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1 Biosignal Research Center, Kobe University, Kobe 657-8501, Japa
2 CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
3 Department of Geriatric Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan
4 Department of Molecular Biology and the Diabetes Unit, Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA
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Abstract |
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Introduction |
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Recent work has established that the TOR polypeptide functions in a complex with two other proteins, raptor (Hara et al. 2002; Kim et al. 2002)/Kog1p (Loewith et al. 2002) and GßL (also known as mLST8) (Kim et al. 2003; Loewith et al. 2002). The mTOR/raptor interaction requires multiple sites across the length of raptor, which binds predominantly, but not exclusively to the amino terminal half of mTOR (Kim et al. 2002). GßL appears to bind to the mTOR catalytic domain and independently to raptor (Kim et al. 2003).
Raptor is a 150-kD polypeptide whose association with TOR is indispensable for TOR signalling in vivo, as shown by RNA interference (RNAi) in cultured mammalian cells (Kim et al. 2002) and Caenorhabditis elegans (Hara et al. 2002). Raptor serves as a scaffold for the apposition of mTOR with its substrates 4E-BP1 and p70S6k. Raptor does not alter the catalytic activity of mTOR; rather, raptor binds p70S6k and to 4E-BP1 and the association of raptor with mTOR increases mTOR phosphorylation of p70S6k in vitro four to five fold (Kim et al. 2002; Nojima et al. 2003) and is required absolutely for the mTOR-catalysed phosphorylation of 4E-BP1 in vitro (Hara et al. 2002; Nojima et al. 2003). The binding of p70S6k and the 4E-BPs to raptor is mediated by a short conserved segment on these TOR substrates, the TOR signalling motif (TOS motif) (Schalm & Blenis 2002). Mutation of the 4E-BP1 TOS motif greatly inhibits 4E-BP1 phosphorylation in vivo (Schalm & Blenis 2002), and eliminates completely mTOR-catalysed 4E-BP1 phosphorylation in vitro (Choi et al. 2003; Nojima et al. 2003; Schalm et al. 2003). Mutation or deletion of the TOS motif on p70S6k greatly inhibits its activity and the activating phosphorylation at Thr412 in vivo (Schalm & Blenis 2002; Weng et al. 1998). Additional deletion of the p70S6k carboxy terminal tail partially restores p70S6k Thr412 phosphorylation and catalytic activity, however, the doubly mutant enzyme is now resistant to inhibition in vivo by rapamycin (Weng et al. 1995, 1998) and by withdrawal of ambient amino acids (Hara et al. 1998). These results indicate that the ability of p70S6k to bind to raptor and to be sensitive to inhibition by rapamycin and amino acid deficiency are closely linked.
The molecular mechanisms by which the binding of the FKBP12/rapamycin complex to the TOR FRB domain interferes with TOR signalling remain incompletely understood. Rapamycin inhibits p70S6k activity and 4E-BP1 phosphorylation in vivo with an IC50 of 2–5 nM (Chung et al. 1992; Price et al. 1992; Weng et al. 1995). The addition in vitro of rapamycin to mTOR in the presence of excess of FKBP12 does result in inhibition of the mTOR kinase activity toward p70S6k or 4E-BP1, however, nearly one hundred fold higher concentration of rapamycin is required than is necessary for inhibition in vivo (Brown et al. 1995; Brunn et al. 1997; Isotani et al. 1999). Moreover, the phosphorylation in vivo of mTOR Ser2481, a site of mTOR-catalysed autophosphorylation in vitro, is not affected by treatment of cells with rapamycin (Peterson et al. 2000). These findings suggest that primary mechanism by which rapamycin interferes with mTOR function in vivo is other than through the inhibition of mTOR's intrinsic kinase catalytic activity.
