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Molecular and Cellular Biology, November 2002, p. 7428-7438, Vol. 22, No. 21
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.21.7428-7438.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Rapamycin-Binding Domain Governs Substrate Selectivity by the Mammalian Target of Rapamycin

Lloyd P. McMahon,¹ Kin M. Choi,¹ Tai-An Lin,² Robert T. Abraham,³ and John C. Lawrence, Jr.^1,4*

Departments of Pharmacology,¹ Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908,⁴ Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543,² Signal Transduction Program, Burnham Institute, La Jolla, California 92037³

Received 30 April 2002/ Accepted 24 July 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70^S6K. Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70^S6K, were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mammalian target of rapamycin (mTOR, also known as FRAP or RAFT1) is a Ser/Thr (S/T) protein kinase that functions in a signaling pathway that has been implicated in the control of a wide variety of metabolic and transcriptional processes that lead to cell growth and/or proliferation (18, 36). Defining processes in cells that are mediated by mTOR has been greatly facilitated by the highly specific inhibitor rapamycin, a lipophilic macrolide originally isolated from a strain of Streptomyces hygroscopicus (1). Rapamycin binds mTOR with high affinity when presented in a complex with the FK506 binding protein with a molecular weight of 12,000 (FKBP12). The FKBP12-rapamycin binding domain (FRB) is located upstream of the catalytic domain between residues 2025 and 2114 (18, 36). This domain is essential for the protein kinase activity of mTOR, and mTOR lacking the FRB is not capable of supporting G₁ progression in cells (43). Ser2035 is critical for rapamycin binding, as mutation of this residue to any amino acid larger than Ala markedly reduces the binding affinity of the isolated FRB for FKBP12-rapamycin (12).

Studies with rapamycin provided the first evidence that mTOR was involved in the control of mRNA translation (6). Rapamycin blocks the activation of p70^S6K by growth factors or nutrient stimuli (13, 32), an action that has been proposed to decrease the translation of mRNAs containing a polypyrimidine motif (22). Rapamycin also promotes the dephosphorylation of PHAS-I (also known as 4E-BP1), thereby increasing PHAS-I binding to eIF4E and decreasing cap-dependent mRNA translation (3, 26). Both p70^S6K and PHAS-I are controlled by multisite phosphorylation. Of the eight phosphorylation sites in p70^S6K, Thr229, Ser371, Thr389, Ser404, and Ser411 are sensitive to rapamycin (2, 15). These sites conform to one of two motifs. Thr229, Thr389, and Ser404 are flanked by hydrophobic residues. Ser371 and Ser411 are followed by proline residues, as are three other sites located in the autoinhibitory domain in the COOH terminal region of the kinase. Both Thr229 in the activation loop and Thr389 must be phosphorylated for the kinase to be active (2, 15). The remaining sites have less critical modulatory roles. PHAS-I is phosphorylated in the following five sites, all of which conform to an (S/T)P motif: Thr36, Thr45, Ser64, Thr69, and Ser82 (16, 28). Except for Ser82, all are sensitive to rapamycin, although in some cells, the phosphorylation of Thr36 and Thr45 is inhibited less by rapamycin than the phosphorylation of Ser64 and Thr69 (17).

mTOR phosphorylates both PHAS-I and p70^S6K in vitro (7, 8, 10, 17, 21). The rate of PHAS-I phosphorylation is markedly enhanced by the mTOR antibody mTAb1, whose epitope is located near the COOH terminus of mTOR (7). Deleting the mTAb1 epitope also increases the PHAS-I kinase activity of mTOR, suggesting that the epitope is located in an inhibitory regulatory domain (RD) (39). The effects of mTAb1 or of deleting the inhibitory domain on the phosphorylation of p70^S6K by mTOR have not been investigated. There are conflicting reports concerning the relative rates of phosphorylation and the sites phosphorylated in the two substrates. Under some assay conditions p70^S6K is phosphorylated much more rapidly than PHAS-I by mTOR (10), an observation that has led to the argument that mTOR phosphorylates p70^S6K in cells but not PHAS-I. Some investigators have concluded that Thr36 and Thr45 are the only sites in PHAS-I phosphorylated by mTOR (10, 17), whereas we have detected significant phosphorylation of Ser64 and Thr69 as well (7, 27). The lack of agreement with respect to the sites phosphorylated by mTOR is worrisome since there is little precedent for phosphorylation by a single protein kinase of sites as different as those conforming to the hydrophobic and proline motifs in p70^S6K and PHAS-I (23).

The present study addresses issues relating to the phosphorylation site specificity of mTOR. The findings are consistent with a model in which substrate selectivity is controlled by the FRB and the COOH-terminal region of mTOR.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies. mTAb1 was generated by immunizing rabbits with keyhole limpet hemocyanin conjugated to the NH₂-terminal cysteine of a peptide having a sequence identical to that of residues 2431 to 2451 in mTOR (7). A monoclonal antibody to the AU1 epitope was purchased from Berkeley Antibody Company. The phospho-specific antibodies, P-Thr36/45 and P-Thr69, were prepared as described previously (27). The sequence of amino acids immediately surrounding Thr36 and Thr45 is identical (20). Consequently, P-Thr36/45 binds to PHAS-I phosphorylated in either Thr36 or Thr45 (27). Phospho-specific antibody to the Thr389 site in p70^S6K was purchased from New England Biolabs.

Preparation of recombinant proteins. PHAS-I, T36-PHAS-I, T45-PHAS-I, T69-PHAS-I, and 5A-PHAS-I were expressed in bacteria and purified as described previously (28). These proteins lack an epitope tag and were only used in experiments for Fig. 1.

