Rapamycin is a natural product with both antifungal and immunosuppressive activities (reviewed in (1, 2, 3) ). In mammalian T-lymphocytes, rapamycin blocks an unknown step in the signal transduction pathway initiated by interleukin-2 (IL-2), ()leading to G cell cycle arrest via inhibition of cyclin D- and cyclin E-dependent p33 and p34kinase activities(4, 5, 6, 7) . This arrest may result from a block in IL-2-stimulated Cdk-inhibitor p27 degradation(7) . Rapamycin also prevents the phosphorylation and activation of the p70 S6 kinase(8, 9) .

Several studies have shown that rapamycin and the structurally related macrolide FK506 are mutually antagonistic inhibitors of T-cell activation(10, 11) . The intracellular receptor for FK506 is the cytoplasmic 12-kDa cis-trans peptidyl-prolyl isomerase FKBP12 (for FK506 Binding Protein; Refs. 12 and 13). Prolyl isomerization is a rate-limiting step in protein folding, and two classes of enzymes catalyze this reaction, the FKBPs and cyclophilins. Despite their similar activity, cyclophilins and FKBPs share no homology; remarkably, though, cyclophilin A is the intracellular receptor for the immunosuppressant cyclosporin(14, 15, 16) . FKBP12 also binds rapamycin(17) , but FK506 and rapamycin inhibit distinct T-cell signaling pathways; FK506 prevents IL-2 expression in response to antigen presentation to the T cell receptor while rapamycin prevents the subsequent autocrine response to IL-2(10, 11, 17) .

Early models suggested that immunosuppression resulted from inhibition of FKBP12 enzymatic activity; however, several lines of evidence argue against this. First, several FK506 analogs inhibit isomerase activity but are not immunosuppressive(18, 19) . Additionally, rapamycin is toxic to the yeast Saccharomyces cerevisiae, but mutants lacking yeast FKBP12 (fpr1) are viable and drug resistant (20) . The relevant target of both FK506 and cyclosporin, the Ca/calmodulin-regulated protein phosphatase calcineurin, was identified using FKBP12-FK506 and cyclophilin-cyclosporin affinity chromatography(16) . Inhibition of calcineurin by these complexes prevents T-cell activation by blocking the nuclear import of the cytoplasmic subunit of NFAT, a transcription factor that regulates transcription of genes involved in T-cell activation(21, 22, 23) .

Genetic screens in yeast identified mutations in three genes, FPR1 (encodes FKBP12), TOR1, and TOR2, which confer rapamycin resistance(20) . TOR1 and TOR2 (for Target Of Rapamycin) encode large proteins related to phosphatidylinositol and protein kinases(24, 25, 26) , and TOR2 has been shown to have an associated phosphatidylinositol 4-kinase activity (27) . TOR2 is essential whereas TOR1 is not. Depletion of both TOR genes leads to a G cell cycle arrest resembling that imposed by rapamycin(20, 25) . The genetic finding of nonallelic noncomplementation between tor1, tor2, and fpr1 mutations led to a model in which the FKBP12-rapamycin complex physically interacts with the TOR proteins(20) , analogous to the interaction between FKBP12-FK506 and calcineurin. This model has recently been confirmed by the discoveries that TOR2 and the mammalian TOR homologue (mTOR, also known as FRAP, RAFT1, and RAPT1) bind FKBP12-rapamycin in vitro(27, 28, 29, 30, 31) and that FKBP12-rapamycin interacts with a small piece of TOR2 or mTOR in the two-hybrid system (32, 33) . It is not yet clear how binding of FKBP12-rapamycin to TOR arrests the cell cycle.

To characterize molecular interactions between FKBP12-rapamycin and TOR proteins, we identified additional rapamycin-resistant yeast mutants. We describe here several novel TOR2 mutations and employ the two-hybrid system to examine interactions between wild-type and mutant TORs and the FKBP12-rapamycin complex. Our studies confirm that the TORs physically interact with FKBP12-rapamycin, that TOR mutations confer rapamycin resistance by preventing this interaction, and that a composite FKBP12-rapamycin surface contacts TOR.

