TSC1�TSC2 and Rheb have different effectson TORC1 and TORC2 activityQian Yang*†, Ken Inoki*, Eunjung Kim*, and Kun-Liang Guan*†‡§

*Life Sciences Institute, †Department of Biological Chemistry, and ‡Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109

Communicated by Jack E. Dixon, University of California at San Diego School of Medicine, La Jolla, CA, March 21, 2006 (received for reviewJanuary 23, 2006)

Target of rapamycin (TOR) plays a central role in cell growthregulation by integrating signals from growth factors, nutrients,and cellular energy levels. TOR forms two distinct physical andfunctional complexes, termed TOR complex 1 (TORC1) and TORcomplex 2 (TORC2). TORC1, which is sensitive to rapamycin, reg-ulates translation and cell growth, whereas TORC2, which isinsensitive to rapamycin, regulates cell morphology and cellgrowth. The Ras homology enriched in brain (Rheb) small GTPaseis known to be a key upstream activator of TORC1, although themechanism of Rheb in TORC1 activation remains to be determined.However, the function of Rheb in the TORC2 regulation has notbeen elucidated. By measuring Akt and S6K phosphorylation as afunctional assay for TORC1 and -2, here, we report that dRheb hasan inhibitory effect on dTORC2 activity in Drosophila S2 cells. Thisnegative effect of dRheb on dTORC2 is possibly due to a feedbackmechanism involving dTORC1 and dS6K. We also observed thatRheb does not activate TORC2 in human embryonic kidney 293cells, although it potently stimulates TORC1. Furthermore, tuber-ous sclerosis complex 1 (TSC1) and TSC2, which are negativeregulators of Rheb, have negative and positive effects on TORC1and -2, respectively. Our observations suggest that TSC1�2 andRheb have different effects on the activity of TORC1 and -2, furthersupporting the complexity of TOR regulation.

Akt � S6K � target of rapamycin � tuberous sclerosis complex � Drosophila

Target of rapamycin (TOR) is a protein kinase that belongs to aphosphatidylinositol kinase-related kinase family and is highly

conserved in all eukaryotes (1). The TOR signaling pathway has awide array of functions to regulate cell growth, proliferation,apoptosis, and autophagy by integrating multiple signals fromgrowth factors, energy, and nutrients. Emerging evidence suggeststhat TOR exists in two distinct physical and functional complexes,termed TOR complex 1 (TORC1) and TORC2. TORC1 and -2were initially identified in the budding yeast Saccharomyces cerevi-siae, which has two TOR genes (2). Recently, TORC1 and -2 havealso been characterized in mammalian and Drosophila cells thathave a single TOR gene, termed mTOR and dTOR, respectively(3–6). Both of the mammalian TOR complexes contain mTOR andmammalian homologue of LST8 (mLST8)�G�-like (G�L) (2, 7).However, regulatory associated protein of mTOR (Raptor) andrapamycin-insensitive companion of mTOR (Rictor)�mAVO3 areexclusively associated with TORC1 and -2, respectively (5, 6).Importantly, only TORC1, but not TORC2, is sensitive to inhibitionby rapamycin (2, 5). The prevailing view is that TORC1 mainlyregulates cell growth and protein synthesis by phosphorylating twokey translational regulators eukaryote initiation factor 4E-bindingprotein (4EBP1) and S6K1 (8).

The function of TORC2 is much less well characterized. How-ever, it has been reported that TORC2 regulates the actin cytoskel-eton by modulating protein kinase C and Rho-family small GT-Pases in a rapamycin-insensitive manner (5, 6, 9, 10). One of theexciting findings about TORC2 is that it directly phosphorylatesAkt�PKB at the hydrophobic motif site Ser-473, which is thephosphorylation site of a presumed PDK2, an enzyme not identi-fied until recently (11). Akt�PKB plays essential roles in many

aspects of cellular regulation, including cell growth and cell survival(12). The demonstration that TORC2 has PDK2 activity placesTORC2 at the center stage of cellular signaling and multiplecellular processes. TORC2 does not phosphorylate the wild-typeS6K1 under normal physiological conditions. But, interestingly,TORC2 can phosphorylate a mutant S6K1 that has a deletion of theC-terminal region (Fig. 1A) (13). Both Akt and S6K1 belong to theAGC kinase family. Many AGC kinases, such as Akt, do not containthe C-terminal extension present in S6K1. Therefore, it is specu-lated that TORC2 may also phosphorylate other members of theAGC kinases, such as PKC and SGK (5, 14).