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FIG. 1. Phosphorylation of wild-type and mutant PHAS-I proteins by mTOR. 293T cells were transfected with pcDNA3 containing an AU1-mTOR insert. After 18 h extracts were prepared and immunoprecipitations were conducted with anti-AU1 antibody bound to protein G-agarose beads. After extensive washing, immune complexes were incubated for 90 min at 22°C without additions (open symbols) or with 0.25 mg of mTAb1 (filled symbols)/ml before the beads were rinsed and suspended in 20 µl of buffer A. The kinase reactions were initiated by adding 20 µl of buffer A supplemented with 0.2 mM [-32P]ATP (2,000 mCi/mmol), 20 mM MnCl2, and 40 µg of wild-type PHAS-I, T36-PHAS-I, T45-PHAS-I, T69-PHAS-I, or 5A-PHAS-I per ml. After increasing times of incubation at 30°C, the reactions were terminated by adding SDS sample buffer. Samples were subjected to SDS-PAGE. (A) 32P incorporation into the proteins was determined by scintillation counting of gel slices. The small amounts of 32P incorporated into 5A-PHAS-I were subtracted from the values presented. The results for 32P incorporation are expressed relative to the phosphorylation of wild-type PHAS-I that had been incubated for 60 min with mTAb1-activated mTOR. At this time the substrate was phosphorylated to a stoichiometry of 0.08 mol/mol. Immunoblots were prepared with P-Thr36/45 antibodies (B) or P-Thr69 antibodies (C). The results represent signal intensities determined by laser densitometry and are expressed relative to the intensity of the wild-type PHAS-I signal after the 60-min incubation.

Histidine-tagged PHAS-I ([H⁶]PHAS-I) was used in most experiments. This protein (M_r, 15,000) was purified as described previously (19). In experiments with wortmannin and fluorosulfonylbenzoyl adenosine (FSBA) (described later), [H⁶]PHAS-I was reduced and alkylated before use. This was accomplished by incubating the protein for 30 min at 37°C with 1 mM dithiothreitol (DTT), followed by a 30-min incubation with 3 mM N-ethylmaleimide (NEM) and exhaustive dialysis.

To prepare a histidine-tagged carboxyl-terminal fragment of p70^S6K (CT-p70^S6K), cDNA encoding amino acids from 332 to 502 of p70^S6K-II (accession no. M60725) was inserted between the EcoRI and XhoI sites in pProEX HT (Invitrogen). Escherichia coli [BL21(DE3) containing pLysS] was transformed with pProEX HT-CT-p70^S6K and grown to an optical density at 600 nm of 0.8 before expression of CT-p70^S6K was induced by incubating the bacteria with 0.2 mM isopropyl-ß-D-thiogalactopyranoside for 4 h at 37°C. The recombinant protein (M_r, 22,600), which was recovered in inclusion bodies, was dissolved in 8 M urea and applied to a column containing Ni²⁺-nitrilotriacetic acid resin (Qiagen). After stepwise removal of urea, CT-p70^S6K was eluted with 200 mM imidazole and dialyzed against 100 mM NaCl and 50 mM HEPES, pH 7.4.

Glutathione S-transferase (GST)-FKBP12 was expressed in bacteria and purified as described previously (19, 34).

mTOR expression constructs and site-directed mutagenesis of Ser2035. cDNAs encoding mTOR proteins with NH₂-terminal AU1 epitope tags were inserted into pcDNA3. The constructs used to express wild-type mTOR (wt), Ser2035Ile mTOR (SI), Asp2338Ala mTOR (KD), (2433-2451) mTOR (), (2433-2451), Ser2035Ile mTOR (SI/), (2433-2451), Asp2338Ala mTOR (KD/), Ser2448Glu mTOR, and Ser2448Glu, Ser2035Ile mTOR were generated as described previously (previous designations are in parentheses) (39).

To generate cDNA encoding additional mutant mTOR proteins, the KpnI-XhoI fragment was excised from wild-type pcDNA3-mTOR and inserted between the KpnI and XhoI sites in pBluescript SK(-). This plasmid was used as a template for oligonucleotide-directed mutagenesis (Transformer Site-Directed Mutagenesis Kit; Clontech). Appropriately designed mutant oligonucleotides were used to convert the Ser2035 codon into Ala, Arg, Asp, Glu, Thr, and Trp codons and to convert the Cys2546 codon into Ala. In each case, an oligonucleotide that destroyed the unique ScaI site in pBluescript was used for selection. After mutagenesis the mTOR fragments were sequenced to confirm that the correct mutations were present and that no unexpected changes had been introduced. The mutant fragments were excised with KpnI and NotI, which cuts just downstream of XhoI in pBluescript, and were used to replace the corresponding KpnI-NotI fragment in wild-type pcDNA3-mTOR.

Cell culture and transfections. 293T cells were seeded into plastic tissue culture dishes (2 x 10⁴ cells/cm²; Falcon) and cultured in a humidified atmosphere of 5.5% CO₂ in air for 24 h in growth medium composed of 10% (vol/vol) fetal bovine serum in Dulbecco's modified Eagle medium. The cells were transfected using TransIT-LT2 polyamine transfection reagent (Mirus, Madison, Wis.) and 5 µg of DNA per 100-mm-diameter dish.

Measurements of mTOR kinase activities. After culturing of cells for 18 h, extracts were prepared and AU1-mTOR proteins were immunoprecipitated as described previously by using anti-AU1 antibody bound to protein G-agarose beads (27). Prior to the kinase assay, the exhaustively washed beads were incubated at 22°C for 90 min without additions or with 2 to 5 µg of mTAb1 in 20 µl of buffer A (50 mM NaCl, 0.1 mM EGTA, 1 mM DTT, 0.5 mM microcystin LR, 10 mM Na-HEPES, and 50 mM ß-glycerophosphate, pH 7.4) and were then rinsed twice. In experiments with wortmannin and FSBA, DTT was omitted from the reaction mixture. Unless otherwise indicated, the beads were suspended in 20 µl of buffer A, and the kinase reactions were initiated by adding 20 µl of buffer A supplemented with 0.2 mM [-³²P]ATP (2,000 mCi/mmol), 20 mM MnCl₂, and 40 µg of PHAS-I protein or CT-p70^S6K per ml. The reactions were terminated by adding sodium dodecyl sulfate (SDS) sample buffer.

