©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of a Protein Target of the FKBP12-Rapamycin Complex in Mammalian Cells (*)

(Received for publication, September 30, 1994)

Candace J. Sabers (1)(§) Mary M. Martin (2)(§) Gregory J. Brunn (1) Josie M. Williams (3) Francis J. Dumont (2) Gregory Wiederrecht (2) Robert T. Abraham (1) (3)(¶)

From the  (1)Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905, the (2)Department of Immunology Research, Merck Research Laboratories, Rahway, New Jersey 07065, and the (3)Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G(1)-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G(1) phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G(1)- to S-phase progression in T-lymphocytes and other sensitive cell types.


INTRODUCTION

The structurally related macrolides, rapamycin and FK506, are potent immunosuppressants that inhibit signal transduction pathways required for T-cell activation and growth (for review, see (1) ). FK506 interferes with a Ca-dependent signaling event that couples T-cell antigen receptor occupancy to transcription of the genes encoding interleukin-2 (IL-2) (^1)and several other cytokines. In contrast, rapamycin inhibits IL-2-stimulated T-cell proliferation by blocking cell cycle progression from late G(1) into S phase(2, 3) .

FK506 and rapamycin bind to a family of intracellular receptors termed FK506 binding proteins (FKBPs). The most well characterized member of the FKBP family is a 12-kDa isoform, FKBP12(4, 5, 6) . Studies in yeast and mammalian cells have established a novel mechanism of action for rapamycin and FK506. According to this model, the binding of rapamycin or FK506 to FKBP12 generates a toxic complex that interferes with a specific component of the intracellular signaling machinery. For example, the FKBP12bulletFK506 complex binds to and inhibits the Ca-calmodulin-regulated protein serine-threonine phosphatase, calcineurin(7, 8) , which catalyzes an event necessary for IL-2 gene transcription(9, 10) . In contrast, the FKBP12-rapamycin complex does not interact with calcineurin, and the molecular target(s) of this complex in lymphoid cells remains undefined.

Rapamycin's inhibitory action on G(1)-phase progression in yeast (11) parallels its anti-proliferative effect on IL-2-stimulated T-lymphocytes. Genetic studies in yeast may therefore provide clues regarding the mechanism of action of rapamycin in mammalian cells. As predicted from the gain-of-function model described above, disruption of the yeast FKB1 locus, which encodes FKBP12, yields viable cells that are resistant to rapamycin(11) . A screen for additional rapamycin-resistant yeast mutants led to the discovery of two novel genes, TOR1 and TOR2(12, 13) . The TOR1 and TOR2 genes encode related proteins containing putative lipid kinase domains homologous to those found in both yeast and mammalian phosphatidylinositol 3-kinases. Whether the FKBP12-rapamycin complex binds targets in mammalian cells that are homologous to the yeast TOR proteins remains an important but unsettled question.

In this study, we have isolated from mammalian tissue extracts a high molecular mass protein that binds specifically to the FKBP12-rapamycin complex. The amino acid sequence of this protein, which we have designated a mammalian target of rapamycin (mTOR), suggests that it represents a mammalian homolog of the yeast TOR proteins. Furthermore, comparisons of rapamycin-sensitive and -resistant mutant subclones of a murine T-lymphoma cell line indicate that drug resistance is highly correlated with reduced binding of mTOR to the FKBP12-rapamycin complex. These studies strongly suggest that mTOR represents a relevant target of the FKBP12-rapamycin complex in T-lymphocytes.


MATERIALS AND METHODS

Cell Lines

YAC-1 T-lymphoma cell lines were maintained in 10 mM Hepes-buffered RPMI 1640 medium (pH 7.2) supplemented with 10% fetal bovine serum, 2 mML-glutamine, and 50 µM 2-mercaptoethanol. The derivation of rapamycin-resistant and -sensitive YAC-1 subclones has been described previously(14) .

Production of Glutathione S-Transferase (GST)-FKBP12 Fusion Protein

A cDNA encoding the open reading frame of human FKBP12 was cloned into the BamHI site of pGEX2T (Pharmacia). The recombinant plasmid was transformed into E. coli strain JM101. To produce the GST-FKBP12 fusion protein, one-liter cultures of transformed bacteria were grown at 37 °C with shaking to an optical density (A) of 0.8. The culture was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside, and the incubation was continued for an additional 3 h. The bacteria were harvested and lysed by sonication according to the protocol supplied with the pGEX2T vector. GST-FKBP12 was purified from the cleared bacterial extract by chromatography over a glutathione (GSH)-agarose column. The column was washed with phosphatebuffered saline (PBS) containing 1% Triton X-100 (PBST), and the fusion protein was eluted with PBST containing 30 mM GSH. Peak fractions were pooled and dialyzed into PBS at 4 °C. The purified GST-FKBP12 displayed FK506 binding and peptidyl-prolyl isomerase activities equivalent to those observed with native FKBP12.

Preparation of Tissue Extracts

Frozen rat brains were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and were homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) in two volumes of 50 mM Tris-HCl, 100 mM NaCl, 15 mM beta-glycerophosphate, 1 mM CaCl 1 mM MgCl(2) (pH 7.4), containing 10% glycerol, 2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin. The homogenate was centrifuged for 30 min at 14,500 times g, and the resulting supernatant was recentrifuged for 1 h at 140,000 times g. Proteins in the supernatant were precipitated by dropwise addition of saturated ammonium sulfate solution to a final concentration of 30% (v/v). Precipitated proteins were collected by centrifugation and were resolubilized in 2 ml of homogenization buffer/brain equivalent.