It has been well established that TOR activity is positivelyregulated by the phosphatidylinositol 3-kinase (PI3K) signaling inhigher eukaryotes, such as Drosophila and mammals (8, 15).Furthermore, recent studies have identified that the tumor sup-pressors tuberous sclerosis complex 1 (TSC1) and TSC2 function askey negative regulators of TOR in both Drosophila and mammals(16, 17). Mutation of either the TSC1 or TSC2 gene results in TSC(17). TSC1 and -2 form a physical complex that is important fortheir physiological functions (18). Phosphorylation of S6K1 andeukaryote initiation factor 4E-binding protein is highly elevated inTSC tumor cells, indicating constitutive activation of TORC1 inTSC1�/� or TSC2�/� cells (8, 17). These results demonstrate thatTSC1 and -2 negatively regulate TORC1 activity. The connectionbetween TOR and PI3K is revealed by the observation that Akt,which is activated by the PI3K pathway, phosphorylates and inhibitsTSC2 (19–21). Furthermore, the understanding of the molecularfunction of TSC1�2 in regulating TORC1 was significantly ad-vanced when Ras homology enriched in brain (Rheb) was identifiedas a key component acting downstream of TSC1�2 and upstream ofTOR (22–27). TSC2 inhibits Rheb activity by functioning as aGTPase-activating protein toward Rheb. In addition, Rheb stim-ulates phosphorylation of S6K1 and eukaryote initiation factor4E-binding protein in a TOR-dependent manner. Rheb may di-rectly bind to and stimulate TORC1 function (28). Therefore, asignaling pathway of PI3K-Akt-TSC2-Rheb-TORC1 has been pro-posed. Interestingly, active S6K1, in turn, suppresses the PI3K-Aktpathway by inactivating insulin receptor substrate (IRS) (29, 30).S6K1 has been shown to directly phosphorylate IRS1 and, thereby,attenuates PI3K activation in response to growth factors, such asinsulin. Therefore, S6K1 is a key component in the feedbackregulation of PI3K by TORC1.

The function of TSC1�2 and Rheb in TORC1 regulation is wellestablished. However, no conclusion has been reached as towhether and how TSC1�2 and Rheb regulate TORC2. Here, weshow that, in Drosophila S2 cells, knockdown of dRheb expression

Conflict of interest statement: No conflicts declared.

Abbreviations: G�L, G�-like; HEK, human embryonic kidney; IRS, insulin receptor substrate;MEF, mouse embryonic fibroblast; mLST8, mammalian homologue of LST8; mTOR, mam-malian TOR; PI3K, phosphatidylinositol 3-kinase; Raptor, regulatory associated protein ofTOR; Rheb, Ras homology enriched in brain; Rictor, rapamycin-insensitive companion ofTOR; RNAi, RNA interference; TOR, target of rapamycin; TORC, TOR complex; TSC, tuberoussclerosis complex; HA, hemagglutinin.

§To whom correspondence should be addressed. E-mail: kunliang@umich.edu.

© 2006 by The National Academy of Sciences of the USA

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by RNA interference (RNAi) ablates the phosphorylation ofdS6K1 but enhances the phosphorylation of dAkt. Knockdown ofother components of the TORC1 signaling pathway, includingdRaptor and dS6K1, also increases dAkt phosphorylation. Incontrast, knockdown of dTSC1 or -2 enhances dS6K1 phosphory-lation but inhibits dAkt phosphorylation, suggesting that dRhebpositively regulates dTORC1 while negatively affecting dTORC2.Consistent with these observations, S6K1 phosphorylation is ele-vated, whereas Akt phosphorylation is inhibited in TSC1�/� andTSC2 �/� mammalian cells. Furthermore, Rheb activates TORC1kinase activity but not TORC2, as determined by in vitro kinaseassays. Together, our data demonstrate that TSC1�2 and Rhebhave different effects on TORC1 and -2.

ResultsdRheb, dTSC1, and dTSC2 Have Opposite Effects on the Phosphoryla-tion of dAkt and dS6K. TORC1 and -2 phosphorylate differentsubstrates and have distinct cellular functions. However, TORC1and -2 share two common components, TOR and LST8�G�L.TORC1 has been demonstrated to be activated by Rheb in both

Drosophila and mammalian cells (22–25, 31). However, it is notclear whether Rheb directly activates TORC1 or requires interme-diate molecules. To determine whether Rheb activates TORC2, weused RNAi in cultured Drosophila S2 cells to knock down Dro-sophila Rheb (dRheb) expression. TORC2 activity is examined bymeasuring the phosphorylation of the hydrophobic motif site,Ser-505, of Drosophila Akt (dAkt), which is a direct physiologicalsubstrate of TORC2 (Fig. 1A) (11). In parallel, TORC1 activity ismeasured by the phosphorylation of the hydrophobic motif site,Thr-398, of Drosophila S6K (dS6K). Knockdown of dRheb expres-sion with dsRNAs promoted the phosphorylation of dAkt andinhibited the phosphorylation of dS6K (Fig. 1B). In addition,knockdown of dRheb enhanced insulin-stimulated dAkt phosphor-ylation but blocked dS6K phosphorylation (Fig. 1B). These datashow that dRheb displays opposite effects on dTORC1 and -2.dRheb plays a positive role in the phosphorylation of the dTORC1substrate dS6K and a negative role in the phosphorylation of theTORC2 substrate dAkt.