Electrophoretic analyses. Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) by using the method of Laemmli (25). Typically, 25% of each reaction (250 ng of substrate) was loaded. Relative amounts of ³²P incorporated into proteins were determined by phosphorimaging, and absolute amounts were determined by scintillation counting of gel slices. Immunoblots were prepared to detect mTOR and phosphorylated proteins as described previously (8, 27). Proteins were electrophoretically transferred to Immobilon (Millipore) membranes. Alkaline phosphatase-conjugated secondary antibodies and the Tropix Western-Star Chemiluminescence Kit were used to detect binding of the primary antibodies. Relative signal intensities were determined by scanning laser densitometry.

Other materials. cDNA encoding hemagglutinin (HA)-tagged p70^S6K in pRK7 was a generous gift of John Blenis. Rapamycin, wortmannin, and LY294002 were from Calbiochem-Novabiochem International. Caffeine, DTT, NEM, and FSBA were from Sigma Chemical Co. [-³²P]ATP was from Perkin-Elmer Life Science.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phosphorylation of sites in PHAS-I by mTOR. To investigate the phosphorylation of sites in PHAS-I by mTOR, AU1-tagged mTOR was overexpressed in 293T cells and immunopurified with anti-AU1 antibodies. The washed immune complexes were incubated without or with mTAb-1 before initiating the protein kinase reactions by adding Mn:[-³²P]ATP and wild-type PHAS-I, T36-PHAS-I, T45-PHAS-I, or T69-PHAS-I. The latter three proteins retain Thr36, Thr45, or Thr69, respectively, but have Ser/ThrAla in the other four (S/T)P sites. Thus, ³²P incorporation into the mutant proteins should provide an index of the phosphorylation of Thr36, Thr45, and Thr69. mTAb-1 increased the rate of phosphorylation of all four PHAS-I substrates, determined both by ³²P labeling (Fig. 1A) and by immunoblotting with phospho-specific antibodies (Fig. 1B and C).

The reactivity of wild-type PHAS-I, T36-PHAS-I, and T45-PHAS-I with the P-Thr36/45 antibodies (Fig. 1B) and that of wild-type and T69-PHAS-I with the P-Thr69 antibodies (Fig. 1C) increased linearly with time when the proteins were phosphorylated with mTAb1-activated mTOR. The ratio of the immunoblotting signals obtained with T36-PHAS-I and T45-PHAS-I was very similar to the ratio of ³²P incorporation into the two proteins, indicating that the P-Thr36/45 antibody provides a reliable index of the relative level of phosphorylation of Thr36 and Thr45. The amounts of the P-Thr36/45 antibody, as well as P-Thr69 antibody, bound decreased in a linear manner with dilution of phosphorylated PHAS-I (results not presented), supplying additional evidence that binding of the phospho-specific antibodies is proportional to the amount of the phosphorylated sites.

The absolute rates of phosphorylation of the PHAS-I proteins assessed by ³²P incorporation differed considerably, with phosphorylation of wild-type PHAS-I > T45-PHAS-I > T36-PHAS-I >> T69 PHAS-I (Fig. 1A). The very low rate of ³²P incorporation into T69-PHAS-I underestimates the phosphorylation of Thr69 in the wild-type protein, as immunoblotting with P-Thr69 antibodies indicated that phosphorylation of Thr69 was approximately eightfold higher in wild-type PHAS-I than in T69-PHAS-I (Fig. 1C).

Rapamycin sensitivity of the phosphorylation of PHAS-I and CT-p70^S6K by mTOR. Increasing concentrations of rapamycin, in the presence of 10 µM GST-FKBP12, inhibited the phosphorylation of PHAS-I by mTOR; however, the maximum effect represented a decrease of only about 50% (Fig. 2A and B). Surprisingly, sites in PHAS-I differed markedly with respect to the extent of inhibition produced by rapamycin (Fig. 2C and D). In this experiment, Thr36/45 phosphorylation was decreased by only 30%, even at the highest concentrations of rapamycin tested. Due to the relatively small inhibitory response, it was difficult to determine precisely the 50% inhibitory concentration (IC₅₀) for inhibition of Thr36/45 phosphorylation, but significant inhibition occurred in the low-nanomolar range of rapamycin concentrations. Thr69 phosphorylation was almost completely inhibited by concentrations of rapamycin above 1 µM.

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FIG. 2. Inhibitory effects of increasing concentrations of rapamycin on the phosphorylation of PHAS-I and CT-p70S6K by mTOR. AU1-mTOR was overexpressed, immunoprecipitated, and incubated without and with mTAb1 as described in the legend to Fig. 1. The samples were rinsed twice and incubated in 20 µl of buffer containing no additions or 10 µM GST-FKBP12 and increasing concentrations of rapamycin. Control experiments verified that, in the absence of GST-FKBP12, rapamycin was without effect on the phosphorylation of either [H6]PHAS-I or CT-p70S6K. After 20 min 20 µl of reaction mixtures containing 200 µM [-32P]ATP and 50 µg of either [H6]PHAS-I or CT-p70S6K per ml was added, and the samples were incubated for either 30 or 10 min, respectively, before the reactions were terminated by adding SDS sample buffer. Samples were subjected to SDS-PAGE and immunoblots were prepared with phospho-specific antibodies to sites in PHAS-I. (A) Autoradiograms of 32P-labeled [H6]PHAS-I and p70S6K are presented. (B) The relative amounts of 32P incorporated into [H6]PHAS-I and CT-p70S6K were determined by using a phosphorimager. The results are expressed as percentages of the respective amounts of 32P incorporated in the absence of rapamycin. The stoichiometries of phosphorylation of [H6]PHAS-I and CT-p70S6K after incubation with mTAb-1-treated mTOR in the absence of rapamycin were 0.12 and 0.05 mol/mol, respectively. (C) P-Thr36/45 and P-Thr69 immunoblots are shown. (D) The relative intensities of the bands in the immunoblots were determined by optical density scanning. The results are expressed as percentages of intensities of the bands from samples that had been incubated in the absence of rapamycin.