YAC-1 cells (2 times 10^8 cells/sample) were harvested from culture medium, washed 2 times in PBS, and resuspended in hypotonic buffer (10 mM Tris-HCl, 5 mM MgCl(2) (pH 7.4)) containing 50 µM digitonin and the protease inhibitor mixture described above. After 20 min on ice, the cells were lysed in an all-glass Dounce homogenizer. The hypotonic buffer was subsequently converted to brain homogenization buffer by the addition of the appropriate amounts of the solutes described above. All remaining steps in the preparation of T-cell extracts were identical to those described above.

Affinity Purification of Drug-FKBP12 Binding Proteins

To prepare the affinity matrix, GST-FKBP12 (100 µg) was immobilized on 20 µl of GSH-coupled agarose beads. The beads were washed 4 times with homogenization buffer containing 0.02% Nonidet P-40. Brain or YAC-1 cell extracts (0.4-4 mg of protein/ml) were precleared with GSH-agarose loaded with GST-FKBP12. The cleared extracts were rotated for 1 h with immobilized GST-FKBP12 in the absence or presence of 10 µM rapamycin or FK506. The precipitates were washed 4 times with homogenization buffer, and bound proteins were eluted with 2 times Laemmli sample buffer(15) . Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and detected by silver staining (Bio-Rad).

Preparative scale purification of the FKBP12-rapamycin-binding protein was carried out for amino acid sequence analysis. GST-FKBP12 was immobilized on GSH-agarose and incubated with brain extract (3-5 mg of protein) in the presence of 10 µM rapamycin as described above. After the batch adsorption step, the remaining supernatant was discarded, and a fresh aliquot of brain extract was added to the same tube. The serial adsorption process was repeated until approximately 20 mg of extract protein had been incubated with each aliquot of GST-FKBP12-coupled beads. The bound proteins were eluted and separated by SDS-PAGE as described above.

Amino Acid Sequencing

After SDS-PAGE, the proteins were fixed and stained in 20% methanol, 0.5% glacial acetic acid containing 0.2% (w/v) Coomassie Blue. Based on the staining intensities of the target protein-containing gel fragments relative to that of a known standard (rabbit skeletal muscle myosin), we estimate that approximately 6 µg of purified protein was submitted to the Mayo Peptide Synthesis and Protein Sequencing Facility for further analysis. The appropriate regions of the gel were excised, and the protein was subjected to in-gel digestion with modified sequencing grade trypsin (BoehringerMannheim)(16) . The tryptic peptides were injected onto 2.1 times 220-mm Aquapore 300 high performance liquid chromatography column (Applied Biosystems) and were eluted from the column at a solvent flow rate of 0.2 ml/min. The initial eluting solvent was 1% solvent B (40% acetonitrile in 0.1% trifluoracetic acid) and 99% solvent A (5% acetonitrile in 0.1% trifluoracetic acid). After 8 min, a linear gradient to 10% solvent B was performed over 5 min. At 13 min, a second linear gradient to 100% solvent B was imposed over the next 67 min. The optical density of the eluate was monitored at 215 nm, and 0.1-ml fractions were collected. Amino acid microsequencing was performed on an Applied Biosystems Model 475A protein sequencer.

Isolation of a cDNA Clone Encoding a Mammalian TOR Homolog

Amino acid microsequencing yielded an unambiguous LELAVPG sequence that was also present in the yeast TOR proteins but not in any other known protein. To isolate the full-length cDNA encoding mTOR, primers based upon the LELAVPG sequence and upon regions of conservation between the yeast TOR proteins and the phosphatidylinositol 3-kinase catalytic subunit were used to generate a PCR fragment that could be used to probe a cDNA library. The first set of four sense primers were degenerate 17-mers covering the amino acid sequence ELAVPG obtained from the purified protein. The primers were GAR YTN GCN GTN CCT GG, GAR YTN GCN GTN CCC GG, GAR YTN GCN GTN CCA GG, and GAR YTN GCN GTN CCG GG. The first set of three antisense primers covered the conserved amino acid sequence (I/F)HIDFG, from the yeast TOR proteins(12, 13) . The primers were: NCC RAA RTC (A/G/T)AT RTG AA, NCC RAA RTC (A/G/T)AT RTG GA, and NCC RAA RTC (A/G/T)AT RTG TA. The primers were used in PCR reactions in all 12 possible combinations with rat brain cDNA as the template and with Ultima Taq Polymerase (Perkin-Elmer) as the polymerase. The PCR conditions were 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s.

The products of the first reaction were used as templates in a second PCR reaction to generate a nested product. The second set of four degenerate sense primers corresponded to the amino acid sequence (D/E)D(I/L)RQD(12, 13) . The primers were GAN GAY ATN CGN CAA GA, GAN GAY ATN CGN CAG GA, GAN GAY ATN AGN CAA GA, and GAN GAY ATN AGN CAG GA. The second set of four degenerate antisense primers covered the conserved sequence DRH(P/N)SN(12, 13) . The four primers were (A/G)TT N(G/C) (A/T) N(G/T) (G/T) (A/G)TG NCG ATC, (A/G)TT N(G/C) (A/T) N(G/T) (G/T) (A/G)TG NCG GTC, (A/G)TT N(G/C) (A/T) N(G/T) (G/T) (A/G)TG NCT ATC, and (A/G)TT N(G/C) (A/T) N(G/T)(G/T) (A/G)TG NCT GTC. Sixteen independent PCR reactions were performed using 0.5 µl of each of the 12 initial PCR reaction products as templates (192 reactions total). Eleven reactions yielded products of the predicted size (about 450 base pairs), and these fragments were subcloned into pUC19 and sequenced.