We (Hara et al. 2002) and Kim et al. (Kim et al. 2002) have observed previously that the association between raptor and mTOR is diminished in rapamycin-treated cells. We now further characterize the effects of rapamycin on the stability of the mTOR/raptor complex. Our results indicate that the ability of the FKBP12/rapamycin to inhibit the interaction of raptor with mTOR, thereby reducing raptor-dependent mTOR-catalysed substrate phosphorylation, is likely to be a dominant mechanism by which rapamycin acts in vivo to interfere with mTOR function.
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Results |
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HEK293 cells were treated with rapamycin or by withdrawal of serum or both serum and amino acids and the association of raptor and GßL with endogenous mTOR was assessed by immunoblotting of mTOR immunoprecipitates (Fig. 1A). Withdrawal of serum or amino acids had no significant effect on the recovery of raptor (Hara et al. 2002; Kim et al. 2002) or GßL with mTOR (Fig. 1A, second and third panels, lanes 2–4). By contrast, rapamycin severely diminished the recovery of raptor with mTOR (Fig. 1A, second panel, lane 5), as shown previously (Hara et al. 2002; Kim et al. 2002), whereas the recovery of GßL with mTOR was unaffected (Fig. 1A, third panel, lane 5). We next examined the ability of rapamycin treatment to affect the association of endogenous raptor with recombinant wild-type or mutant mTOR polypeptides (Fig. 1B). In the absence of rapamycin, equivalent amounts of endogenous raptor was recovered with wild-type mTOR (WT), the rapamycin-resistant Ser2035Thr mTOR (ST) and the kinase-inactive Asn2343Lys mTOR (NK) polypeptides (Fig. 1B, second panel, lanes 3, 5 and 7). Treatment of cells with rapamycin prior to extraction substantially diminished the recovery of raptor with both WT-mTOR (Fig. 1B, second panel, lane 4) and NK-mTOR (Fig. 1B, second panel, lane 8) whereas the recovery of raptor with ST-mTOR, which lacks the ability to bind to the FKBP12/rapamycin complex (Brown et al. 1995; Hara et al. 1997), was undiminished (Fig. 1B, second panel, lane 6). Thus the ability of rapamycin to inhibit the association of raptor with mTOR depends on the ability of mTOR to bind the FKBP12/rapamycin complex, but is independent of mTOR's catalytic activity. Moreover, despite the extensive interaction surface between mTOR and raptor (Kim et al. 2002 and unpublished observations), the binding of FKBP12/rapamycin to the mTOR FRB is sufficient to promote substantial dissociation of raptor from mTOR.
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The binding of raptor to mTOR is critical for the mTOR-catalysed phosphorylation of 4E-BP1 and p70S6k in vitro (Hara et al. 2002; Nojima et al. 2003). We therefore treated cells with various concentrations of rapamycin in vivo, immunoprecipitated the mTOR complex, and examined the binding of raptor to mTOR and the mTOR-catalysed phosphorylation of p70S6k and 4E-BP1. The recovery of raptor with mTOR decreased progressively as the concentration of rapamycin was increased (Fig. 2A,B, fourth panel), accompanied by a progressive reduction in mTOR-catalysed phosphorylation of GST-p70S6k in vitro (Fig. 2A, second panel). Similar results were observed when GST-4E-BP1 was employed as substrate (Fig. 2B, second panel). Notably, at no concentration of rapamycin was an inhibition of mTOR autophosphorylation evident (Fig. 2A,B, first panel).
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We next inquired whether incubation of FKBP12/rapamycin with mTOR complex in vitro is sufficient to promote the dissociation of raptor from mTOR. As shown in Fig. 3, mTOR was immunoprecipitated from HEK293 cell extracts, and the washed immobilized mTOR complex was incubated in the presence of an excess (5 µM) of prokaryotic recombinant FKBP12, either alone or with increasing amounts of rapamycin. After 90 minutes, the supernatant was separated from the immobilized mTOR and the content of raptor in both fractions was analysed by immunoblotting. Raptor was specifically dissociated from the mTOR complex in vitro by rapamycin (in the presence of GST-FKBP12) in a dose-dependent manner. Rapamycin alone had no effect on the binding of raptor to mTOR (data not shown). These in vitro results clearly indicate that FKBP12/rapamycin directly displaces raptor from the mTOR complex.
