(Received for publication, September 30, 1994)
From the
The immunosuppressive drug, rapamycin, interferes with an
undefined signaling pathway required for the progression of
G-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
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
- to S-phase progression
in T-lymphocytes and other sensitive cell types.
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) (
)and several other cytokines. In contrast, rapamycin
inhibits IL-2-stimulated T-cell proliferation by blocking cell cycle
progression from late G
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 FKBP12FK506 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-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.
YAC-1 cells (2
10
cells/sample) were harvested from culture medium, washed
2 times in PBS, and resuspended in hypotonic buffer (10 mM Tris-HCl, 5 mM MgCl
(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.
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.
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 [-
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.
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.
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.
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.
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. ()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.
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.
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-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. ()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
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. (
)
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-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
-phase progression
in eukaryotic cells. However, studies with rapamycin as a pharmacologic
probe suggest that TOR function is rate limiting for passage through
G
-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) .
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 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
-phase progression on mTOR
function. Notably, exposure of cycling T-cells to rapamycin appears to
slow, rather than prohibit, the entry of late G
-phase cells
into S phase. (
)In yeast, a double disruption of TOR1 and TOR2 results in overt growth-arrest in G
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-phase progression and other cellular responses.
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.