mTOR activity was also assessed by using a fragment of p70^S6K. CT-p70^S6K lacks the protein kinase domain but contains three sites (Ser371, Thr389, and Ser404) that are phosphorylated in a rapamycin-dependent manner in cells (2). Burnett et al. (10) demonstrated that a GST-tagged form of this fragment of p70^S6K was phosphorylated as efficiently as full-length p70^S6K by mTOR. When incubations were conducted without mTAb1, CT-p70^S6K was phosphorylated much more heavily than PHAS-I (Fig. 2A). mTAb1 increased the ³²P incorporation into CT-p70^S6K and the phosphorylation of Thr389, but the effects were much smaller than those on PHAS-I (Fig. 2A). The relatively small effect of mTAb1 on CT-p70^S6K phosphorylation was not due to lack of available phosphorylation sites. Control experiments indicated that the phosphorylation reaction proceeded in a linear manner for at least 30 min and that only a small fraction of the CT-p70^S6K protein was phosphorylated (0.05 mol of ³²P/mol of CT-p70^S6K in Fig. 2A).

Increasing concentrations of rapamycin progressively decreased CT-p70^S6K phosphorylation (Fig. 2A and B). The maximum effect represented almost complete inhibition of phosphorylation. Moreover, the dose-response curve for inhibition of CT-p70^S6K phosphorylation (IC₅₀ 20 nM) was almost superimposable on that for inhibition Thr69 phosphorylation (IC₅₀ 25 nM) (Fig. 2B and D), supporting the conclusion that mTOR mediates the phosphorylation of Thr69 and CT-p70^S6K.

Effects of inhibitors on mTOR activity. Because Thr36/45 phosphorylation was only partially inhibited by rapamycin, we considered the possibility that a portion of Thr36/45 phosphorylation was catalyzed by a kinase other than mTOR. If this were the case, then phosphorylation of PHAS-I would be expected to be partly resistant to inhibition by other mTOR inhibitors.

Wortmannin is an active site inhibitor that has been shown to abolish mTOR autophosphorylation (9, 31). Another active site inhibitor is FSBA, an ATP analogue that binds covalently in the active site of many kinases and inhibits kinase activity (41). Both wortmannin and FSBA are unstable in the presence of sulfhydryl-reducing agents (9, 41). For this reason it was necessary to omit DTT from the reaction mixtures in experiments with these inhibitors. Interestingly, this omission markedly decreased PHAS-I phosphorylation. Reduction followed by alkylation of sulfhydryls in PHAS-I with N-ethylmaleimide obviated the need for DTT (results not presented), indicating that the effect of the DTT was primarily on the substrate. Thus, it was possible to investigate the effects of increasing concentrations of wortmannin and FSBA on mTOR activity by using NEM-treated PHAS-I as the substrate. At concentrations above 1 µM, wortmannin abolished the phosphorylation of both PHAS-I and CT-p70^S6K (Fig. 3A). Half-maximal inhibition of the phosphorylation of both PHAS-I and CT-p70^S6K (Fig. 3A), as well as inhibition of phosphorylation of Thr36/45 and Thr69 in PHAS-I (Fig. 3B) and Thr389 in CT-p70^S6K (results not presented), occurred at 200 nM, which is the concentration of the drug previously shown to inhibit half maximally mTOR autophosphorylation (9). FSBA did not fully inhibit the phosphorylation of either PHAS-I or CT-p70^S6K (Fig. 3A). The dose-response curves for inhibition of ³²P incorporation into PHAS-I with wortmannin and FSBA were almost identical to those for inhibition of the overall phosphorylation of CT-p70^S6K by the respective inhibitors.

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FIG. 3. Effect of increasing concentrations of mTOR inhibitors on the phosphorylation of PHAS-I and CT-p70S6K. (A) After immunoprecipitation of AU1-mTOR, immune complexes were incubated with 0.25 mg of mTAb1/ml for 90 min. Samples were rinsed twice to remove DTT and were incubated in 20 µl with increasing concentrations of wortmannin or FSBA, which were assessed in separate experiments. Reaction mixtures (20 µl) containing 200 µM [-32P]ATP and 40 µg of either NEM-alkylated [H6]PHAS-I or CT-p70S6K per ml were added. After 10 min the reactions were terminated by adding SDS sample buffer. The amounts of 32P incorporated into [H6]PHAS-I and CT-p70S6K are expressed as percentages of the amount of 32P incorporated into the respective proteins in the absence of inhibitors. (B) mTOR immune complexes were incubated with 2.5 mg of mTAb1/ml before the samples were rinsed twice to remove DTT and were incubated with increasing concentrations of inhibitors. Separate experiments were performed to assess the effects of wortmannin, LY294002, or caffeine. After termination of the reactions, immunoblots were prepared using P-Thr36 and P-Thr69 antibodies. The relative intensities of the bands in immunoblots prepared with P-Thr36/45 and P-Thr69 antibodies were determined by optical density scanning. The results are expressed as percentages of intensities of the bands from samples that had been incubated in the absence of inhibitors.

The effects of increasing concentrations of two other mTOR inhibitors, LY294002 (9) and caffeine (38), on the phosphorylation of sites in PHAS-I were also assessed by immunoblotting with phospho-specific antibodies (Fig. 3B). Above concentrations of 10 µM and 1 mM, respectively, LY294002 and caffeine abolished the phosphorylation of both Thr36/45 and Thr69. Dose-response curves for inhibition of Thr36/45 and Thr69 by LY294002 were essentially identical. Likewise, the phosphorylation of Thr36/45 and Thr69 exhibited nearly identical sensitivity to caffeine. The half-maximal inhibition of phosphorylation by LY294002 occurred at a concentration of 2 µM. Caffeine was approximately 100 times less potent than LY294002. The nearly identical sensitivity of Thr36/45 and Thr69 phosphorylation to the various inhibitors supports the conclusion that these sites are phosphorylated by mTOR.

