Identification of a conserved motif required for mTOR signaling

BACKGROUND: The mammalian target of rapamycin (mTOR) controls the translation machinery via activation of S6 kinases 1 and 2 (S6K1/2) and inhibition of the eukaryotic initiation factor 4E (eIF4E) binding proteins 1, 2, and 3 (4E-BP1/2/3). S6K1 and 4E-BP1 are regulated by nutrient-sensing and mitogen-activated pathways. The molecular basis of mTOR regulation of S6K1 and 4E-BP1 remains controversial. RESULTS: We have identified a conserved TOR signaling (TOS) motif in the N terminus of all known S6 kinases and in the C terminus of the 4E-BPs that is crucial for phosphorylation and regulation S6K1 and 4E-BP1 activities. Deletion or mutations within the TOS motif significantly inhibit S6K1 activation and the phosphorylation of its hydrophobic motif, Thr389. In addition, this sequence is required to suppress an inhibitory activity mediated by the S6K1 C terminus. The TOS motif is essential for S6K1 activation by mTOR, as mutations in this motif mimic the effect of rapamycin on S6K1 phosphorylation, and render S6K1 insensitive to changes in amino acids. Furthermore, only overexpression of S6K1 with an intact TOS motif prevents 4E-BP1 phosphorylation by a common mTOR-regulated modulator of S6K1 and 4E-BP1. CONCLUSIONS: S6K1 and 4E-BP1 contain a conserved five amino acid sequence (TOS motif) that is crucial for their regulation by the mTOR pathway. mTOR seems to regulate S6K1 by two distinct mechanisms. The TOS motif appears to function as a docking site for either mTOR itself or a common upstream activator of S6K1 and 4E-BP1.     

Requirement for the eIF4E Binding Proteins for the Synergistic Down-Regulation of Protein Synthesis by Hypertonic Conditions and mTOR Inhibition

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.     

The rapid activation of protein synthesis by growth hormone requires signaling through mTOR

An important function of growth hormone (GH) is to promote cell and tissue growth, and a key component of these effects is the stimulation of protein synthesis. In this study, we demonstrate that, in H4IIE hepatoma cells, GH acutely activated protein synthesis through signaling via the mammalian target of rapamycin (mTOR) and specifically through the rapamycin-sensitive mTOR complex 1 (mTORC1). GH treatment enhanced the phosphorylation of two targets of mTOR signaling, 4E-BP1 and ribosomal protein S6. Phosphorylation of S6 and 4E-BP1 was maximal at 30-45 min and 10-20 min after GH stimulation, respectively. Both proteins modulate components of the translational machinery. The GH-induced phosphorylation of 4E-BP1 led to its dissociation from eIF4E and increased binding of eIF4E to eIF4G to form (active) eIF4F complexes. The ability of GH to stimulate the phosphorylation of S6 and 4E-BP1 was blocked by rapamycin. GH also led to the dephosphorylation of a third translational component linked to mTORC1, the elongation factor eEF2. Its regulation followed complex biphasic kinetics, both phases of which required mTOR signaling. GH rapidly activated both the MAP kinase (ERK) and PI 3-kinase pathways. Signaling through PI 3-kinase alone was, however, sufficient to activate the downstream mTORC1 pathway. Consistent with this, GH increased the phosphorylation of TSC2, an upstream regulator of mTORC1, at sites that are targets for Akt/PKB. Finally, the activation of overall protein synthesis by GH in H4IIE cells was essentially completely inhibited by wortmannin or rapamycin. These results demonstrate for the first time that mTORC1 plays a major role in the rapid activation of protein synthesis by GH.     

Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.     

TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function

BACKGROUND: The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. RESULTS: Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. CONCLUSIONS: Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.     

Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.     

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.     

Rapamycin-insensitive regulation of 4e-BP1 in regenerating rat liver

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppressant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due to dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial hepatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-BP1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active kinase from partially hepatectomized animals was highly phosphorylated. Phosphorylated 4E-BP1 from regenerating liver was unable to form an inhibitory complex with initiation factor 4E. Rapamycin blocked the activation of p70 S6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphorylation of 4E-BP1 induced by injection of cycloheximide or growth factors was partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resistant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver regeneration, but not functional phosphorylation of 4E-BP1, in response to partial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in cultured cells.     

Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3′-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.     

