the turnover number of the enzyme complex wouldbe much too low. Therefore, the finding of Hara et al. (6) thatphosphorylation of 4EBP1 abolishes binding to raptor fulfills an
important requirement for raptor as a substrate-bindingsubunit.The enhanced binding to 4EBP1 may serve a function not
directly related to increasing the kinase activity of mTORC1.For example, the interaction with the 4EBP1-eIF4E complexmight localize mTORC1 at the 5�-end of mRNAs to which the4EBP1-eIF4E complex is bound. In this connection, the recentfindings that insulin increases the association of bothmTORC1and eIF4G with eIF3 (41, 42) are intriguing. These interactions(41, 42) and the increase in binding of mTORC1 to 4EBP1observed in the present study might serve to place eIF4G in afavorable position to bind to eIF4E after 4EBP1 has been phos-phorylated by mTOR.A much more speculative hypothesis is that mTORC1 and
4EBP1 might actually replace eIF4G in an alternative initiationcomplex. By binding to both eIF3 and 4EBP1-eIF4E, mTORC1would be expected to recruit the small ribosomal subunit(bound to eIF3) to the 5�-end of the message. Whether such acomplex would be competent to initiate scanning is of coursehypothetical, but most of the essential initiation factors wouldbe expected to be bound to either eIF3 or the 40 S ribosomalsubunit. This complex would lack the helicase, eIF4A, if eIF4Gwas not bound to eIF3. eIF4A is essential for translation ofmes-sages having secondary structure in the 5�-untranslated region(10); however, messages with unstructured 5�-untranslatedregions can be translated without eIF4A. An interesting impli-cation is that by recruiting mTORC1 and eIF3 to the 5�-endof certain mRNAs, 4EBP1might facilitate translation insteadof acting as a translational repressor. Such a role would helpto understand why 4EBP1, like eIF4G (50), actually increasesbinding of eIF4E to the mRNA cap (51). Future studies areneeded to investigate the role of mTORC1 in the control oftranslation initiation and to identify the modifications in thecomplex that lead to increased 4EBP1 binding and kinaseactivity.
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Activation of Dimeric mTORC1
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