Of the eleven PCR products sequenced, one yielded a deduced amino acid sequence homologous to the yeast TOR proteins. That PCR product was labeled by random priming (Prime-It, Stratagene) with hexamers in the presence of [alpha-P]dATP and was used to screen 1.2 million recombinant phage from an oligo(dT) and randomly primed rat brain cDNA library (Stratagene). Prehybridization, high stringency hybridization, and washing conditions were performed according to the manufacturer's specifications. Positive phage were plaque-purified, and the EcoRI cDNA inserts were excised from DNA purified from plate lysates of the cloned phage. The EcoRI cDNA inserts were subcloned into pUC19 and sequenced with either Sequenase or Taq polymerase on an Applied Biosystems 373A automated DNA sequencer. Each strand was sequenced a minimum of 4 times each.

A 7.1-kilobase pair partial cDNA was isolated from the first screen. After sequencing, EcoRI-linked primers were synthesized and used in a PCR reaction to isolate the first 450 base pairs in the 7.1-kilobase pair partial cDNA. The sense primer was GAA TTC ATG AAY CCN GCN TTT GT, and the antisense primer was GAA TTC GTN GKR TAR WAY TCG TC. The product was digested with EcoRI and subcloned into pUC19. The product was excised from EcoRI, labeled as described above, and used to rescreen the same library. Several clones were isolated that overlapped with the 5` end of the 7.1-kilobase pair clone by as much as 4000 nucleotides and extended the sequence upstream by as much as 1500 nucleotides.

Because the open reading frame (ORF) still extended to the 5` end of the contiguous sequence, the remainder of the cDNA was isolated by RACE (rapid amplification of cDNA ends). The template was 5` Race-Ready rat brain cDNA (Clontech). The 5` anchor primer was supplied by the manufacturer. The antisense primers corresponding to the rat mTOR gene were GAA TTC CCA CCT TCC ACT CCA ATG for the primary reaction and GAA TTC CAT ATG CTT GGG ACA GGC CCT GCC ACG in the secondary PCR reaction. Sequencing of the RACE products demonstrated that we had obtained the remainder of the ORF.

In Vitro Transcription and Translation of mTOR cDNA

The 7.1-kilobase pair mTOR cDNA clone was excised from pUC19 by digestion with EcoRI, and the gel-purified insert was cloned into the EcoRI site of pSP72 (Promega). A recombinant pSP72 plasmid containing the 7.1-kilobase pair cDNA in the proper orientation relative to the T7 RNA polymerase promoter site was identified by restriction mapping and purified by cesium chloride gradient centrifugation. The cDNA was transcribed and translated in the presence of [S]methionine using the TnT T7 coupled reticulocyte lysate system (Promega). One-tenth volume (5 µl) of the translation reaction was denatured in Laemmli sample buffer and subjected to SDS-PAGE. The remainder of the translation reaction was diluted with homogenization buffer containing 1 mg/ml bovine serum albumin. The diluted sample was divided into aliquots and incubated with GSH-agarose-bound fusion proteins in the absence or presence of rapamycin or FK506, and protein-binding assays were performed as described above. The bound proteins were separated by SDS-PAGE. The gels were treated with EN^3HANCE (DuPont NEN), and the S-labeled translation products were detected by fluorography at -70 °C.


RESULTS

Detection of Drug-FKBP12 Binding Proteins in Tissue Extracts

Our strategy for the detection of rapamycin-FKBP12 ligands was based on the affinity purification technique previously employed for the identification of calcineurin as an intracellular target for the FK506bulletFKBP12 complex(7) . Tissue extracts were incubated with GSH-agarose-coupled GST-FKBP12 in the absence or presence of rapamycin or FK506, and bound proteins were analyzed by SDS-PAGE. As shown in Fig. 1, rapamycin-loaded GST-FKBP12 precipitated a high molecular mass (>200 kDa) protein from rat brain extracts. The interaction of this protein with GST-FKBP12 was specifically dependent on the presence of rapamycin, as the protein was not found in precipitates prepared without drug or with FK506. As predicted, the GST-FKBP12bulletFK506 complex recovered relatively abundant 57- and 61-kDa proteins corresponding to known isoforms of the calcineurin A subunit(7) . Both the calcineurin B subunit (15 kDa) and calmodulin (17 kDa) were also evident when the latter precipitates were electrophoresed through higher percentage acrylamide gels (data not shown).


Figure 1: Binding of rat brain-derived proteins to GST-FKBP12-drug complexes. Rat brain extracts (1 mg of protein/sample) were incubated with 100 µg of GST-FKBP12 coupled to GSH-agarose beads. Precipitations were performed in the absence of drug (-) or in the presence of 10 µM rapamycin (RAP) or 10 µM FK506 (FK). Precipitated proteins were eluted, resolved by SDS-PAGE through a 8.75% gel, and visualized by silver staining. Lane1 shows the molecular mass calibration standards, with actual masses (kDa) designated on the left. Unlabeled arrow on the right indicates the GST-FKBP12-rapamycin-specific binding protein. Labeled arrows on right indicate the locations of the 57- and 61-kDa isoforms of the calcineurin A (CnA) subunit and the GST-FKBP12 fusion protein.