MATERIALS AND METHODS

Media and Strains

Yeast strains MLY10 (tor1::LEU2 TRP1 MAT), MLY11a (tor1::LEU2 HIS4 MATa), MLY58-3a (tor1::LEU2 tor2-1 TRP1 MAT), MLY60 (tor1::LEU2 tor2-1 HIS4 MATa), and MH346-1a (tor2::ADE2 ade2 MATaTOR2-URA3-2µ) ((25) ) were isogenic with strain JK9-3d (ura3-52 leu2-3, 112 his4 trp1 rme1 HMLa) except where indicated. Strain SMY4 (TOR1-3 fpr1::ADE2) was isogenic with two-hybrid host strain Y190 (38) and has been described(39) .

Mutant Isolation

DNA Manipulations

Oligonucleotide Primers

Site-directed Mutagenesis

Two-hybrid Plasmid Constructions

The GAL4(AD)-FKBP12 plasmid was created by subcloning FPR1 from pSBH1 (39) into pGAD424. The GAL4(BD)-FKBP12 mutants described above were subcloned into pGAD424 to produce GAL4(AD)-FKBP12 plasmids pML68 (R49I), pML69 (F94V), pML70 (R49I F94V), pML71 (D48V), and pML73 (F43Y).

The GAL4(BD)-TOR1 fusion plasmids were constructed by PCR amplification of genomic DNA from strain JK9-3da (TOR1 wt) or R1 (TOR1-1, S1972R; (20) ) using primers 370 and 374 (above) and cloning into pGBT9, producing plasmids pML80 (wt) and pML82 (S1972R). The GAL4(BD)-mTOR constructs were amplified from rat cDNA (wt) or a subclone bearing a site-directed mutation (S2035I; a gift of C. Alarcon) using primers 398 and 399 and cloning into pGBT9, producing plasmids pML88 (wt) and pML90 (S2035I).

Full-length TOR2 Plasmid Construction

The plasmids used in the minimum inhibitory concentration experiment (Table 1) were pSEY18-TOR2 (2µ-URA3-TOR2; (25) ) and pML48 (CEN URA3-TOR2), which was created by subcloning TOR2 from pML40 into plasmid pRS316(43) .

Western Blotting

-Galactosidase Assays

RESULTS

TOR Gene Expression Levels Alter Rapamycin Sensitivity

Isolation of Rapamycin-resistant Mutants

Spontaneous mutants resistant to 10 ng/ml rapamycin were isolated in two isogenic parental strains with convenient markers for genetic analyses (MLY10a and MLY11, see ``Materials and Methods''). TOR2 mutations usually confer dominant rapamycin resistance, precluding complementation tests. TOR2 mutations were identified by segregation analysis following a cross to a tor2-1 rapamycin-resistant mutant. In this cross, TOR2 mutations segregated 4R:0S at meiosis, whereas mutations unlinked to TOR2 exhibited 4R:0S, 3R:1S, and 2R:2S (in a 1:2:1 ratio) segregation events. In this screen, mutations in TOR2 and one other gene were identified. We characterize here the TOR2 mutations.

Identification of TOR2 Mutations

Figure 1: Rapamycin resistance of tor2 alleles. Isogenic strain expressing wild-type and mutant TOR2 alleles were assayed for growth on YPD medium containing the indicated concentration of rapamycin at 30 °C for 3 days.

The remaining TOR2 mutations conferred only partial rapamycin resistance (Fig. 1). To identify these mutations, the TOR2 genes were amplified and cloned from genomic DNA by long range PCR (see ``Materials and Methods''). The cloned TOR2 genes were shown to functionally complement, restoring viability and conferring rapamycin resistance, by a plasmid shuffle in a tor2 strain (see ``Materials and Methods''). Restriction fragment exchange between mutant and wild-type TOR2 plasmid clones delimited the mutations to a 2-kb region surrounding Ser-1975. Sequence analysis identified single nucleotide changes resulting in substitutions of W2042L (3 times), W2042C(1) , and F2049L (1) (Fig. 2A). The three identified residues, Ser-1975, Trp-2042, and Phe-2049, are all conserved between TOR1, TOR2, and mTOR.