As expected, knockdown of dTSC1 or -2 significantly increaseddS6K phosphorylation. Interestingly, knockdown of either dTSC1or -2 expression decreased dAkt phosphorylation, especially inresponse to insulin stimulation (Fig. 1C), indicating that dTSC1 and-2 stimulate dTORC2. The above observations are consistent withdata obtained with dRheb knockdown, therefore further support-ing the notion that dRheb activates dTORC1 and inhibits dTORC2.However, the above data cannot distinguish whether dRheb inhibitsdTORC2 directly or indirectly.

TORC2 Mediates the Inhibitory Effect of dRheb on dAkt Phosphory-lation. We examined the effects of components in the TORsignaling pathway on phosphorylation of dAkt and dS6K. Knock-down of dS6K increased dAkt phosphorylation; however, knock-down of dAkt did not significantly inhibit dS6K phosphorylation(Fig. 2A). These results are consistent with two reports that dAktdoes not play a major role in dS6K phosphorylation in Drosophilacells (11, 32). However, Lizcano et al. (33) reported that knockdownof dAkt inhibited the phosphorylation of dS6K. The reasons for thisdiscrepancy are not obvious; therefore, further studies are neededto clarify the function of dAkt in dTORC1 regulation in DrosophilaS2 cells.

As expected, knockdown of dRaptor decreased dS6K phosphor-ylation but increased dAkt phosphorylation (Fig. 2A). These resultsare consistent with the current model that Raptor is a key compo-nent present in only TORC1 (3–6). Furthermore, knockdown ofdRictor decreased dAkt phosphorylation and reproducibly causeda moderate increase in dS6K phosphorylation. Moreover, knock-down of dTOR decreased phosphorylation of both dAkt and dS6K,consistent with the role of dTOR in both dTORC1 and -2. It shouldbe noted that knockdown of dRictor and dTOR caused only partialreductions in dAkt phosphorylation, although the results are readilyreproducible (Fig. 2A). These results suggest the presence of anadditional source of PDK2 activity in S2 cells or an incompleteknockdown by RNAi experiments. Consistent with the later pos-sibility, knockdown of dTOR did not completely eliminate phos-phorylation of dS6K (Fig. 2A), whereas rapamycin completelyeliminated dS6K phosphorylation under the same conditions (datanot shown). We also noticed that dLST8 RNAi did not inhibit dS6Kphosphorylation, whereas mLST8 RNAi inhibits S6K1 phosphor-ylation in human embryonic kidney 293T (HEK293T) cells (7). Areasonable explanation could be that dLST8 is not as potent fordTORC1 function in Drosophila S2 cells as it is in HEK293T cells.Knockdown of dPTEN increased phosphorylation of both dAktand dS6K (Fig. 2A), supporting the fact that both TORC1 and -2are stimulated by PI3K signaling.

PDK1 phosphorylates the activation loop of S6K1 and Akt (34,35). It has been reported that knockout of PDK1 eliminatesphosphorylation of S6K1 on both the activation loop Thr-229 andthe hydrophobic motif site Thr-389 in mammalian cells (36),

Fig. 1. Rheb and TSC1�2 show opposite effects on TORC1 and -2 in Drosoph-ila S2 cells. (A) Schematic illustration of S6K1 and Akt as functional readoutsof TORC1 and -2, respectively. S6K1 was used as a functional readout of TORC1activity, whereas S6K1 3A��C and Akt were used as functional indicators ofTORC2 activity. Phosphospecific antibodies recognizing Ser-473 of Akt andThr-389 of S6K1 were used to detect Akt and S6K1 activity, respectively. (B)dRheb RNAi promotes dAkt phosphorylation and inhibits dS6K phosphoryla-tion. Drosophila S2 cells were treated with 4 �g of dRheb dsRNA as indicated.Insulin stimulation (600 ng�ml insulin for 30 min) is indicated. The phosphor-ylation of dAkt and dS6K were detected by the phosphospecific antibodiesrecognizing mammalian Akt Ser-473 and S6K1 Thr-389, respectively. To avoidconfusion, we kept the labeling as p-dAkt(S505) and p-dS6K(T398) for Dro-sophila proteins and p-Akt(S473) and p-S6K1(T389) for mammalian proteins inall figures, although the same phosphospecific antibodies were used. dS6Kprotein was detected by Drosophila anti-dS6K antibody, and dAkt protein wasmonitored by mammalian anti-Akt antibody. The two Akt isoforms generatedby alternative splicing are indicated by arrows. The phospho-Akt antibody alsodetected a nonspecific band between the two Akt splicing forms. It should benoted that the phospho-Akt Western blot of the samples treated with insulinwas exposed for a much shorter time than the corresponding Western blot ofsamples without insulin treatment. (C) Knockdown of dTSC1 or -2 by RNAidecreased dAkt phosphorylation but enhanced dS6K phosphorylation. Exper-iments were performed similarly to those described in B.