Expression of Drug-FKBP12 Binding Proteins in T-lymphoma Cells

The results described above prompted additional studies to determine whether similar FKBP12-rapamycin binding proteins were expressed in T-lymphocytes. Earlier reports documented that rapamycin inhibits both the proliferation and the cytokine responsiveness of the murine T-lymphoma cell line, YAC-1(17, 18, 19) , indicating that YAC-1 cells express a functionally important target of rapamycin. The results in Fig. 2depict a comparison of the proteins recovered from rat brain and YAC-1 cell extracts by the GST-FKBP12-rapamycin complex. In the presence of rapamycin, the immobilized GST-FKBP12 specifically precipitated proteins with similar electrophoretic mobilities from both YAC-1 cell and rat brain extracts. Henceforth, these high molecular mass binding proteins will be provisionally designated as mTOR. Interestingly, the GST-FKBP12-rapamycin precipitates from YAC-1 cells contained two high molecular mass proteins, with the lower band co-migrating with mTOR isolated from rat brain. The recovery of the higher molecular mass species from different YAC-1 cell extracts appeared to correlate with that of the lower band (see Fig. 3), suggesting that these are either posttranslationally modified versions of the same protein or that the upper band represents a distinct protein whose interaction with the FKBP12-rapamycin complex is contingent on the binding of mTOR.


Figure 2: Isolation of mTOR from YAC-1 T-lymphoma cells. Extracts were prepared from rat brain and YAC-1 T-lymphoma cells, and equal amounts of total protein (0.45 mg/sample) were precipitated with GSH-agarose-coupled GST-FKBP12 in the absence(-) or presence (+) of 10 µM rapamycin (RAP). Bound proteins were separated by SDS-PAGE in a 7.5% gel and were visualized by silver staining as described in the Fig. 1legend. Doublearrow on the right indicates the location of brain and YAC-1 cell-derived mTOR.




Figure 3: Recovery of mTOR from wild-type and mutant YAC-1 clones. Left panel shows equivalent amounts of extract (0.45 mg of protein/sample) from wild-type (WT) YAC-1 cells, and the mutant clones R103 (103), R125 (125), 4R16, and 10R13 were precipitated with GST-FKBP12 in the absence(-) or presence (+) of 10 µM rapamycin (RAP). Bound proteins were separated and visualized as described in the legend to Fig. 2. Doublearrow on right indicates the location of mTOR. Right panel shows extracts that were prepared from rat brain, wild-type (WT) YAC-1 cells, a rapamycin-sensitive revertant subclone derived from R19 (R19), and the original, rapamycin-resistant R19 clone (R19). Recovery of mTOR was determined as described above.



Recovery of Putative Targets of Rapamycin from Mutant T-lymphoma Sublines

Recent studies characterized a panel of rapamycin-resistant and -sensitive clones derived from the YAC-1 T-lymphoma cell line(14) . The mutant clones were generated by treatment of YAC-1 cells with ethylmethanesulfonate, followed by limiting dilution cloning in rapamycincontaining culture medium. Clonal populations were selected for resistance to the antiproliferative effect of rapamycin. Further characterization of these clones revealed that several (R19, 4R16, and 10R13) displayed a stable, drug-resistant phenotype, whereas others (R103, R125) were fully sensitive to rapamycin in short term cultures. The three rapamycin-resistant clones expressed normal amounts of FKBP12 and retained wild-type levels of sensitivity to FK506, suggesting that the resistant phenotype was due to an alteration located downstream of FKBP12(14) . By analogy to the previously described yeast TOR mutants(11, 12, 13) , we reasoned that one or more of the rapamycin-resistant YAC-1 clones might express mutant mTOR with altered binding to the GST-FKBP12-rapamycin complex.

The silver-stained gel shown in Fig. 3, left panel, compares the recoveries of mTOR from wild-type (WT) YAC-1 cells, and rapamycin-sensitive (R103, R125) or -resistant (4R16, 10R13) clones. The immobilized GST-FKBP12-rapamycin complexes precipitated mTOR from both of the rapamycin-sensitive clones, R103 and R125, at levels comparable with those obtained from wild-type cells. In contrast, substantially lower levels of mTOR were consistently precipitated from the rapamycin-resistant clones 4R16 and 10R13. These results indicated that the reduced recovery of mTOR by the GST-FKBP12-rapamycin complex was correlated with the rapamycin-resistant phenotype in the mutant YAC-1 clones (see ``Discussion'').

The correlation between decreased binding of mTOR to the FKBP12-rapamycin complex and resistance to rapamycin was further substantiated by studies performed with a spontaneous revertant of the rapamycin-resistant R19 clone. After over 6 months in culture medium without rapamycin, a stock culture of R19 cells was found to express an increased level of sensitivity to rapamycin. A drug-sensitive revertant clone, designated R19, was subsequently isolated from this variant population. Binding experiments revealed that the GST-FKBP12-rapamycin complex consistently precipitated a higher amount of mTOR from R19 cells than from parental, rapamycin-resistant R19 cells (Fig. 3, right panel). Thus, the reacquisition of rapamycin sensitivity in the R19 clone was accompanied by an increase in the amount of mTOR precipitated by the GST-FKBP12-rapamycin complex.

mTOR Is a Mammalian Homolog of Yeast TOR1 and TOR2

The observation that little or no mTOR binds to the FKBP12-rapamycin complex in the rapamycin-resistant YAC-1 mutants strongly suggested that mTOR is a relevant target of rapamycin in mammalian cells. The yeast TOR1 and TOR2 genes had been cloned genetically from rapamycin-resistant yeast strains by their ability to confer rapamycin resistance to wild-type strains. The high molecular masses of both the yeast TOR gene products and purified mTOR suggested that mTOR might be a mammalian counterpart to yeast TOR1 and TOR2. We therefore hypothesized that mTOR might be related to the proteins encoded by yeast TOR1 and TOR2. To confirm this, the cDNA encoding rat mTOR was isolated from a rat brain cDNA library.