Effect of mutations in the FRB and RD on mTOR activity. To investigate features in mTOR responsible for the quantitatively different effects of rapamycin and mTAb1 on the phosphorylation of sites in PHAS-I, we compared the kinase activity of wild-type mTOR with the activities of several mutant mTOR proteins (Fig. 4). These mutations include removal of the mTAb1 epitope and the following point mutations: Asp2338Ala, Ser2448Glu, and Ser2035Ile. An Asp in the position corresponding to residue 2338 in mTOR is highly conserved in protein kinases, and it is essential for kinase activity (5, 8). Ser2448 is phosphorylated in response to insulin (29, 33, 37, 39), and the negative charge supplied by Glu is sometimes able to mimic the effect of phosphorylation. The Ser2035Ile point mutation decreases markedly the affinity for rapamycin-FKBP12 (5) but has been otherwise assumed to be silent with respect to mTOR function. All of the mTOR proteins had NH₂ terminal AU1 epitope tags, and after expression in 293T cells, approximately equal amounts of the proteins were recovered with anti-AU1 antibody (Fig. 4). Table 1 summarizes results from four experiments in which relative levels of ³²P incorporation and phosphorylation of Thr36/45 and Thr69 were corrected for the slight differences in mTOR recovery.

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FIG. 4. Relative rates of phosphorylation of sites in PHAS-I by wild-type and mutant mTOR proteins. 293T cells were transfected with the pcDNA3 vector alone (Vector) or with pcDNA3 containing inserts for expression of the following AU1-tagged mTOR proteins: wild type, Ser2035Ile mTOR (Ser2035Ile), Asp2338Ala mTOR (D2338A), (2433-2451) mTOR, (2433-2451) forms of mTOR having an additional mutation in either Ser2035 [(2433-2451), S2035I] or Asp2338 [(2433-2451), D2338A]. After preparation of extracts, the mTOR proteins were immunoprecipitated with AU1 antibody and were then incubated at 22°C without or with mTAb1 for 90 min before [H6]PHAS-I phosphorylation was assessed. An autoradiogram of 32P-labeled [H6]PHAS-I is presented (32P). Also shown are immunoblots prepared with P-Thr36/45 or-P-Thr69 antibodies.

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TABLE 1. Phosphorylation of Thr36/45 and Thr69 by mutant mTORsa

Deleting the mTAb1 epitope markedly increased the phosphorylation of PHAS-I (Fig. 4; Table 1). When incubated without mTAb1, the rates of phosphorylation of Thr36/45 and Thr69 by (2433-2451) mTOR were approximately 4- and 10-fold higher, respectively, than the phosphorylation of the sites by wild-type mTOR (Table 1). However, removal of the mTAb1 epitope did not fully activate mTOR, since the rates of Thr36/45 and Thr69 phosphorylation by (2433-2451) mTOR were still only 60 and 10%, respectively, of the rates at which these sites were phosphorylated by wild-type mTOR after incubation with mTAb1. As expected, mTAb1 was without effect on PHAS-I phosphorylation by (2433-2451) mTOR, which lacks the mTAb1 epitope (Table 1).

mTOR harboring an Asp2338Ala mutation exhibited little, if any, PHAS-I kinase activity, even after incubation with the activating antibody (Table 1). Likewise, introducing the Asp2338Ala mutation essentially abolished the kinase activity of (2433-2451) mTOR. That a point mutation in the catalytic domain of mTOR abolishes activity provides independent confirmation that Thr36/45 and Thr69 sites are both phosphorylated by mTOR. Mutating Ser2448 to Glu had relatively little effect on the phosphorylation of either Thr36/45 or Thr69 in PHAS-I.

The rate of phosphorylation of PHAS-I by Ser2035Ile mTOR was much less than that by wild-type mTOR (Fig. 4; Table 1). Strikingly, almost no phosphorylation of Thr36/45 was detected by the rapamycin-resistant form of mTOR, even after it had been incubated with mTAb1. The phosphorylation of Thr36/45 by (2433-2451) mTOR was also markedly decreased by the Ile substitution. This point mutation did not simply cripple the kinase, as Thr69 phosphorylation was still observed after wild-type mTOR was incubated with mTAb1 and as introducing the Ser2035Ile mutation into either (2433-2451) mTOR or Ser2448Glu mTOR actually enhanced Thr69 phosphorylation (Table 1).

Effect of mutating residue 2035 on the phosphorylation of sites in PHAS-I and CT-p70^S6K. Because of the striking effect that mutating Ser2035Ile had on inhibiting the phosphorylation of Thr36/45 in PHAS-I, we investigated the effect of other amino acid substitutions at this position on phosphorylation of sites in PHAS-I and CT-p70^S6K (Fig. 5). Phosphorylation of sites in PHAS-I by Ser2035Ala mTOR was indistinguishable from that by wild-type mTOR (Fig. 5A, C, and D). CT-p70^S6K phosphorylation assessed by ³²P incorporation from [-³²P]ATP was somewhat higher in the Ala2035 mTOR incubated without the activating antibody (Fig. 5B); however, there was no difference between the rates of phosphorylation of CT-p70^S6K by the mutant and wild-type mTORs after incubation with mTAb1. The Ala mutation did not prevent inhibition by rapamycin-FKBP12. This was not unexpected, since the Ala substitution did not significantly decrease the affinity of the isolated FRB fragment for rapamycin (12).