Figure 2: Schematic of TOR mutations and two-hybrid constructs with their ability to bind to FKBP12. Panel A, Full-length TOR2 depicting the region used in the two-hybrid system, including the specific mutations analyzed in this study (left). The interaction of these fusions with GAL4(AD)-FKBP12 in medium with or without 1.0 µg/ml rapamycin as measured by the activity of the -galactosidase reporter is shown in the table on the right. Panels B and C, similar to panel A, diagramming the regions and mutations of TOR1 (B, left) and mTOR (C, left) and their interaction with GAL4(AD)-FKBP12 (right). Panel D, a schematic of full-length TOR2, indicating other segments of TOR2 that were tested in the two-hybrid system (left) and their interaction (+) or lack of interaction(-) with GAL4(AD)-FKBP12 in the presence or absence of 1.0 µg/ml rapamycin (right). PI, phosphatidylinositol.

FKBP12-Rapamycin Binds Wild-type but Not Mutant TORs

Rapamycin stimulated a complex between wild-type TOR2 and FKBP12 in the two-hybrid assay (Fig. 2A and Fig. 4), and this interaction was competed with excess FK506 (data not shown). Larger pieces of TOR2 failed to interact with FKBP12 or FKBP12-rapamycin in the two-hybrid assay (Fig. 2D). Introduction of TOR2 rapamycin resistance mutations (S1975I, S1975R, W2042L, W2042C, and F2049L) prevented FKBP12-rapamycin binding (Fig. 2A). Western blot analysis confirmed that the wild-type and mutant GAL4-TOR2 fusion proteins were expressed to approximately equivalent extents (Fig. 3). Thus, these mutations act by blocking the TOR2-rapamycin-FKBP12 complex and do not, for example, destabilize the GAL4-TOR2 fusion protein. Because the W2042C, W2042L, and F2049L mutations confer only partial rapamycin resistance in vivo but do not bind FKBP12-rapamycin in the two-hybrid assay, other regions of TOR2, or other interacting proteins, likely participate in vivo.

Figure 4: Two-hybrid interactions of TOR1, TOR2, and mTOR with FKBP12-rapamycin. -Galactosidase activity was measured in strain SMY4, expressing the GAL4(AD)-FKBP12 fusion and GAL4(BD)-TOR1 (), GAL4(BD)-TOR2 (), or GAL4(AD)-mTOR in the presence of increasing concentrations of rapamycin. Vertical bars represent the variation between two separate experiments; where absent, the error was smaller than the symbol on the graph.

Figure 3: Wild-type and mutant GAL4-TOR2 and GAL4-FKBP12 fusion proteins are stably expressed. Panel A, GAL4(BD)-TOR wt and mutant fusion proteins were expressed in strain SMY4 and detected by Western blot with -GAL4(BD) antiserum in highly concentrated, trichloroacetic acid-precipitated extracts. Due to the low steady state levels of the fusions, about 600 µg total protein were loaded per lane. Panel B, the same extracts as in panel A were detected using -cyclophilin A antiserum as a control for protein loading. Panel C, extracts of strains expressing GAL4(AD)-FKBP12 fusions were detected by Western blot with -FKBP12 antiserum. The extracts were prepared as in panel A. Panel D, extracts from panel C were detected with -cyclophilin A as a control for protein loading. The portions of the gels shown represent molecular masses from 22 to 35 kDa (A), 7 to 24 kDa (B, D), and 25 to 50 kDa (C).

We created similar GAL4(BD)-TOR1 and GAL4(BD)-mTOR fusions and found that both TOR1 and mTOR bind to FKBP12-rapamycin (Fig. 2, B and C, and 4). Mutations at Ser-1972 of TOR1 and Ser-2035 of mTOR eliminated interaction. To examine the relative affinities of TOR proteins for FKBP12-rapamycin, two-hybrid reporter gene expression was measured with increasing concentrations of rapamycin in strains co-expressing the GAL4(AD)-FKBP12 and a GAL4(BD)-TOR fusion protein. Half-maximal binding of FKBP12-rapamycin to TOR1 occurred at 10-fold lower rapamycin concentration compared to TOR2 and for mTOR was intermediate between TOR1 and TOR2 (Fig. 4).