In order to clone the cDNA, mTOR was purified from rat brain as described previously and digested with trypsin, and the resulting tryptic peptides were subjected to amino acid sequencing. One of the peptides yielded an unambiguous heptapeptide sequence, LELAVPG. This heptapeptide is 100% identical to an amino acid sequence found in both the yeast TOR1 and TOR2 proteins(12, 13) . In contrast, the heptapeptide is not found in any other known protein in the protein (NBRF-PIR release 40, Swiss-Prot release 28) or translated nucleic acid (GenBank release 83) data bases. In particular, the LELAVPG sequence is not contained within any of the regions of homology between the yeast TOR proteins and the mammalian phosphatidylinositol 3-kinase catalytic subunits (p110). We designed a cloning strategy based in part on the mTOR- and yeast TOR-specific LELAVPG sequence. We further postulated that those regions in the yeast TOR proteins that share homology with p110 would also be conserved in mTOR. A set of ``sense'' PCR primers was designed that corresponded to the LELAVPG sequence. The use of these primers insured that cDNAs encoding mammalian phosphatidylinositol 3-kinase catalytic subunits would not be amplified because the encoded heptapeptide is not present in any phosphatidylinositol 3-kinase nor is it present in any other known protein. A set of ``antisense'' PCR primers was designed that corresponded to the amino acid sequence (I/F)HIDFG, which is well conserved between the yeast TOR proteins and p110. These sets of primers were used in PCR reactions (see ``Materials and Methods'' for details) with rat brain cDNA as the template. A small amount of the product from this primary PCR reaction was used in a secondary PCR reaction. The ``sense'' primers in the secondary reaction corresponded to the sequence ED(I/L)RQD, which is conserved between the yeast TOR proteins and the phosphatidylinositol 3-kinases. The ``antisense'' primers corresponded to another region of conservation, DRH(P/N)SN. One of the products of the secondary PCR reaction yielded a fragment of the size predicted assuming that mTOR is homologous to the yeast TOR1 and TOR2 proteins.

The PCR product was subcloned, and subsequent nucleotide sequencing revealed that it encoded a protein fragment with 70% identity to regions in TOR1 and TOR2 that correspond to their putative lipid kinase domains. In contrast, alignment of the mTOR fragment with the catalytic domain of bovine p110 demonstrated a lower level (32.5%) of amino acid identity. Therefore, we concluded that the PCR product we had isolated encoded a mammalian homolog of the yeast TOR proteins rather than a phosphatidylinositol 3-kinase catalytic subunit or related protein.

The PCR product was used to screen a randomly-primed rat brain cDNA library (see ``Materials and Methods''), and a 7.1-kilobase pair clone was isolated. Sequencing of the 7.1-kilobase pair clone revealed that it encoded a protein highly homologous to the yeast TOR proteins. Moreover, the encoded protein contained the LELAVPG sequence obtained by protein sequencing. The ORF extended to the 5` end of the cDNA indicating that it might not be complete. This was confirmed by the results of Northern analysis of rat brain mRNA with the 7.1-kilobase pair clone as a probe. The Northern suggested that the full-length mRNA might be as large as 9.5 kilobase pairs. (^2)A PCR product derived from the extreme 5` end of the 7.1-kilobase pair clone was used to rescreen the library. Three additional cDNAs were isolated that overlapped with the 7.1-kilobase pair clone and that extended the sequence in the 5` direction. The ORF still extended to the 5` end of the contiguous cDNA; therefore, the remainder of the cDNA was isolated by RACE. The complete cDNA contains 8554 nucleotides and encodes a protein of 2549 amino acids (Fig. 4) with a predicted molecular mass of 289 kDa. The presence of an in-frame stop codon upstream of the first methionine in the ORF indicates that it is probably the initiator methionine and that a full-length cDNA clone has been isolated.


Figure 4: Alignment of the translation product of the mTOR ORF with the translation products of the yeast TOR genes. Identical residues are boxed. Amino acid number is indicated at the right of each line. The LELAVPG sequence obtained from peptide sequencing is indicated by the heavyoverline. The locations of the primers used in the first PCR reaction (a and b) and the second PCR reaction (c and d) are indicated by arrows. The partial cDNA used to probe the cDNA libraries is contained between the c and d primers. Alignments were performed using GCG (Genetics Computer Group, University of Wisconsin, Madison) software(27) .



The translated ORF is shown aligned with the yeast TOR1 and TOR2 proteins in Fig. 4. The deduced amino acid sequence of mTOR is 42.4% and 44.9% identical to TOR1 and TOR2, respectively. The location of the LELAVPG heptapeptide (amino acids 2136-2142), which was obtained by protein sequencing and was used to design the first ``sense'' PCR primer (the a primer) is shown overlined. The deduced amino acid sequence of mTOR also contains the conserved sequences from yeast TOR proteins and the p110 subunit of mammalian phosphatidylinositol 3-kinase that were used to design the other three PCR primers: EDLRQD (residues 2190-2195), DRHPSN (residues 2338-2343), and LHIDFG (residues 2354-2359). The deduced amino acid sequence of mTOR proved to be identical to both yeast TOR proteins in all of these regions except for the conservative leucine for isoleucine substitutions in the EDLRQD and LHIDFG peptides. The original PCR product that was isolated encodes the region located between the c and d PCR primers (residues 2190 and 2343).