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FIG. 5. Effect of introducing different mutations at position 2035 on the phosphorylation of sites in PHAS-I and CT-p70S6K. Wild-type and mutant AU1-mTOR proteins were immunoprecipitated before being incubated for 90 min without or with 0.25 mg of mTAb1/ml and were then rinsed twice and incubated with or without 10 µM FKBP12 and 1 µM rapamycin for 20 min. Samples were then incubated for 10 min with [-32P]ATP and either [H6]PHAS-I or CT-p70S6K. The reactions were terminated, and samples were subjected to SDS-PAGE. Autoradiograms were prepared to allow detection of 32P-labeled PHAS-I (A) and CT-p70S6K (B). Immunoblots were prepared with P-Thr36/45 (C) and P-Thr69 (D) antibodies. The results are expressed relative to the respective signals obtained with AU1-mTOR that had been incubated with mTAb1 and are mean values + standard errors from three experiments.

Mutating Ser2035 to Glu decreased the mTAb1-stimulated phosphorylation of PHAS-I, although the phosphorylation that was observed was resistant to inhibition by rapamycin (Fig. 5A). The decrease in phosphorylation was not simply a matter of an acidic substitution, since an Asp substitution did not decrease Thr36/45 phosphorylation. The acidic substitutions inhibited the overall phosphorylation of CT-p70^S6K by 80 to 90% (Fig. 5B). The effects of an Ser2035Arg mutation on both PHAS-I and CT-p70^S6K were comparable to those produced by the Glu mutation. The relatively conservative Ser2035Thr mutation decreased the mTAb1-stimulated phosphorylation of Thr36/45 in PHAS-I (Fig. 5C). The Ser2035Thr mutation also decreased the phosphorylation of CT-p70^S6K, although the effect was less than that produced by the acidic and basic substitutions.

The Ser2035Ile mutation substantially inhibited the ability of mTOR to phosphorylate CT-p70^S6K (Fig. 5B), but it had a much more dramatic effect on decreasing Thr36/45 phosphorylation (Fig. 5C). The marked inhibitory effect on Thr36/45 could not be attributed to a bulky hydrophobic residue, since substituting Trp for Ser2035 had little, if any, effect on PHAS-I phosphorylation when incubations were conducted without rapamycin (Fig. 5A, C, and D). Interestingly, the Trp substitution had a relatively modest inhibitory effect on the phosphorylation of CT-p70^S6K (Fig. 5B), while conferring marked rapamycin resistance to the phosphorylation of both PHAS-I and CT-p70^S6K. Thus, of the rapamycin-resistant mTORs, the activity spectrum of Ser2035Trp protein was closest to that of wild-type mTOR incubated without rapamycin.

Effect of Ser2035 mutations on mTOR activity in vivo. To determine whether the in vitro measurements of mTOR activities were of value in predicting activities of mTOR in vivo, wild-type and mutant mTOR proteins were coexpressed with HA-tagged, full-length p70^S6K in 293T cells. The cells were then treated with rapamycin to inhibit endogenous mTOR before the HA-tagged kinase was immunoprecipitated and before the phosphorylation of Thr389 was assessed by immunoblotting (Fig. 6A). Rapamycin abolished the phosphorylation of Thr389 in cells transfected with wild-type mTOR. The range of protection to inhibition by rapamycin in vivo ranged from almost none with Ser2035Ala mTOR to over 60% protection with the Ser2035Trp mTOR. In Fig. 6B, the rapamycin-resistant Thr389-phosphorylating activity of the different rapamycin-resistant mTOR proteins in vitro is plotted against the relative level of Thr389 phosphorylation detected in cells. Linear regression analysis revealed a strong positive correlation (r = 0.97) between the in vivo and in vitro measurements (Fig. 6B).

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FIG. 6. Effect of Ser2035 mutations in mTOR on the phosphorylation of p70S6K in cells. 293T cells were cotransfected with cDNA encoding HA-p70S6K and either wild-type AU1-mTOR (Ser) or with mTOR proteins harboring different mutations at position 2035. After 18 h the cells were incubated with 200 nM rapamycin for 1 h before extracts were prepared. HA-p70S6K was immunoprecipitated with antibodies to the epitope. (A) Immunoblots prepared with antibodies to P-Thr389 or to the HA and AU1 epitopes. (B) The relative band intensities from the Thr389 blots were determined and expressed relative to the signal observed with wild-type mTOR from cells incubated without rapamycin. After correction for levels of expression of the different AU1-mTOR proteins, these values were plotted versus those obtained from P-Thr389 immunoblots of CT-p70S6K samples that had been phosphorylated by different mTOR proteins in the presence of FKBP12 and rapamycin as described in the legend to Fig. 5. The results are means ± standard errors of three in vitro kinase experiments and means ± one-half the range of two experiments involving overexpression of HA-p70S6K. The line and correlation coefficient (r) were determined by linear regression analysis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although it is well established that mTOR has a central role in the control of cell growth, the mechanisms involved in regulating the activity of this protein kinase are not well understood. The present study yielded several novel and unexpected findings that provide mechanistic insight into substrate recognition by mTOR and/or that have important practical implications for studies of mTOR function in cells.

Nature of the phosphorylation sites. After incubation with mTAb1, mTOR phosphorylated PHAS-I and CT-p70^S6K at comparable rates (Fig. 2, for example). The fact that all of the sites known to be phosphorylated by mTOR conform to either an (S/T)P motif, such as those in PHAS-I, or an h(S/T)h motif (where h = a hydrophobic residue), such as Thr389 in p70^S6K, is consistent with the interpretation that the amino acid sequence context provides one of the determinants for phosphorylation by mTOR. Many Ser/Thr protein kinases also utilize primary structure in selecting a particular Ser or Thr for phosphorylation (23). However, few, if any, of these kinases recognize two motifs as different as those represented by the mTOR phosphorylation sites. The kinase domain of mTOR is more closely related to the lipid kinase phosphatidylinositol (PI) 3-kinase than to the larger family of Ser/Thr protein kinases (18, 36). Perhaps it should not be surprising that mTOR exhibits more flexibility in its substrate recognition than the typical protein kinase, since PI 3-kinase is able to phosphorylate both lipid and protein (4). Such flexibility does not appear to extend to other members of the PI 3-kinase-related protein kinases, which exhibit a strong preference for (S/T)Q sites (24).