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We have shown previously that a mutation of the p70S6k1 TOS motif, Phe28Ala (p70S6k-F28A), eliminates the binding of p70S6k to raptor (Nojima et al. 2003). The phosphorylation of p70S6k-F28A by mTOR in vitro was diminished by 75–80% as compared with wild-type p70S6k (p70S6k-WT), an inhibition similar in extent to that observed for the phosphorylation of p70S6k-WT when mTOR was rendered raptor-free by detergent washes (Nojima et al. 2003). Thus the phosphorylation of p70S6k-F28A by mTOR in vitro provides a measure of the raptor-independent kinase activity of mTOR. We therefore inquired whether rapamycin, in the presence of excess FKBP12, can inhibit mTOR-catalysed phosphorylation in vitro of p70S6k-F28A. The immobilized mTOR complex was incubated alone or in the presence of FKBP12 (5 µM) and various concentrations of rapamycin; after 90 minutes the beads were washed and assayed for their ability to phosphorylate GST-p70S6k-F28A (Fig. 4, lanes 1–8) or GST-p70S6k-WT (Fig. 4, lanes 9–16). As noted above, the phosphorylation of GST-p70S6k-F28A by mTOR was approximately 25% that observed for an equivalent amount of GST-p70S6k-WT (Fig. 4, compare lanes 2, 3, 10 and 11). Importantly, whereas rapamycin inhibited the phosphorylation of GST-p70S6k-WT by a further 75–80% (Fig. 4, lanes 15 and 16), no inhibition of mTOR-catalysed phosphorylation of GST-p70S6k-F28A was observed (Fig. 4, lanes 3–8).
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Discussion |
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Despite the close correlation observed here between the displacement of raptor and the inhibition of mTOR-catalysed substrate phosphorylation in vitro, it is possible that other factors contribute to the inhibitory effects of rapamycin on mTOR signalling in vivo. For example, the FKBP12/rapamycin complex may interfere with the effective coupling between mTOR and its substrates in vivo without engendering an actual displacement of raptor from TOR. We (Hara et al. 1998) and others (Peterson et al. 1999) have suggested previously that rapamycin inhibits mTOR's function in part by interfering with mTOR's negative regulation of protein phosphatase activity. The present data raise the possibility that, in addition to deinhibition of phosphatase, the distortion of the mTOR/raptor association induced by rapamycin may increase the susceptibility of mTOR substrates to dephosphorylation. The FRB domain, in addition to its ability to bind the FKBP12/rapamycin complex, has also been shown to bind phosphatidic acid (PA) in vitro (Fang et al. 2001), and the interaction between PA and mTOR in vivo appears to be important for mTOR signalling (Fang et al. 2001). Based on the ability of the FKBP12/rapamycin complex to compete with PA for binding to the FRB domain in vitro, Fang et al. (2001) have suggested that inhibitory effects of rapamycin are due to the dissociation of PA from mTOR. We have not yet directly examined the relation between raptor and PA, but treatment of HEK293 cells with 0.3% 1-butanol or 0.3% 2-butanol, conditions that sequester PA generated by phospholipase D as inactive esters, did not affect the stability of mTOR/raptor complex (data not shown), suggesting that the effect of PA on mTOR function is mediated independently of the mTOR/raptor interaction.