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suggesting that PDK1 activity is required for TORC1 activity or thatthe phosphorylation of the activation loop by PDK1 is necessary forThr-389 phosphorylation by TORC1. In contrast, PDK1 knockoutseliminate only the activation loop Thr-308 phosphorylation in Aktbut not the hydrophobic motif site Ser-473 phosphorylation, indi-cating that the PDK1-dependent phosphorylation of activation loopof Akt is not required for phosphorylation of Ser-473 by PDK2 (37).Consistent with the mammalian model, we observed that knock-down of dPDK1 inhibited TORC1-dependent dS6K phosphoryla-tion but did not inhibit dAkt phosphorylation (Fig. 2A). In fact,knockdown of dPDK1 increased TORC2-dependent dAkt phos-phorylation. Together, our data suggest that TORC1 and -2 areregulated differently, and the two TOR complexes may negativelyaffect each other.

To further test the relationship between Rheb and TORCcomponents in dS6K and dAkt phosphorylation, we used a com-bination of different RNAis. Knockdown of dRheb elevated dAktphosphorylation. The dRheb RNAis-induced dAkt phosphoryla-tion was blocked by a simultaneous knockdown of any of theTORC2 components, including dTOR, dLST8, or dRictor (Fig.2B). In contrast, knockdown of dRaptor, which is only present inTORC1, or dS6K, which is a downstream target of TORC1, had

little effect on dAkt phosphorylation induced by Rheb RNAi.These results show that dAkt phosphorylation induced by dRhebknockdown depends on TORC2. As expected, down-regulation ofdTOR and dRaptor further decreased dS6K phosphorylation in thepresence of dRheb RNAi. These observations support our modelthat dRheb has opposite effects on TORC1 and -2. However, theabove data do not distinguish whether dRheb inhibits TOR2directly or indirectly. We performed a similar combinatory RNAiof dRaptor and other TORC components. Knockdown of dRaptorincreased dAkt phosphorylation, and this effect was blocked byknockdown of dLST8, dTOR, and dRictor (Fig. 2C), supporting theimportance of TORC2 in dAkt phosphorylation.

A possible mechanism for Rheb to inhibit TORC2 is the feed-back inhibition of IRS by S6K1. Rheb activates TORC1, which thenactivates S6K1, which, in turn, phosphorylates and inhibits IRS. Weexamined the effect of the Drosophila IRS gene, Chico. When ChicodsRNA was combined with dRheb, dRaptor, dS6K, dPDK1, ordPTEN, the phosphorylation of dAkt was inhibited (Fig. 2D). Ourresults obtained in Drosophila cells suggest that dRheb regulatesTORC1 and -2 oppositely. It is worth noting that knockdown ofeither dRheb or dRaptor produced a stronger dAkt phosphoryla-tion than the knockdown of dS6K. These data indicate that a