Contributing to the complexity of the problem of substrate recognition by mTOR is evidence that prior phosphorylation of the TP sites in PHAS-I increases the rate of Ser64 phosphorylation by mTOR (17, 28). The low rate of phosphorylation by mTOR of Thr69 in T69-PHAS-I (Fig. 1C), which lacks Thr36 and Thr45, relative to that of Thr69 in wild-type mTOR is consistent with the hypothesis that prior phosphorylation also enhances Thr69 phosphorylation. However, prior phosphorylation of Thr36 and Thr45 cannot be strictly required for the phosphorylation of Thr69, since T69 PHAS-I was phosphorylated by mTOR. Also, mutating Ser2035 to Ile markedly decreased Thr36 and Thr45 phosphorylation while only modestly decreasing Thr69 phosphorylation (Fig. 5).

Mutations in Ser2035 affect more than sensitivity to rapamycin. mTOR proteins harboring different mutations in Ser2035 in the FRB exhibited a spectrum of activities with respect to phosphorylation of sites in PHAS-I and p70^S6K (Fig. 5). In a previous study, introducing a Trp2027Phe mutation just upstream of Ser2035 abolished the phosphorylation of PHAS-I by mTOR (43). Thus, it is clear that certain mutations in the FRB are not silent with respect to mTOR function. The dramatic inhibition of Thr36/45 phosphorylation resulting from the Ser2035Ile mutation was the most striking example of a switch in substrate selectivity. However, there were many other instances in which mutations at position 2035 exerted more pronounced effects on the phosphorylation of one substrate than on that of another. For example, mutating Ser2035Arg markedly decreased the phosphorylation of Thr389 in p70^S6K, both in vitro and in vivo, but had relatively little effect on the phosphorylation of PHAS-I (Fig. 5 and 6). There is precedence for differential effects of Arg mutations at the highly conserved Ser2035 equivalents in target of rapamycin proteins in yeasts. Mutating Ser1834 to Arg in tor1⁺ in Schizosaccharomyces pombe essentially abolished the function of tor1⁺ in sexual development (45), but Saccharomyces cerevisiae tor1p with such a mutation retains at least part of its functionality. Indeed, Ser1972Arg was one of the original mutations in tor1p that led to the discovery of the target of rapamycin proteins by allowing the budding yeast to grow in the presence of rapamycin (11).

Initial studies of the mTOR kinase demonstrated that autophosphorylation, now known to occur on Ser2481 (31), was not fully inhibited by rapamycin (9). Similarly, rapamycin had a modest inhibitory effect on the phosphorylation of Thr36/45 in PHAS-I by mTOR (Fig. 2C and D). In contrast, the phosphorylation of CT-p70^S6K and Thr69 in PHAS-I was almost completely inhibited by sufficiently high concentrations of the drug (Fig. 2A and B). Because the rates of phosphorylation of the different sites were not affected equally, it is clear that rapamycin does not inhibit the catalytic center of mTOR. Rapamycin could potentially inhibit activity either by competitively inhibiting substrate binding or by inducing conformational changes that reduce substrate affinity.

Role of the FRB: a working hypothesis. The findings with rapamycin, as well as results of experiments with mTOR having mutations within the FRB, suggest that the FRB participates in substrate recognition. This hypothesis would account for the differential effects of Ser2035 mutations and rapamycin on the phosphorylation of sites in PHAS-I and CT-p70^S6K. It is unlikely that the FRB recognizes the proline adjacent to Thr36/45 and Thr69, since the sensitivities of these sites to rapamycin and Ser2035 mutations differ greatly. Instead, we propose that the FRB functions as an initial substrate discriminator. This domain might recognize motifs in p70^S6K and PHAS-I that directly facilitate binding of substrates to mTOR. Alternatively, the FRB may bind protein cofactors, such as those postulated by Nishiuma et al. (30), that present substrate to mTOR. The substrate or cofactor binding motifs are predicted to be distinct from the (S/T)P and h(S/T)h determinants. A correlative prediction of our working hypothesis is that interactions with the FRB position the substrates for phosphorylation by the catalytic domain, which phosphorylates those sites having the appropriate proline or hydrophobic determinants.

Recent reports describe two motifs, well removed from the phosphorylation sites, which facilitate the phosphorylation of PHAS-I and/or p70^S6K in cells. Tee and Proud (42) demonstrated that mutations of residues 13 to 16 in PHAS-I (Arg, Ala, Ile, and Pro, designated the RAIP motif) markedly decreased phosphorylation of the protein in cells. The RAIP motif is also present in PHAS-II but not in p70^S6K. Schalm and Blenis (35) identified a motif, designated TOS (for TOR signaling), which is found at the COOH termini of all PHAS isoforms and at the NH₂ terminus of p70^S6K. Mutating the TOS motif in p70^S6K significantly inhibited the activation of the kinase in cells and prevented the inhibitory effect of p70^S6K overexpression on PHAS-I phosphorylation. von Manteuffel et al. (44) had previously found that overexpressing either wild-type or kinase-dead forms of p70^S6K inhibited PHAS-I phosphorylation, an observation suggesting that the two proteins were phosphorylated by the same kinase, possibly mTOR. The TOS motif cannot be strictly required for phosphorylation of Thr389, since mTOR phosphorylated CT-p70^S6K (Fig. 2 and 7) which lacks the motif. Nevertheless, it will be interesting to determine how mutations to the RAIP and TOS motifs affect the phosphorylation of substrates by mTOR in vitro.