Kim et al. (2002) reported previously that the binding between mTOR and raptor becomes ‘tight’ under nutrient poor conditions and ‘loose’ when nutrients are adequately supplied. The transition to this ‘tight’ complex was correlated with the inhibition of mTOR-dependent signalling in cells and with the repression of mTOR kinase activity in vitro. Although we studied the endogenous mTOR/raptor complex rather than over-expressed mTOR and raptor, we employed the lysis buffer containing 0.3% CHAPS used by Kim et al. (2002); nevertheless, we did not find that the stability of the mTOR/raptor complex is weakened in amino acid-replete as compared to amino acid-deficient cells, using extraction/washing conditions that enabled the demonstration of rapamycin-induced dissociation of raptor from mTOR. Moreover, we found that the dissociation of raptor from mTOR induced by rapamycin in vivo and in vitro led to the reduction of mTOR-catalysed substrate phosphorylation in vitro. These results support our view that raptor functions as a scaffold protein that binds both mTOR and mTOR substrates, the latter through their TOS motif, and that the ability of raptor to physically couple mTOR substrates to mTOR is necessary for effective mTOR-catalysed substrate phosphorylation.
Amino acid deficiency and rapamycin both inhibit selectively the phosphorylation of p70S6k (Thr412) and 4E-BP1, and the ability of both perturbations to inhibit p70S6k (Thr412) phosphorylation can be abrogated by deletion/mutation of the p70S6k amino terminal TOS motif together with deletion of the carboxy terminal autoinhibitory domain (Hara et al. 1998). Despite these similarities, the present finding that amino acid withdrawal does not alter the association of raptor with mTOR under conditions that reveal a marked inhibition by rapamycin, provides evidence that the site and mode of action of amino acid deficiency and rapamycin in the negative regulation of mTOR are distinct. This is consistent with the demonstration that deficiency of the tuberous sclerosis complex (TSC), a potent negative regulator of the TOR/p70S6k pathway, results in a constitutive activation of p70S6k that is resistant to amino acid withdrawal but sensitive to inhibition by rapamycin (Marygold & Leevers 2002). Similarly, over-expression of the small GTPase Rheb, a direct target of the intrinsic GTPase-activating function of TSC2, is able to overcome the inhibition of p70S6k engendered by amino acid withdrawal, but the effects of Rheb are completely inhibited by rapamycin (Manning & Cantley 2003). Thus Rheb functions as a positive regulator downstream of TSC and upstream of mTOR; the ability of Rheb to overcome amino acid deficiency suggests that the site of amino acid action lies at or upstream of Rheb whereas rapamycin acts further downstream, directly on mTOR. The molecular mechanisms by which depletion of amino acid is sensed and transduced to mTOR remain obscure, but the present data indicate that they do not include interference with the mTOR/raptor interaction.
In conclusion we propose that the ability of the FKBP12/rapamycin complex to promote the dissociation of the scaffold protein raptor from mTOR and thereby disrupt the coupling between mTOR with its substrates is a major component of the mechanism by which rapamycin inhibits TOR signalling in vivo. The factors that underlie the exceptional potency of rapamycin's inhibitory actions in vivo however, remain to be fully elucidated.
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Experimental procedures |
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The anti-mTOR antibody (Ab) and the anti-raptor Ab were produced as previously described (Hara et al. 2002; Nishiuma et al. 1998). A rabbit polyclonal anti-GßL/mLST8 Ab was produced against a peptide corresponding to the residues 17–44 of human GßL/mLST8. Other materials were obtained from commercial sources.
Purification and identification of GßL
mTOR immunoprecipitates prepared from frozen 5 x 109 HeLa cells were prepared as previously described (Hara et al. 2002) and separated by SDS-PAGE and visualized by silver staining or negative staining. The p39 polypeptide was cut out from negative-stained gel and digested with Achromobactor Protease I. An in-gel digest of this polypeptide was subjected to mass spectrometric analysis for protein identification by the peptide mass fingerprinting. The p39 polypeptide was identified as GßL/mLST8 (accession number AAH01313), which have been recently reported by two groups as a binding partner of mTOR (Kim et al. 2003; Loewith et al. 2002).
cDNAs
The expression vectors of FLAG-tagged wild-type (WT) mTOR, rapamycin-resistant (ST) mTOR, kinase-inactive (NK) mTOR, GST-tagged FKBP12 (GST-FKBP12), GST-tagged 4E-BP1 (GST-4E-BP1), GST-tagged p70 S6 kinase (GST-p70S6k) and GST-tagged p70 S6 kinase TOS motif mutant (GST-p70S6k-F28A) were constructed as previously described (Alessi et al. 1998; Hara et al. 2002, 1997; Nojima et al. 2003).