FKBP12-Rapamycin-TOR Complexes Do Not Require FKBP12 Prolyl Isomerase Activity

Figure 5: FKBP12 mutations alter interactions with the TOR proteins. Panels A-C, GAL4(AD)-FKBP12 fusion proteins encoding the indicated mutation were tested for interaction with wild-type TOR2 (A), TOR1 (B), or mTOR (C) GAL4(BD) fusion proteins. , 0.0 µg/ml rapamycin; , 0.1 µg/ml; &cjs2108;, 1.0 µg/ml.

FKBP12 Surface Residues Bind TOR1 and TOR2

A hydrophobic substitution of an FKBP12 acidic surface residue, D48V, had only a minor 2-4-fold effect on FKBP12-rapamycin binding to TOR1 or TOR2 (Fig. 5, A and B). Substitution of two other surface residues, R49I and F94V, alone and in combination, had a more severe impact on binding of the mutant FKBP12-rapamycin complex to TOR1 and TOR2. There are, however, subtle differences between the TOR1 and TOR2 complexes with FKBP12-rapamycin (Fig. 5, A and B). For example, the F94V mutation impaired formation of the FKBP12-rapamycin-TOR2 complex, and this defect was more pronounced with the R49I,F94V double mutant. In contrast, the FKBP12-rapamycin-TOR1 complex was more sensitive to the R49I mutation, and the F94V mutation on its own had little or no effect on association with TOR1. Rapamycin-stimulated binding of GAL4(AD)-FKBP12 to GAL4(BD)-mTOR was less affected by these mutations (Fig. 5C), possibly as a result of the relatively stronger interaction (7-fold; Fig. 2C) with FKBP12-rapamycin compared with TOR1 or TOR2.

The GAL4(AD)-FKBP12 mutant fusion proteins were also expressed in an FKBP12 mutant strain and tested for functional complementation. The degree to which these fusion proteins complemented to restore rapamycin sensitivity in vivo (Fig. 6) correlated with ability to bind to GAL4(BD)-TOR2 fusion proteins (Fig. 5), indicating that binding to TOR2 is directly correlated with toxicity. Taken together, these findings reveal that a composite FKBP12-drug surface contacts the TOR proteins.

Figure 6: FKBP12 mutants that fail to bind the TOR proteins do not complement fpr1. The FKBP12 mutant fusion proteins were expressed in the FKBP12-deficient strain JHY2-1c (fpr1::ADE2) and grown on synthetic medium lacking leucine (SD-Leu) and supplemented with the indicated concentration of rapamycin for 2 days at 30 °C.

DISCUSSION

Our finding that rapamycin stimulates the formation of complexes between FKBP12 and both the TOR1 and TOR2 proteins, taken together with other recent reports of FKBP12-rapamycin binding to mTOR(28, 29, 30, 33) , to a TOR2 fragment(32) , or to intact TOR2 (27) confirms the original model, based on genetic evidence, that the TOR proteins are the direct targets of FKBP12-rapamycin(2, 20) . Moreover, the finding that drug-resistant TOR mutants no longer bind to FKBP12-rapamycin further implicates FKBP12-rapamycin binding to the TOR proteins (especially to TOR2, which is essential) as the critical event in rapamycin toxicity.

Our findings reveal that while FKBP12 prolyl isomerase activity is dispensable, FKBP12 surface residues are important for FKBP12-rapamycin binding to TOR1 and TOR2. FKBP12-rapamycin binding to TOR2 was more sensitive to perturbation by FKBP12 mutations compared with binding to TOR1, suggesting that although TOR1 and TOR2 are highly related, there are structural differences in the FKBP12-rapamycin-TOR interfaces. Notably, residues Arg-49 and Phe-94 of yeast FKBP12, and the corresponding residues Arg-42 and His-87 of human FKBP12, have been previously implicated in FKBP12-FK506 binding to calcineurin(39, 51, 52, 53) . Taken together, these findings suggest that some of the same FKBP12 surface residues are important for the two distinct FKBP12-drug inhibitor complexes to interact with different targets, calcineurin, and the TOR proteins.