Repression of protein synthesis and mTOR signaling in rat liver mediated by the AMPK activator aminoimidazole carboxamide ribonucleoside

The studies described herein were designed to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), an activator of the AMP-activated protein kinase (AMPK), on the translational control of protein synthesis and signaling through the mammalian target of rapamycin (mTOR) in rat liver. Effects of AICAR observed in vivo were compared with those obtained in an in situ perfused liver preparation to investigate activation of AMPK in the absence of accompanying changes in hormones and nutrients. AMPK became hyperphosphorylated, as assessed by a gel-shift analysis, in response to AICAR both in vivo and in situ; however, increased relative phosphorylation at the Thr172 site on the kinase was observed only in perfused liver. Phosphorylation of AMPK either in vivo or in situ was associated with a repression of protein synthesis as well as decreased phosphorylation of a number of targets of mTOR signaling including ribosomal protein S6 kinase 1, eukaryotic initiation factor (eIF)4G, and eIF4E-binding protein (4E-BP)1. The phosphorylation changes in eIF4G and 4E-BP1 were accompanied by a reduction in the amount of eIF4E present in the active eIF4E.eIF4G complex and an increase in the amount present in the inactive eIF4E.4E-BP1 complex. Reduced insulin signaling as well as differences in nutrient availability may have contributed to the effects observed in vivo as AICAR caused a fall in the serum insulin concentration. Overall, however, the results from both experimental models support a scenario in which AICAR directly represses protein synthesis and mTOR signaling in the liver through an AMPK-dependent mechanism.     

The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites.

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent stimulator of Erk, leads to the phosphorylation of 4E-BP1 and its dissociation from eIF4E. In contrast to agonists such as insulin, this occurs independently of PKB activation. In this report, we investigate the mechanism by which TPA regulates 4E-BP1 phosphorylation. Treatment of HEK293 cells with TPA was found to result in the phosphorylation of 4E-BP1 at Ser(64), Thr(69), and Thr(36/45). The TPA-stimulated phosphorylation of all these sites is sensitive to inhibitors of MEK and to the inhibitor of mTOR, rapamycin, indicating that inputs from both mTOR and MEK are required for the regulation of 4E-BP1 phosphorylation by TPA. Indeed, evidence is presented that mTOR may initially be required for the phosphorylation of Thr(45) in a priming step, which is necessary for the subsequent phosphorylation of Ser(64) and Thr(69) through an Erk-dependent pathway. Overexpression of constitutively active MEK in HEK293 cells resulted both in the phosphorylation of 4E-BP1 at Ser(64) and Thr(36/45) and its release from eIF4E. In this case, the phosphorylation of these sites was also blocked by inhibitors of MEK or by rapamycin. In conclusion, the Erk pathway, via mechanisms also requiring mTOR, regulates the phosphorylation of multiple sites in 4E-BP1 in vivo and this is sufficient for the release of 4E-BP1 from eIF4E.     

Myogenic differentiation is dependent on both the kinase function and the N-terminal sequence of mammalian target of rapamycin

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase known to control initiation of translation through two downstream pathways: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic initiation factor 4E and ribosomal p70 S6 kinase (S6K1). We previously showed in C2C12 murine myoblasts that rapamycin arrests cells in G(1) phase and completely inhibits terminal myogenesis. To elucidate the pathways that regulate myogenesis, we established stable C2C12 cell lines that express rapamycin-resistant mTOR mutants (mTORrr; S2035I) that have N-terminal deletions (Delta10 or Delta91) or are full-length kinase-dead mTORrr proteins. Additional clones expressing a constitutively active S6K1 were also studied. Our results show that Delta10mTORrr signals 4E-BP1 and permits rapamycin-treated myoblasts to differentiate, confirming the mTOR dependence of the inhibition of myogenesis by rapamycin. C2C12 cells expressing either Delta91mTORrr or kinase-dead mTORrr(D2338A) could not phosphorylate 4E-BP1 in the presence of rapamycin and could not abrogate the inhibition of myogenesis. Taken together, our results indicate that both the kinase function of mTOR and the N terminus (residues 11-91, containing part of the first HEAT domain) are essential for myogenic differentiation. In contrast, constitutive activation of S6K1 does not abrogate rapamycin inhibition of either proliferation or myogenic differentiation.     

The Akt/mTor signaling cascade is modified during placentation in the porcine uterine tissue

Recently we showed that essential components for the initiation of protein synthesis, namely the eukaryotic initiation factor 4E (eIF4E, mRNA-cap-binding protein) and its repressors 4E-BP1 as well as 4E-BP2, are proteolytically processed in the porcine endometrium during implantation. Here, the situation during placentation was compared with ovariectomized (OVX) animals and animals on pregnancy day 1 (PD1). Furthermore, the research was extended to factors which phosphorylate eIF4E and 4E-BPs and regulate their activities. These are the protein kinase B/mammalian target of rapamycin kinase (Akt/mTor) with the regulators Raptor and Rictor as well as the mitogen activated protein kinases (MAPKs): extra cellular-signal regulated kinase 1 and 2 (ERK1 and ERK2). Striking differences in the placentation site (PS) and the areas aside from PS (peri-PS) were observed. EIF4E and 4E-BP2 truncation as well as 4E-BP1 degradation took place in the endometrium of the peri-PS on PD24. Accompanied by a fragmentation of Akt/mTor, no expression of Rictor was observed, whereas the abundance of Raptor was not altered. On the contrary, MAPKs expression and phosphorylation remained almost stable in the peri-PS. In conclusion, the results indicated that on PD24 the translational regulation was shifted to 4E-BP2 control. Furthermore, the Akt/mTor signaling cascade seemed to be down regulated which suggest reduced phosphorylation of 4E-BP2. Whereas Akt was proteolyzed, the observed mTor fragments represented most likely splicing variants. The results indicate that translational control of gene expression is an important feature in the porcine endometrium during early pregnancy.     