In summary, there are at least three features about the clone that confirm that we have isolated the cDNA encoding rat mTOR. First, the LELAVPG sequence obtained by peptide sequencing is contained within the ORF. Second, the calculated molecular weight of the encoded protein, 289 kDa, is similar to that of the protein that we have purified. Third, mTOR is highly similar in sequence to both of the yeast TOR proteins. Searches of two protein data bases (Swiss-Prot release 28 and NBRF-PIR release 40) and a translated nucleic acid data base (GenBank release 83) with the mTOR amino acid sequence confirmed that mTOR is more highly homologous to the yeast TOR proteins than it is to any other known proteins.

mTOR cDNA Encodes a Ligand for the FKBP12-Rapamycin Complex

To determine whether the isolated mTOR cDNA encodes a FKBP12-rapamycin-binding protein, the 7.1-kilobase pair cDNA clone described above was transcribed and translated in vitro in the presence of [S]methionine. The 5` terminus of this cDNA contains an in-frame AUG codon (corresponding to residue 488 in Fig. 4) located in a reasonable Kozak consensus context (20) for the initiation of translation. The full-length translation product encoded by the 7.1-kilobase pair cDNA would be a 234-kDa polypeptide containing mTOR residues 488-2549 (Fig. 4). Direct electrophoretic analysis of the mTOR cDNA-primed translation mixture revealed a closely spaced doublet of S-labeled polypeptides with apparent molecular masses of greater than 200 kDa (Fig. 5, lane 2). Incubation of the translation mixture with immobilized GST only or GST-FKBP12 resulted in the recovery of barely detectable amounts of this radiolabeled polypeptide (lanes 3 and 4). The binding of the mTOR-derived polypeptides to GST-FKBP12 was not enhanced by the addition of FK506 to the precipitation reaction (lane 5). In contrast, rapamycin dramatically increased the binding of the S-labeled translation products to the GST-FKBP12-coupled beads (lane 6). These results demonstrate that the isolated mTOR cDNA encodes a protein that binds directly and specifically to the FKBP12-rapamycin complex.


Figure 5: Binding of FKBP12-drug complexes to mTOR cDNA-derived translation products. A partial cDNA clone encoding mTOR residues 488-1259 (see Fig. 4) was transcribed and translated in vitro in the presence of [S]methionine (see ``Materials and Methods''). Five µl of the translation mixture was analyzed directly by SDS-PAGE in a 7.5% gel (lane 2). The translation products from a parallel reaction primed with a control luciferase cDNA are shown in lane 1. In lanes 3-6, the mTOR translation products were precipitated with the indicated GST-fusion proteins, in the absence or presence of FK506 or rapamycin (RAP). Bound polypeptides were solubilized and separated by SDS-PAGE. S-labeled polypeptides were detected by fluorography. The locations of molecular mass calibration standards are indicated on the left.




DISCUSSION

In this study, we used an affinity purification technique to isolate a direct ligand of the FKBP12-rapamycin complex from rat brain and murine T-lymphoma cells. Amino acid sequencing of the rat brain-derived target protein, designated mTOR, led to the isolation of a full-length cDNA whose deduced amino acid sequence displayed a high degree of identity to the yeast TOR1 and TOR2 gene products. TOR1 and TOR2 are highly related proteins that perform both overlapping and distinct functions in the regulation of the yeast cell cycle(11) . The present data do not allow us to determine whether the mTOR cDNA encodes a functional homolog of TOR1 or TOR2. Nonetheless, the present findings lend strong support to the hypothesis that the FKBP12-rapamycin complex interferes with G(1)-phase progression in yeast and mammalian cells by acting on a highly homologous set of target proteins.

Nonallelic noncomplementation among recessive, rapamycin-resistant alleles of FPR1, TOR1, and TOR2 in yeast provided the first genetic evidence that these gene products interacted in the presence of rapamycin(11) . The biochemical results presented in this study not only indicate that the FKBP12-rapamycin complex interacts with a mammalian TOR homolog but also demonstrate for the first time that a TOR family member serves as a direct ligand for the immmunophilin-drug complex. The available data are consistent with a model that proposes that the FKBP12-rapamycin complex exerts its growth-inhibitory and cytotoxic effects in yeast by inhibiting the functions of TOR1 and TOR2(21, 22) . Our studies suggest that this model can be extended to mammalian cells, and imply that mTOR represents a novel cell-cycle regulatory protein in rapamycin-sensitive cell types, including the T-lymphocyte.

The notion that mTOR represents a pharmacologically relevant target of rapamycin in T-lymphocytes was substantiated by studies of rapamycin-resistant clones of the YAC-1 T-lymphoma cell line. Earlier results had suggested that the resistant phenotypes of the 4R16, 10R13, and R19 clones were due to mutations located downstream of FKBP12(14) . In this study, we demonstrate that GST-FKBP12-rapamycin complexes precipitate significantly lower amounts of mTOR from rapamycin-resistant YAC-1 clones than from their sensitive counterparts. Northern analyses have shown that the sensitive and resistant cell lines express equivalent levels of mTOR mRNA, indicating that the observed differences in mTOR recovery are not due to alterations in mTOR mRNA turnover. (^3)These results support the hypothesis that the resistant clones express a mutation that leads to a reduction in mTOR binding affinity for the rapamycin-FKBP12 complex, while leaving the essential catalytic function of mTOR intact. T-cells expressing such a mutation would not experience a loss of mTOR function in the presence of rapamycin, and, therefore, would continue to transit G(1) phase at normal rates. A mutation leading to a reduction in the binding affinity of mTOR for the FKBP12-rapamycin complex, with otherwise normal mTOR catalytic activity, would be predicted to act in a dominant fashion in heterozygotes. Consistent with this prediction, heterokaryon fusions between rapamycin-sensitive, parental YAC-1 cells and rapamycin-resistant 4R16, 10R13, or R19 cells displayed mutant-type resistance to rapamycin. (^4)