Cell culture and treatment
HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS). For starvation of amino acids, cells were first incubated in DMEM without FBS for 16 h, washed twice with DMEM lacking amino acids, and further incubated in the same medium for 1.5 h. Then the medium was replaced with other mediums for 30 min as indicated in each experiment.
Transient transfection
Transient transfection was performed by the lipofection method using lipofectAMINE according to the manufacturer's protocol (Life Technologies).
Immunoprecipitation
This procedure was carried out by standard techniques. All cells were lysed in ice-cold buffer A (40 mM HEPES [pH 7.5], 120 mM NaCl, 0.3% CHAPS, 1 mM EDTA, 50 mM NaF, 10 mMß-glycerophosphate, 1.5 mM Na3VO4, 2 µg/ml aprotinin, 10 µg/ml leupeptin). The immunocomplexes were washed four times in buffer A and twice in buffer B (10 mM HEPES [pH 7.5], 50 mMß-glycerophosphate, 50 mM NaCl). The washed immunocomplexes were then used for immunoblotting, the mTOR kinase assay, or dissociation assay as described below.
mTOR kinase assay
The mTOR kinase assay was performed as previously described (Hara et al. 2002). GST-4E-BP1, GST-p70S6 k and GST-p70S6 k-F28A were prepared for substrates of the mTOR kinase assay, as previously described (Nojima et al. 2003).
Dissociation of raptor from the mTOR/raptor complex by GST-FKBP12/rapamycin in vitro
Endogenous mTOR and raptor complexes were immunoprecipitated with the anti-mTOR Ab as described above. The protein G sepharose beads containing immunocomplexes were incubated with GST-FKBP12 (5 µM) and the indicated concentrations of rapamycin for 90 min at 30 °C in buffer C (10 mM HEPES [pH 7.5], 50 mMß-glycerophosphate, 50 mM NaCl, 0.4% CHAPS). The supernatants were subjected to SDS-PAGE and the dissociated raptor was analysed by immunoblotting with the anti-raptor Ab. Then the beads were washed four times with buffer C and twice with buffer B. The washed immunocomplexes were then used for immunoblotting or the mTOR kinase assay as described above.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yonezawa{at}kobe-u.ac.jp
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References |
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Brown, E.J., Beal, P.A., Keith, C.T., Chen, J., Shin, T.B. & Schreiber, S.L. (1995) Control of p70, s6 kinase by kinase activity of FRAP in vivo. Nature 377, 441–446.[CrossRef][Medline]
Brunn, G.J., Hudson, C.C., Sekulic, A., et al. (1997) Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science
277, 99–101.
Cafferkey, R., Young, P.R., McLaughlin, M.M., et al. (1993) Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol. Cell. Biol.
13, 6012–6023.
Chen, J., Zheng, X.F., Brown, E.J. & Schreiber, S.L. (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. USA
92, 4947–4951.
Choi, J., Chen, J., Schreiber, S.L. & Clardy, J. (1996) Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273, 239–242.[Abstract]
Choi, K.M., McMahon, L.P. & Lawrence, J.J. (2003) Two Motifs in the Translational Repressor PHAS-I Required for Efficient Phosphorylation by Mammalian Target of Rapamycin and for Recognition by Raptor. J. Biol. Chem.
278, 19667–19673.
Chung, J., Kuo, C.J., Crabtree, G.R. & Blenis, J. (1992) Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69, 1227–1236.[CrossRef][Medline]
Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A. & Chen, J. (2001) Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science
294, 1942–1945.