Fig. 2. Knockdown of the TOR signaling pathway components indicates that dRheb inhibits dTORC2 in vivo. (A) Effects of knockdown of TOR signaling pathwaycomponents on phosphorylation of dAkt and dS6K. Drosophila S2 were treated with individual dsRNA as indicated. Where indicated, cells were treated with600 ng�ml insulin for 30 min. The phosphorylation of dS6K and dAkt, as well as the protein levels, were detected by the antibodies described in Fig. 1B. (B) Theenhancement of dAkt phosphorylation by Rheb RNAi depends on TORC2 components. Drosophila S2 cells were treated with Rheb dsRNA and other componentsof the TOR pathway as indicated. Phosphorylation of dS6K and dAkt, as well as the protein levels, were detected by the antibodies described in Fig. 1B. (C) Theeffect of dRaptor RNAi on dAkt phosphorylation depends on TORC2. Drosophila S2 cells were cultured and treated similarly to that described in A. Fourmicrograms of each dsRNA was used individually or in combination with dRaptor. The phosphorylation of dS6K and dAkt and the protein levels were detectedby the antibodies described in Fig. 1B. (D) Chico RNAi inhibits TORC2 activity. Drosophila S2 cells were cultured in 12-well plates and treated for 4 days withindicated dsRNA(s). Four micrograms of dsRNA was used for each target gene and added to each well on days 1 and 3. Two Chico dsRNAs, targeting differentcoding regions of the Chico gene, were used to determine the RNAi effect. dAkt phosphorylation and dS6K phosphorylation were determined by pAkt (Ser-473)antibody and pS6K1 (Thr-389) antibody, respectively. Protein levels were monitored by anti-Akt and anti-dS6K antibodies. (E) Amino acids have opposite effectson the phosphorylation of dAkt and dS6K. S2 cells were starved for amino acids for 30 min and restimulated with amino acids for 10 min as indicated. The aminoacid-absent medium was made based on the published Schneider’s Drosophila medium (GIBCO BRL) recipe, by removing the amino acids and yeastolate.Phosphorylation and protein levels of dAkt and dS6K were determined. RNAi of dRheb and dRas were indicated as controls. The Drosophila dAkt phospho-antibody, pdAkt(Ser-505), was used in the Western blot of E. The Drosophila dAkt phosphoantibody does not detect the nonspecific band between the two dAktisoforms. Western blotting with the Drosophila pdAkt(Ser-505) antibody produced results similar to those obtained by an anti-mammalian pAkt(Ser-473)antibody.

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negative feedback inhibition by dS6K cannot be the sole factormediating the inhibitory effect of dRheb on dAkt (Fig. 2D). dRhebmay affect the balance between the two TOR complexes, therebyexerting its positive effect on dTORC1 and negative effect ondTORC2.

TORC1 is known to be regulated by nutrients, such as aminoacids (8, 38). We observed that removal of amino acids induced adramatic dephosphorylation of dS6K (Fig. 2E). Interestingly,amino acid starvation increased dAkt phosphorylation. Addition ofamino acids stimulated dS6K and, at the same time, reversed thenutrient starvation-induced dAkt phosphorylation. It is worth not-ing that the effects of amino acids on dS6K and dAkt can be readilyobserved within 10 minutes. Rapamycin effectively blocked theeffects of amino acids on the phosphorylation of both dS6K anddAkt (Fig. 5, which is published as supporting information on thePNAS web site). These results demonstrate that TORC1 and -2 areregulated differently by amino acids.

Rheb Stimulates Phosphorylation of the TORC1 Substrate S6K1 but Notthe TORC2 Substrates Akt and S6K1 3A��C. To examine the regula-tion of the TOR complexes by Rheb in mammalian cells, we useda S6K1 mutant containing both a TOS motif mutation and aC-terminal deletion (S6K1 3A��C) (13, 39). Mutation of the TOSmotif renders this S6K1 unphosphorylatable by TORC1 (Fig. 1A).As a result, phosphorylation of S6K1 3A��C on Thr-389 would beresistant to rapamycin inhibition. Interestingly, S6K1 3A��C canstill be phosphorylated by TORC2 as a result of the deletion of theC-terminal extension. We used the phosphorylation of S6K1 as theindicator of TORC1 activity and the phosphorylation of S6K13A��C and Akt as the indicators of TORC2 activity (Fig. 1A). Asexpected, rapamycin inhibited phosphorylation of S6K1 but not Aktor S6K1 3A��C in HEK293 cells (Fig. 3A). Rheb stimulated S6K1phosphorylation (Fig. 3A), and the stimulatory effect of Rheb onS6K1 was completely inhibited by rapamycin. Rheb, however, alsoslightly activated S6K1 3A��C. We suspected that the modestincrease in phosphorylation of S6K1 3A��C was due to a slightresponsiveness of S6K1 3A��C to TORC1. Therefore, we treatedthe cells with rapamycin to exclude the TORC1 effect. Indeed, thephosphorylation of S6K1 3A��C reduced to the basal level afterrapamycin treatment. In agreement with the results obtained withS6K1 3A��C, Akt phosphorylation was not stimulated by Rheb(Fig. 3A). In addition, LY294002, a PI3K inhibitor, inhibited thephosphorylation of wild-type S6K1, S6K1 3A��C, and Akt (Fig.3A). These results are consistent with the notion that both TORC1and -2 are regulated by the PI3K pathway. The above data show thatRheb activates TORC1 but not TORC2 in vivo, as determined bythe phosphorylation of S6K1, S6K1 3A��C, and Akt. A noticeabledifference from the Drosophila S2 results is that Rheb does notinhibit TORC2 in HEK293 cells (Fig. 3A).