Previous studies showed that the resistance of the 4R16, 10R13, and R19 clones to rapamycin was not absolute. In short term cultures, the proliferation of these cells was inhibited by 20-40% in the presence of micromolar concentrations of rapamycin(14) . The residual sensitivities of the 4R16, 10R13, and R19 clones to rapamycin suggests that these cells express mutations that reduce, but do not abrogate, the ability of the FKBP12-rapamycin complex to interact with mTOR. Sequencing of mTOR cDNAs from rapamycin-sensitive and -resistant YAC-1 clones might begin to localize the binding site for the FKBP12-rapamycin complex. However, resistance-conferring mutations may not be restricted to the mTOR gene locus. A recent report suggests that the lipid kinase domains of yeast TOR1 and TOR2 contain homologous serine residues that may require phosphorylation to interact with the rapamycin-FKBP12 complex(22) . Expression of a mutant kinase with decreased phosphotransferase activity toward mTOR might explain the observed decrease in mTOR recovery from the rapamycin-resistant YAC-1 clones.

The function of the TOR family members in cellular regulation remains enigmatic. The C-terminal 600 amino acid residues of mTOR are approximately 65% identical to TOR1 and TOR2. This region bears significant homology to the lipid kinase domains of mammalian p110 and yeast VPS34. Deletion experiments indicate that the putative lipid kinase domains of yeast TOR1 and TOR2 play essential but interchangeable roles in cell cycle regulation(22) . Nonetheless, biochemical proof that the TOR proteins possess phosphoinositide kinase activity is currently unavailable. Recent studies have shown that mammalian p110 is actually a dual specificity phosphotransferase(23) . In addition to the classically-defined phosphatidylinositol 3-kinase activity, p110 catalyzes the phosphorylation of a regulatory serine residue in the p85 subunit of the phosphatidylinositol 3-kinase heterodimer. It is conceivable that the TOR family members also function as dual specificity, lipid-protein kinases in yeast and mammalian cells.

The inhibitory effect of rapamycin on G(1)-phase progression in yeast parallels its antiproliferative action on IL-2-stimulated T-cells. Given the apparent conservation of TOR catalytic function in yeast and mammalian cells, it is tempting to speculate that the TOR proteins function as ``universal'' regulators of G(1)-phase progression in eukaryotic cells. However, studies with rapamycin as a pharmacologic probe suggest that TOR function is rate limiting for passage through G(1)-phase in some, but not all, mammalian cell types. Rapamycin strongly inhibits the growth of most hematopoietic cell lines, but exerts marginal to negligible growth-inhibitory effects on many mesenchymal or epithelial cell lines (24) .^3 A possible explanation is that rapamycin-insensitive cell types compensate for the loss of mTOR function through the expression of redundant signaling pathways that are not affected by the FKBP12-rapamycin complex.

An alternative hypothesis is that mTOR plays a permissive rather than an obligate role in G(1) to S phase progression in mammalian cells. The sensitivities of different cell types to rapamycin might therefore reflect intrinsic variations in the dependence of the rate of G(1)-phase progression on mTOR function. Notably, exposure of cycling T-cells to rapamycin appears to slow, rather than prohibit, the entry of late G(1)-phase cells into S phase. (^5)In yeast, a double disruption of TOR1 and TOR2 results in overt growth-arrest in G(1) phase, whereas disruption of TOR1 only yields a ``slow-growth phenotype''(22) . The growth-inhibitory effect of rapamycin on lymphoid and other sensitive mammalian cell types might resemble the phenotype of the yeast TOR1 mutant more closely than that of doubly-disrupted TOR1/TOR2 mutant.

The relatively tissue-specific effect of rapamycin on proliferation contrasts sharply with the ability of this drug to completely block p70 ribosomal S6-kinase (p70S6K) activation in most, if not all, mammalian cell types(24, 25, 26) . Indeed, earlier studies of mutant YAC-1 clones demonstrated that resistance to the anti-proliferative effect of rapamycin segregated with resistance to the inhibitory effect of this drug on p70S6K activity(14) . Thus, the protein kinase/phosphatase cascade that regulates p70S6K activity appears universally dependent on an upstream function performed by rapamycin-sensitive mTOR(s). Whether inhibition of p70S6K activity is related to the growth-inhibitory effect of rapamycin remains unclear. Perhaps mTOR functions as an upstream regulator of additional signal transduction pathways that are unrelated to cell cycle control. The expression of high levels of mTOR in adult brain, a tissue with a relatively low proliferative potential, supports this notion. Clearly, further biochemical characterization of this protein will be required to elucidate its roles in triggering G(1)-phase progression and other cellular responses.


FOOTNOTES

*
This work was supported by the Mayo Foundation and by Grant GM47286 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors made equivalent contributions to this work.

Leukemia Society of America Scholar. To whom correspondence should be addressed: Dr. Robert T. Abraham, Dept. of Immunology, Rm. 342B, Guggenheim Bldg., Mayo Clinic, Rochester, MN 55905. Tel.: 507-284-4095; Fax: 507-284-1637.

(^1)
The abbreviations used are: IL-2, interleukin-2; FKBPs, FK506 binding proteins; ORF, open reading frame; mTOR, mammalian target of rapamycin; GST, glutathione S-transferase; GSH, glutathione; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; p70S6K, p70 S6 kinase; PCR, polymerase chain reaction.

(^2)
M. Martin and G. Wiederrecht, unpublished results.

(^3)
R. T. Abraham, unpublished observations.

(^4)
F. J. Dumont, manuscript in preparation.

(^5)
R. T. Abraham and F. J. Dumont, unpublished observations.