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FIG. 7. Model for control of mTOR activity. Details are described in the text.

Role of the COOH-terminal RD. Binding of mTAb1 or deletion of the mTAb1 epitope had much more pronounced effects on increasing the phosphorylation of sites in PHAS-I than on sites in p70^S6K. Our results and earlier findings by Sekuli et al. (39) suggest that the mTAb1 epitope is found within an inhibitory regulatory domain, which inhibits substrate interactions with mTOR. Binding of mTAb1 is proposed to change the conformation, removing the barrier to substrate binding, as does deletion of the mTAb1 epitope (Fig. 7). Presumably, binding of p70^S6K is less restricted by RD in the basal state than binding of PHAS-I, since mTAb1 has a relatively small effect on the phosphorylation of p70^S6K. Because of the relatively low activity in the absence of mTAb1, we have been unable to determine whether the antibody decreases the K_m of mTOR for PHAS-I.

PI 3-kinase-related kinases contain a region of homology referred to as the FAT domain (for FRAP, ATM, and TRRAP), which is located between amino acids 1382 and 1982 in mTOR (18). All of these kinases also contain a short, very highly conserved, COOH-terminal domain, designated FATC (FAT COOH-terminal domain), which is critical for the function of mTOR (40). It has been suggested that, because the FAT and FATC domains are always found together, molecular interactions occur between the two (18). Since the FAT domain is adjacent to the FRB, such interactions could be accommodated by slight modification of the working hypothesis in Fig. 7. However, the barrier to phosphorylation of sites in PHAS-I by the RD need not be the COOH terminus itself, as drawn in Fig. 7, nor must the RD directly interact with the substrate recognition motif, since the effects of an interaction with other regions of mTOR could in principle be propagated via conformation changes to the catalytic domain or to other regions of mTOR involved in substrate recognition.

Implications for studies of the phosphorylation of mTOR. Ser2035 in the FRB represents a potential phosphorylation site. The inhibitory effects of acidic substitutions at this position predict that phosphorylation would inhibit activity towards p70^S6K. The finding that the Ser2035Ala mTOR immunoprecipitated from 293T cells exhibited somewhat higher activity towards CT-p70^S6K would be consistent with such an action (Fig. 5B). While there is no direct evidence that Ser2035 is phosphorylated, several studies have now demonstrated that Ser2448 may be phosphorylated by PKB in vitro and/or in response to PKB activation in cells (29, 37, 39). As phosphorylation occurs within the mTAb1 epitope, it has been postulated that phosphorylation mimics the effect of the activating antibody. If this were true, then our findings indicate that phosphorylation of Ser2448 should have a much larger effect on PHAS-I than on p70^S6K (Fig. 1). In this connection, it is interesting that PHAS-I phosphorylation is very sensitive to changes in PKB activity, whereas p70^S6K activity is affected only by membrane-targeted forms of PKB, which exhibit abnormally high activities (14).

Ser2448Glu mTOR exhibited little, if any, difference in activity from wild-type mTOR (Table 1). This negative finding does not eliminate the possibility that phosphorylation of Ser2448 activates mTOR, since acidic substitutions are only sometimes able to substitute for phosphorylation in proteins. Because of the inability to efficiently phosphorylate Ser2448 in vitro, it has not been feasible to determine the effect of directly phosphorylating Ser2448 on mTOR activity. The potential role of Ser2448 phosphorylation on p70^S6K and PHAS-I in cells has been investigated by comparing the effects of Ser2035Ile mTOR and an Ser2035Ile mTOR with an Ser2448Ala mutation (39). After expression in HEK293 cells, which were incubated with rapamycin to inhibit endogenous mTOR, the effects of the two mTOR proteins on PHAS-I phosphorylation and p70^S6K activity were indistinguishable. These results argue against a role of Ser2448 in controlling PHAS-I and p70^S6K, but there are potential complications that weaken this argument. For instance, whether it is reasonable to expect to observe much of an effect of preventing Ser2448 phosphorylation on the activity of the severely crippled Ser2035Ile mTOR towards Thr36/45 is debatable.

Practical implications for studies of mTOR in cells. Since mTOR is the only known target affected by low-nanomolar concentrations of rapamycin in cells, inhibition of a process by such concentrations of the drug is believed to be strong evidence for an input from mTOR. The present results indicate that the converse is not true. Rapamycin is a far more subtle and/or selective inhibitor of mTOR function than would be predicted for a direct inhibitor of the mTOR kinase domain. Thus, rapamycin treatment is not the equivalent of an mTOR knockout in mammalian cells, and the failure of rapamycin to block a process does not prove that mTOR is not involved.

The present findings with Ser2035 mutants also raise a cautionary note on the approach in which mTOR function is investigated by overexpressing a rapamycin-resistant mTOR, such as Ser2035Ile mTOR. The presumption has been that rapamycin treatment abolishes the effect of endogenous mTOR and that activity of the rapamycin-resistant mTOR is representative of that of wild-type mTOR. The relative lack of effect of rapamycin on the phosphorylation of Thr36/45 in PHAS-I by mTOR (Fig. 2D) and the relative inability of Ser2035Ile mTOR to phosphorylate Thr36/45 (Fig. 4 and 5; Table 1) indicate that neither assumption is safe. We believe that Ser2035Trp mTOR, whose kinase activity spectrum most closely resembled that of wild-type mTOR (Fig. 5A to D), will be a better option than Ser2035Ile mTOR for investigating mTOR in cells.

 
This research was supported in part by National Institutes of Health grants DK52753 and DK28312 (to J.C.L.) and CA 76193 (to R.T.A.).

 
* Corresponding author. Mailing address: Department of Pharmacology, University of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735. Phone: (434) 924-1584. Fax: (434) 982-3575. E-mail: jcl3p{at}virginia.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, November 2002, p. 7428-7438, Vol. 22, No. 21
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.21.7428-7438.2002
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