We further tested the effect of TSC1�2. Expression of TSC1�2inhibited phosphorylation of the cotransfected S6K1 but not S6K13A��C (Fig. 3B). In fact, TSC1�2 slightly increased the phosphor-ylation of S6K1 3A��C, indicating that TSC1�2 regulate TORC1and -2 oppositely. To further demonstrate the differential regula-tion of TORC1 and -2, the effects of various stimulation conditionson the phosphorylation of TORC1 and -2 substrates were exam-ined. We found that phosphorylation of S6K1 3A��C and Akt aresimilarly regulated by various extracellular stimuli. Consistent withour earlier results, phosphorylation of S6K1 was regulated differ-ently from Akt and S6K1 3A��C (Fig. 6, which is published assupporting information on the PNAS web site). For example,energy starvation induced by 2-deoxy-glucose strongly inhibitedphosphorylation of S6K1 but had little effect on the phosphoryla-tion of S6K1 3A��C and Akt. Collectively, our data indicate thatTORC1 and -2 are regulated differently by TSC1�2 and intracel-lular energy levels.

In TSC mutant mouse embryonic fibroblast (MEF) cells, bothRheb and TORC1 are constitutively active, as determined by the

high S6K1 phosphorylation (Fig. 4 A and B). Both TSC1�/� andTSC2�/� MEF cells have higher basal S6K1 phosphorylation andlower Akt phosphorylation than the wild-type MEF cells. Theseresults suggest that TSC1�2 negatively regulates TORC1 andpositively regulates TORC2. It has been reported that constitutivelyactive S6K1 causes an inhibition of IRS (29, 30). The inhibition ofIRS by S6K1 leads to a decreased response to insulin stimulationand, therefore, decreases TORC2 activity. Prolonged rapamycintreatment, which inhibits S6K1 activity, should restore the insulin

Fig. 3. Regulation of S6K1, Akt, and S6K1 3A��C phosphorylation by Rheband TSC1�2. (A) Rheb stimulates only the phosphorylation of S6K1 but not Aktor the rapamycin-resistant phosphorylation of S6K1 3A��C. HA-S6K1, GST-Akt, and HA-S6K1 3A��C were transfected into HEK293 cells in the presenceor absence of MYC-Rheb (50 ng). The phosphorylation of S6K1 and S6K13A��C were detected by the phospho-specific antibody recognizing S6K1Thr-389, whereas the phosphorylation of Akt was detected by the phospho-Akt antibody, pAkt (Ser-473). Protein levels were determined by using anti-HA(S6K1 and S6K1 3A��C), anti-GST (transfected GST-Akt), anti-Akt (endoge-nous Akt), and anti-MYC (Rheb) antibodies. Cells were treated with either 50�M LY294002 or 25 nM rapamycin for 30 min as indicated. (B) TSC1�2 inhibitonly S6K1 phosphorylation but not S6K1 3A��C. HEK293 cells were trans-fected with HA-S6K1 and HA-S6K1 3A��C. MYC-Rheb (25 ng) or MYC-TSC1(250 ng)�HA-TSC2 (250 ng) was cotransfected where indicated. The phosphor-ylation was detected by pS6K1 (Thr-389) antibody, and the protein level wasdetermined by either anti-HA or anti-MYC antibody.

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response in the TSC�/� MEF cells. As expected, rapamycin treat-ment dramatically enhanced insulin-stimulated Akt phosphoryla-tion in TSC�/� MEF cells to an extent similar to the TSC wild-typeMEF cells (Fig. 4 A and B). These results support the idea that theS6K1-mediated feedback inhibition contributes to the inhibition of

TORC2 by Rheb. However, rapamycin could not enhance the basalAkt phosphorylation in either the TSC1�/� or the TSC2�/� MEFsto the extent as that observed in the TSC wild-type cells, eventhough rapamycin completely blocked S6K1 phosphorylation (Fig.4 A and B). These results suggest that the feedback inhibition byS6K1 only partly contributes to the effect of TSC1�2 on Aktphosphorylation and TORC2 activity, indicating that TSC1�2 mayalso regulate TORC2 in a more direct manner. Consistently,down-regulation of dRheb and dRaptor produced a more dramaticincrease of dAkt phosphorylation than down-regulation of dS6K(Fig. 2D).