Hara, K., Maruki, Y., Long, X., et al. (2002) Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 177–189.
Hara, K., Yonezawa, K., Kozlowski, M.T., et al. (1997) Regulation of eIF-4E BP1 phosphorylation by mTOR. J. Biol. Chem.
272, 26457–26463.
Hara, K., Yonezawa, K., Weng, Q.P., Kozlowski, M.T., Belham, C. & Avruch, J. (1998) Amino acid sufficiency and mTOR regulate p70, S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem.
273, 14484–14494.
Isotani, S., Hara, K., Tokunaga, C., Inoue, H., Avruch, J. & Yonezawa, K. (1999) Immunopurified mammalian target of rapamycin phosphorylates and activates p70, S6 kinase alpha in vitro. J. Biol. Chem.
274, 34493–34498.
Kim, D.H., Sarbassov, D.D., Ali, S.M., et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175.[CrossRef][Medline]
Kim, D.H., Sarbassov, D.D., Ali, S.M., et al. (2003) GßL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient–sensitive interaction between raptor and mTOR. Mol. Cell 11, 895–904.[CrossRef][Medline]
Kunz, J., Henriquez, R., Schneider, U., Deuter Reinhard, M., Movva, N.R. & Hall, M.N. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73, 585–596.[CrossRef][Medline]
Lin, T.A., Kong, X., Saltiel, A.R., Blackshear, P.J. & Lawrence, J.C.
Jr (1995) Control of PHAS-I by insulin in 3T3-L1 adipocytes. Synthesis, degradation, and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-independent pathway. J. Biol. Chem.
270, 18531–18538.
Loewith, R., Jacinto, E., Wullschleger, S., et al. (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468.[CrossRef][Medline]
Manning, B.D. & Cantley, L.C. (2003) Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–576.[CrossRef][Medline]
von Manteuffel, S.R., Gingras, A.C., Ming, X.F., Sonenberg, N. & Thomas, G. (1996) 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Proc. Natl. Acad. Sci. USA
93, 4076–4080.
Marygold, S.J. & Leevers, S.J. (2002) Growth signaling: TSC takes its place. Curr. Biol. 12, R785–R787.[CrossRef][Medline]
Nishiuma, T., Hara, K., Tsujishita, Y., Kaneko, K., Shii, K. & Yonezawa, K. (1998) Characterization of the phosphoproteins and protein kinase activity in mTOR immunoprecipitates. Biochem. Biophys. Res. Commun. 252, 440–444.[CrossRef][Medline]
Nojima, H., Tokunaga, C., Eguchi, S., et al. (2003) The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70, S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J. Biol. Chem.
278, 15461–15464.
Peterson, R.T., Beal, P.A., Comb, M.J. & Schreiber, S.L. (2000) FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive condition. J. Biol. Chem.
275, 7416–7423.
Peterson, R.T., Desai, B.N., Hardwick, J.S. & Schreiber, S.L. (1999) Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc. Natl. Acad. Sci. USA
96, 4438–4442.
Price, D.J., Grove, J.R., Calvo, V., Avruch, J. & Bierer, B.E. (1992) Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science
257, 973–977.
Schalm, S.S. & Blenis, J. (2002) Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12, 632–639.[CrossRef][Medline]
Schalm, S.S., Fingar, D.C., Sabatini, D.M. & Blenis, J. (2003) TOS Motif-Mediated Raptor Binding Regulates 4E-BP1 Multisite Phosphorylation and Function. Curr. Biol. 13, 797–806.[CrossRef][Medline]
Schmelzle, T. & Hall, M.N. (2000) TOR, a central controller of cell growth. Cell 103, 253–262.[CrossRef][Medline]
Weisman, R. & Choder, M. (2001) The fission yeast TOR homolog, tor1+, is required for the response to starvation and other stresses via a conserved serine. J. Biol. Chem.