ACKNOWLEDGEMENTS

We thank Ben Madden for expert assistance with amino acid sequence analysis. We also thank Dr. Larry Karnitz for helpful discussions and Kathy Jensen for assistance with the preparation of this manuscript.

Addendum-During the review of this manuscript, Brown et al.(28) and Sabatini et al.(29) reported the isolation of human and rat cDNA clones encoding FKBP12-rapamycin target proteins, termed FRAP and RAFT1, respectively. Comparison of the deduced amino acid sequences of FRAP and RAFT1 with that of mTOR indicates that all three cDNAs encode identical homologs of the yeast TOR proteins.


REFERENCES

  1. Sigal, N. H., and Dumont, F. J. (1992) Annu. Rev. Immunol. 10, 519-560 [CrossRef][Medline] [Order article via Infotrieve]
  2. Morice, W. G., Brunn, G. J., Wiederrecht, G., Siekierka, J. J., and Abraham, R. T. (1993) J. Biol. Chem. 268, 3734-3738 [Abstract/Free Full Text]
  3. Terada, N., Lucas, J. J., Szepesi, A., Franklin, R. A., Domenico, J., and Gelfand, E. W. (1993) J. Cell. Physiol. 154, 7-15 [Medline] [Order article via Infotrieve]
  4. Siekierka, J. J., Hung, S. H. Y., Poe, M., Lin, C. S., and Sigal, N. H. (1989) Nature 341, 755-757 [CrossRef][Medline] [Order article via Infotrieve]
  5. Standaert, R. F., Galat, A., Verdine, G., and Schreiber, S. L. (1990) Nature 346, 671-674 [CrossRef][Medline] [Order article via Infotrieve]
  6. Siekierka, J. J., Wiederrecht, G., Greulich, H., Boulton, D., Hung, S. H. Y., Cryan, J., Hodges, P. J., and Sigal, N. H. (1990) J. Biol. Chem. 265, 21011-21015 [Abstract/Free Full Text]
  7. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Cell 66, 807-815 [Medline] [Order article via Infotrieve]
  8. Fruman, D. A., Klee, C. B., Bierer, B. E., and Burakoff, S. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3686-3690 [Abstract]
  9. O'Keefe, S. J., Tamura, J., Kincaid, R. L., Tocci, M. J., and O'Neill, E. A. (1992) Nature 357, 692-694 [CrossRef][Medline] [Order article via Infotrieve]
  10. Clipstone, N. A., and Crabtree, G. R. (1992) Nature 357, 695-697 [CrossRef][Medline] [Order article via Infotrieve]
  11. Heitman, J., Movva, N. R., and Hall, M. N. (1991) Science 253, 905-909 [Medline] [Order article via Infotrieve]
  12. Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., and Hall, M. N. (1993) Cell 73, 585-596 [Medline] [Order article via Infotrieve]
  13. Cafferkey, R., Young, P. R., McLaughlin, M. M., Bergsma, D. J., Koltin, Y., Sathe, G. M., Faucette, L., Eng, W.-K., Johnson, R. K., and Livi, G. P. (1993) Mol. Cell. Biol. 13, 6012-6023 [Abstract]
  14. Dumont, F. J., Altmeyer, A., Kastner, C., Fischer, P. A., Lemon, K. P., Chung, J., Blenis, J., and Staruch, M. J. (1994) J. Immunol. 152, 992-1003 [Abstract/Free Full Text]
  15. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  16. Rosenfeld, J., Capdevielle, J., Guillemont, J. C., and Ferrara, P. (1992) Anal. Biochem. 203, 173-179 [Medline] [Order article via Infotrieve]
  17. Dumont, F. J., Melino, M. R., Staruch, M. J., Koprak, S. L., Fischer, P. A., and Sigal, N. H. (1990) J. Immunol. 144, 1418-1424 [Abstract/Free Full Text]
  18. Altmeyer, A., Staruch, M. J., Cofano, F., Landolfo, S., and Dumont, F. J. (1991) Cell Immunol. 138, 94-107 [Medline] [Order article via Infotrieve]
  19. Altmeyer, A., and Dumont, F. J. (1993) Cytokine 5, 133-143 [Medline] [Order article via Infotrieve]
  20. Kozak, M. (1986) Nucleic Acids Res. 15, 8125-8132 [Abstract]
  21. Kunz, J., and Hall, M. N. (1993) Trends Biochem Sci. 18, 334-338 [CrossRef][Medline] [Order article via Infotrieve]
  22. Helliwell, S. B., Wagner, P., Kunz, J., Deuter-Reinhard, M., Henriquez, R., and Hall, M. N. (1994) Mol. Biol. Cell 5, 105-118 [Abstract]
  23. Dhand, R., Hiles, I., Panayotou, G., Roche, S., Fry, M. J., Gout, I., Totty, N. F., Truong, O., Vicendo, P., Yonezawa, K., Kasuga, M., Courtneidge, S. A., and Waterfield, M. D. (1994) EMBO J. 13, 522-533 [Abstract]
  24. Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J. (1992) Cell 69, 1227-1236 [Medline] [Order article via Infotrieve]
  25. Kuo, C. J., Chung, J., Fiorentino, D. F., Flanagan, W. M., Blenis, J., and Crabtree, G. R. (1992) Nature 358, 70-73 [CrossRef][Medline] [Order article via Infotrieve]
  26. Calvo, V., Crews, C. M., Vik, T. A., and Bierer, B. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7571-7575 [Abstract]
  27. Deveraux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395 [Abstract]
  28. Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758 [CrossRef][Medline] [Order article via Infotrieve]
  29. Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994) Cell 78, 35-43 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.