Rheb has been shown to stimulate S6K1 phosphorylation (anindirect in vivo assay for TORC1 activation) in transfected cells, butan increase in TORC1 kinase activity by Rheb has not beendemonstrated in vitro. We wanted to more directly test the effect ofRheb on the kinase activity of TORC1 and -2. We measuredTORC1 and -2 kinase activity in vitro using purified GST-S6K1 andGST-Akt as substrates. Hypophosphorylated GST-S6K1 was puri-fied from rapamycin-treated HEK293 cells that had been trans-fected with a GST-S6K1 expression vector. Similarly, hypophos-phorylated GST-Akt was purified from transfected HEK293 cellsthat had been treated with LY294002. TORC1 was immunopre-cipitated by Raptor, whereas TORC2 was immunoprecipitated byRictor�mAVO3. We found that TORC1 isolated from transfectedHEK293 cells phosphorylated GST-S6K1 on Thr-389 in vitro butcould not phosphorylate recombinant GST-Akt on Ser-473 (Fig.4C). The phosphorylation of GST-S6K1 was dramatically enhancedwhen TORC1 was isolated from Rheb-coexpressed cells, althoughthe mTOR levels were equivalent in the immunoprecipitates.Therefore, we presented in vitro biochemical evidence that Rhebtransfection, indeed, stimulated TORC1 kinase activity. It is worthnoting that Rheb coexpression caused a mobility shift of Raptor(Fig. 4C), which is due to phosphorylation (data not shown). Futurestudy will be required to determine whether the increased phos-phorylation of Raptor by Rheb contributes to TORC1 activation.Consistent with a previous report (11), TORC2 immunoprecipi-tated from HEK293 cells phosphorylated Ser-473 of GST-Akt invitro but could not phosphorylate Thr-389 of the recombinantGST-S6K1 (Fig. 4C). Importantly, Rheb coexpression did notincrease the ability of TORC2 to phosphorylate GST-Akt in vitro.These results clearly demonstrate that Rheb activates TORC1 butnot TORC2.

DiscussionThe recent identification of TORC1 and -2 significantly increasedthe importance of TOR in cell growth regulation and expanded thecomplexity of the TOR signaling network. Although the two TORcomplexes share common components, they display distinct cellularfunctions and phosphorylate different downstream substrates.Studies have demonstrated that TSC1 and -2 suppress TORC1activity by inhibiting Rheb (22–25, 31). It has been established thatRheb is a key upstream activator of TORC1; however, whetherRheb regulates TORC2 in a manner similar to TORC1 had notbeen answered.

Our data show that Rheb does not activate TORC2, althoughRheb is a potent activator of TORC1 in mammalian cells. More-over, dRheb displays an inhibitory effect on dTORC2 in DrosophilaS2 cells. Consistent with this result, the TSC�/� MEF cells, whichhave elevated activities of both Rheb and TORC1, show a low Aktphosphorylation on S473, indicating a decreased TORC2 activity.In other words, TSC1�2 inhibit TORC1 but display a positive effecton TORC2. Together with the direct regulation of Rheb byTSC1�2, it is consistent that Rheb activates TORC1 and inhibitsTORC2 in MEF cells. However, the inhibitory effect of Rhebtoward TORC2 may not be direct. We propose that the negativeeffect of dRheb on dTORC2 is, in large part, due to a feedbackinhibitory loop involving dS6K in the Drosophila S2. It is worthnoting that overexpression of Rheb did not cause a significant

Fig. 4. TSC and Rheb regulate TORC1 and -2 differently both in vivo and in vitro.(A) TSC1 knockout increases S6K1 phosphorylation and decreases Akt phosphor-ylation. MEF cells were cultured in six-well plates for 48 h before analysis. Cellswere treated with rapamycin (25 nM) for 3 or 24 h where indicated. Insulin (600ng�ml) was added for 30 min as indicated. The phosphorylation of Akt and S6K1was detected by pAkt (Ser-473) and pS6K1 (Thr-389) antibody, respectively.Endogenous protein levels were detected by anti-Akt and Anti-S6K1 antibodies.(B) TSC2 knockout increases S6K1 phosphorylation and inhibits Akt phosphory-lation. Experiments were similar to those described in A, except that TSC2�/� cellswere used. (C) Rheb promotes TORC1 activity but has no effect on TORC2 activity.HEK293 cells were cultured in 10-cm plates and transfected with either TORC1components (MYC-mTOR, HA-Raptor and MYC-mLST8�G�L) or TORC2 compo-nents (MYC-mTOR, HA-Rictor�mAVO3 and MYC-mLST8�G�L). MYC-Rheb (200ng) was cotransfected where indicated. TORC1 and -2 were immunoprecipitatedby anti-HA (HA-Raptor or HA-Rictor�mAVO3) antibody. GST-Akt and GST-S6K1,purified from HEK293 cells, were used as substrates for the in vitro kinase assays.The phosphorylation of GST-Akt and GST-S6K1 was detected by pAkt (Ser-473)and pS6K1 (Thr-389) antibody, respectively. Coimmunoprecipitated mTOR wasdetectedbytheanti-mTORantibody,whereas theprotein levelsofHA-RictorandHA-Raptor were detected by anti-HA antibody. The amount of substrates (GST-Akt and GST-S6K1) was determined by Coomassie blue staining as indicated.