The mammalian target of rapamycin (mTOR) partner,raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Herein we demonstrate that raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs to be required for amino acid- and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency.     

A novel hypoxia-inducible factor-independent hypoxic response regulating mammalian target of rapamycin and its targets

Hypoxia triggers a reversible inhibition of protein synthesis thought to be important for energy conservation in O2-deficient environments. The mammalian target of rapamycin (mTOR) pathway integrates multiple environmental cues to regulate translation in response to nutrient availability and stress, suggesting it as a candidate for O2 regulation. We show here that hypoxia rapidly and reversibly triggers hypophosphorylation of mTOR and its effectors 4E-BP1, p70S6K, rpS6, and eukaryotic initiation factor 4G. Hypoxic regulation of these translational control proteins is dominant to activation via multiple distinct signaling pathways such as insulin, amino acids, phorbol esters, and serum and is independent of Akt/protein kinase B and AMP-activated protein kinase phosphorylation, ATP levels, ATP:ADP ratios, and hypoxia-inducible factor-1 (HIF-1). Finally, hypoxia appears to repress phosphorylation of translational control proteins in a manner analogous to rapamycin and independent of phosphatase 2A (PP2A) activity. These data demonstrate a new mode of regulation of the mTOR pathway and position this pathway as a powerful point of control by O2 of cellular metabolism and energetics.     

Target of rapamycin (TOR)-signaling and RAIP motifs play distinct roles in the mammalian TOR-dependent phosphorylation of initiation factor 4E-binding protein 1

The translational repressor protein eIF4E-binding protein 1 (4E-BP1, also termed PHAS-I) is regulated by phosphorylation through the rapamycin-sensitive mTOR (mammalian target of rapamycin) pathway. Recent studies have identified two regulatory motifs in 4E-BP1, an mTOR-signaling (TOS) motif in the C terminus of 4E-BP1 and an RAIP motif (named after its sequence) in the N terminus. Other recent work has shown that the protein raptor binds to mTOR and 4E-BP1. We show that raptor binds to full-length 4E-BP1 or a C-terminal fragment containing the TOS motif but not to an N-terminal fragment containing the RAIP motif. Mutation of several residues within the TOS motif abrogates binding to raptor, indicating that the TOS motif is required for this interaction. 4E-BP1 undergoes phosphorylation at multiple sites in intact cells. The effects of removal or mutation of the RAIP and TOS motifs differ. The RAIP motif is absolutely required for phosphorylation of sites in the N and C termini of 4E-BP1, whereas the TOS motif primarily affects phosphorylation of Ser-64/65, Thr-69/70, and also the rapamycin-insensitive site Ser-101. Phosphorylation of N-terminal sites that are dependent upon the RAIP motif is sensitive to rapamycin. The RAIP motif thus promotes the mTOR-dependent phosphorylation of multiple sites in 4E-BP1 independently of the 4E-BP1/raptor interaction.     

Suppression of the mTOR-raptor signaling pathway by the inhibitor of heat shock protein 90 geldanamycin

Heat shock protein 90 (Hsp90) was co-immunoprecipitated with raptor, the binding partner of the mammalian target of rapamycin (mTOR) from HEK293 cells. Hsp90 was detected in the anti-raptor antibody immunoprecipitates prepared from the cell extract by immunoblot analysis using the anti-Hsp90 antibody, and the association of these two proteins was confirmed by immunoprecipitation from the cells co-expressing Hsp90 and raptor as epitope-tagged molecules. Geldanamycin, a potent inhibitor of Hsp90, disrupted the in vivo binding of Hsp90 to raptor without affecting the association of raptor and mTOR, and suppressed the phosphorylation by mTOR of the downstream translational regulators p70 S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The protein kinase activity of S6K as well as the phosphorylation of the substrate, 40S ribosomal protein S6, were lowered in the geldanamycin-treated cells. These results indicate that Hsp90 is involved in the regulation of protein translation by facilitating the phosphorylation reaction of 4E-BP1 and S6K catalyzed by the mTOR/raptor complex through the association with raptor, and that the mTOR signaling pathway is a novel target of geldanamycin.