276, 7027–7032.
Weisman, R., Finkelstein, S. & Choder, M. (2001) Rapamycin blocks sexual development in fission yeast through inhibition of the cellular function of an FKBP12 homolog. J. Biol. Chem.
276, 24736–24742.
Weng, Q.P., Andrabi, K., Kozlowski, M.T., Grove, J.R. & Avruch, J. (1995) Multiple independent inputs are required for activation of the p70, S6 kinase. Mol. Cell. Biol. 15, 2333–2340.[Abstract]
Weng, Q.P., Kozlowski, M., Belham, C., Zhang, A.H., Comb, M.J. & Avruch, J. (1998) Regulation of the p70, S6 kinase by phosphorylation in vivo—Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem.
273, 16621–16629.
Received: 7 January 2004
Accepted: 27 January 2004
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 A. Tzatsos and K. V. Kandror Nutrients Suppress Phosphatidylinositol 3-Kinase/Akt Signaling via Raptor-Dependent mTOR-Mediated Insulin Receptor Substrate 1 Phosphorylation Mol. Cell. Biol., January 1, 2006; 26(1): 63 - 76. [Abstract] [Full Text] [PDF]
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 |
|
 G. Ohji, S. Hidayat, A. Nakashima, C. Tokunaga, N. Oshiro, K.-i. Yoshino, K. Yokono, U. Kikkawa, and K. Yonezawa Suppression of the mTOR-Raptor Signaling Pathway by the Inhibitor of Heat Shock Protein 90 Geldanamycin J. Biochem., January 1, 2006; 139(1): 129 - 135. [Abstract] [Full Text] [PDF]
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 |
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 S. R. Kimball and L. S. Jefferson Signaling Pathways and Molecular Mechanisms through which Branched-Chain Amino Acids Mediate Translational Control of Protein Synthesis J. Nutr., January 1, 2006; 136(1): 227S - 231S. [Abstract] [Full Text] [PDF]
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S. Paglin, N.-Y. Lee, C. Nakar, M. Fitzgerald, J. Plotkin, B. Deuel, N. Hackett, M. McMahill, E. Sphicas, N. Lampen, et al. Rapamycin-Sensitive Pathway Regulates Mitochondrial Membrane Potential, Autophagy, and Survival in Irradiated MCF-7 Cells Cancer Res., December 1, 2005; 65(23): 11061 - 11070. [Abstract] [Full Text] [PDF]
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S. M. Ali and D. M. Sabatini Structure of S6 Kinase 1 Determines whether Raptor-mTOR or Rictor-mTOR Phosphorylates Its Hydrophobic Motif Site J. Biol. Chem., May 20, 2005; 280(20): 19445 - 19448. [Abstract] [Full Text] [PDF]
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X. Wang, A. Beugnet, M. Murakami, S. Yamanaka, and C. G. Proud Distinct Signaling Events Downstream of mTOR Cooperate To Mediate the Effects of Amino Acids and Insulin on Initiation Factor 4E-Binding Proteins Mol. Cell. Biol., April 1, 2005; 25(7): 2558 - 2572. [Abstract] [Full Text] [PDF]
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I. Mothe-Satney, N. Gautier, C. Hinault, J. C. Lawrence Jr., and E. Van Obberghen In Rat Hepatocytes Glucagon Increases Mammalian Target of Rapamycin Phosphorylation on Serine 2448 but Antagonizes the Phosphorylation of Its Downstream Targets Induced by Insulin and Amino Acids J. Biol. Chem., October 8, 2004; 279(41): 42628 - 42637. [Abstract] [Full Text] [PDF]
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 N. Hay and N. Sonenberg Upstream and downstream of mTOR Genes & Dev., August 15, 2004; 18(16): 1926 - 1945. [Abstract] [Full Text] [PDF]
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Erratum Genes Cells, May 1, 2004; 9(5): 497 - 497. [Full Text] [PDF]
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