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inhibition of Akt phosphorylation in HEK293 cells. In these cells,PTEN activity is relatively low, and TORC2 activity is high.Therefore, the effect of overexpressed Rheb on TORC2 may beoverridden by the low PTEN activity in HEK293 cells. Consistentwith the hypothesis that Rheb has an inhibitory effect on TORC2,TSC1�2 have a positive effect on the phosphorylation of Akt, asdetermined in both the knockout and overexpression cells (29, 30).Our data support the argument that Rheb activates TORC1 in amore direct manner, possibly by a direct interaction. However, wedo not suggest that Rheb directly inhibits TORC2. On the contrary,the inhibitory effect of Rheb on TORC2 may be rather indirect,certainly including a feedback inhibition involving TORC1 anddownstream targets.

We observed that the TORC1-specific component Raptor has aninhibitory effect on TORC2, whereas the TORC2-specific compo-nent Rictor, reciprocally, has an inhibitory effect on TORC1. Apossible molecular basis for these mutually inhibitory effects is thatRaptor and Rictor may compete for common components of theTOR complexes, such as mTOR and mLST8. Rheb may activateTORC1 and inhibit TORC2 by increasing the ratio of TORC1 toTORC2 in cells. In addition to the negative feedback regulation, theabove model suggests another mechanism by which Rheb inhibitsTORC2. In conclusion, our data from both Drosophila S2 andmammalian cells support a model in which the effect of TSC1�2 andRheb on TORC1 and -2 are different.

Materials and MethodsDrosophila RNAi. Drosophila RNAi experiments were performed asdescribed in ref. 40, with minor modification (see SupportingMaterials and Methods and Table 1, which are published as sup-porting information on the PNAS web site). Drosophila S2 cellswere cultured in 12-well plates for 4 days, with a starting density of2 � 105 cells per well. On days 1 and 3, 4 �g of dsRNA targetingthe gene of interest was added directly to the cells. Cells were lysedat the end of day 4, with 150 �l per well of the mild lysis buffer (10

mM Tris�HCl, pH 7.5�100 mM NaCl�1% Nonidet P-40�50 mMNaF�2 mM EDTA�1 mM DTT�1 mM PMSF�10 �g/ml leupep-tin�10 �g/ml aprotinin). Cell lysates were analyzed by Western blot.

Immunoprecipitations and Kinase Assay. HEK293 cells were culturedin 10-cm plates. When the cell density reached 3 � 106 cells perplate, cells were transfected with either TORC1 components[MYC-mTOR, hemagglutinin (HA)-Raptor, and MYC-mLST8�G�L] or TORC2 components (MYC-mTOR, HA-Rictor�mAVO3, and MYC-mLST8�G�L); MYC-Rheb (200 ng) wascotransfected where indicated. Cells were lysed in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS) lysis buffer as described in ref. 3. To immunoprecipitateTORC1 or -2, 1 �g of anti-HA (HA-Raptor or HA-Rictor�mAVO3) antibody was added to each of the cellular lysates andincubated at 4°C for 90 min. Then 20 �l of protein G Sepharoseslurry (50%) was added to the lysates and incubated for anotherhour. Immunoprecipitates were washed four times in the lysisbuffer. At the last wash, the beads were divided equally for parallelexperiments. For kinase assay, the immunoprecipitate was firstwashed once with kinase buffer and then incubated in 15 �l ofkinase assay reaction mix at 37°C for 30 min. Kinase assay reactionswere designed as reported in refs. 3 and 11. To stop the reaction,5 �l of 4� SDS sample buffer was added to each reaction, whichwas then boiled for 5 min.

We thank Drs. Mary Stewart (North Dakota State University, Fargo,ND), David Kwiatkowski (Harvard University, Cambridge, MA), DavidSabatini (Massachusetts Institute of Technology, Cambridge, MA),Michael Hall (University of Basel, Basel), and John Blenis (HarvardMedical School, Boston) for reagents; Dr. Huira Chong for constructionof plasmid; and Chung-Han Lee for critical reading of the manuscript.Q.Y. is partially supported by a Rackham Predoctoral Fellowship of theUniversity of Michigan. This work is supported by grants from theNational Institutes of Health and the Department of Defense (toK.-L.G.).

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