The rapamycin-resistant TOR2 mutations and FKBP12 residues identified here are likely to be relevant for studies of FKBP12-rapamycin-TOR structures. The minimal FKBP12-rapamycin binding domain of TOR would be amenable for structural determination by either NMR or crystallography. Our studies define Trp-2042 and Phe-2049 as residues that, with Ser-1975, are likely to lie on the TOR interface with FKBP12-rapamycin. Similarly, FKBP12 residues Arg-49 and Phe-94 likely lie on the FKBP12-rapamycin interface with TOR2. These mutations should serve to guide analysis of FKBP12-rapamycin-TOR structures and models.

There are several biological correlates to our analysis of FKBP12-rapamycin-TOR interactions in the two-hybrid system. The relative binding affinities of mutant FKBP12-rapamycin complexes to TOR2 (Fig. 5) were well correlated with functional complementation and restoration of rapamycin sensitivity in vivo (Fig. 6). Importantly, we found that FKBP12-rapamycin binding to TOR1 occurs at 10-fold lower rapamycin concentrations compared to TOR2 (Fig. 4). We believe this comparison of relative binding affinity is valid based on the following findings. First, the same highly homologous domain from TOR1, TOR2, and mTOR was fused to GAL4. Second, by Western blot, the GAL4-TOR1 and GAL4-TOR2 fusion proteins were equivalently expressed (Fig. 3). Importantly, two biological observations also support the interpretation that TOR1 binds to FKBP12-rapamycin with higher affinity than TOR2. First, yeast strains lacking TOR1 are rapamycin hypersensitive, indicating that TOR1 normally effectively competes with TOR2 for FKBP12-rapamycin. Second, in yeast strains in which both TOR1 and TOR2 are required for viability, rapamycin sensitivity is increased 10-fold(27) .

Recent studies from our laboratory reveal that while FKBP12-rapamycin binds TOR2, it does not inhibit the phosphatidylinositol 4-kinase activity associated with TOR2(27) . Importantly, the TOR1 and TOR2 domains that interact with FKBP12-rapamycin are distinct from and do not overlap the putative kinase domain. A data base search using the BLAST program of NCBI with the 1886-2081 region of TOR2 returns strong homology to only TOR1 and mTOR and very weak homology to another yeast protein, ESR1/MEC1(54, 55) . Significantly, this region shows no similarity to any other phosphatidylinositol 3- or phosphatidylinositol 4-kinases, indicating that it is outside the putative active site. FKBP12-rapamycin binding to TORs may inhibit interactions with other proteins that may, for example, be important for intracellular localization(27) .

We also found that yeast FKBP12-rapamycin binds mTOR, the mammalian homologue of the yeast TOR proteins, indicating that the FKBP12-TOR interaction surface is highly conserved from yeast to man. Mutation of the conserved serine residue in mTOR abolished binding by FKBP12-rapamycin. Further studies will be required to establish mTOR functions in rapamycin-sensitive signaling cascades in vivo. One approach would be to test if introduction of the mTOR mutant into T-lymphocytes confers rapamycin-resistant IL-2 signaling. Alternatively, mTOR might provide TOR1 or TOR2 function in yeast or render yeast rapamycin resistant when mutated. Such studies are in progress.

FOOTNOTES

*

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.

§

Supported by National Institutes of Health Training Grant 5T32GM07184-20 to the Duke University Cell and Molecular Biology Program.

¶

Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Genetics, Box 3546, 322 CARL Bldg., Research Dr., Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2824; Fax: 919-684-5458; heitm001@mc.duke.edu.

()

The abbreviations used are: IL, interleukin; kb, kilobase(s); PCR, polymerase chain reaction; wt, wild type.

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