1 Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University
of California, Los Angeles, CA 90095-1489, USA
2 Department of Microbiology, Immunology and Molecular Genetics, Jonsson
Comprehensive Cancer Center, University of California, Los Angeles, CA
90095-1489, USA
3 Department of Molecular, Cell and Developmental Biology, Jonsson Comprehensive
Cancer Center, University of California, Los Angeles, CA 90095-1489, USA
Authors for correspondence (e-mail:
jlengyel{at}ucla.edu;
fuyut{at}microbio.ucla.edu)
Accepted 8 May 2003
![]() |
Summary |
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Key words: Cell cycle, Growth, Drosophila, Rheb, TOR
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Introduction |
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Recently, the insulin and TOR (target of rapamycin) signaling pathways have
been shown to regulate growth in Drosophila
(Kozma and Thomas, 2002;
Oldham and Hafen, 2003
).
Mutants in these pathways affect total organ and body size, as well as
individual cell size. A downstream target of both pathways is dS6K, mutations
in which affect only cell size, not cell number
(Montagne et al., 1999
). Most
known components of the insulin and TOR signaling pathways affect both cell
size and number; current understanding is that these effects on cell number
are primarily via their effects on cell growth
(Coelho and Leevers, 2000
;
Johnston and Gallant, 2002
;
Kozma and Thomas, 2002
).
The Ras-like gene Rheb (Ras homolog enriched in brain) was
recently shown to be required for growth of the yeast Schizosaccharomyces
pombe. The rheb mutants (rhb1) arrest in G0/G1 as
small, rounded cells (Mach et al.,
2000; Yang et al.,
2001
), suggesting a role for Rheb in cell cycle
progression and cell growth. Highly conserved Rheb genes have been
described throughout the metazoa (Yamagata
et al., 1994
; Reuther and Der,
2000
; Urano et al.,
2000
; Urano et al.,
2001
; Im et al.,
2002
; Panepinto et al.,
2002
). To investigate the cellular role of Rheb, we have taken
advantage of the unique suitability of Drosophila for both genetic
and biochemical studies. Here, we show that Drosophila dRheb has both
GTP binding and GTPase activities. Overexpression of dRheb results in
tissue overgrowth and increased cell size in the whole organism, and
transition into S phase and cell growth in culture. Conversely, reduction of
dRheb activity results in reduced tissue growth and smaller cell size
in the whole organism, as well as a G1 arrest and smaller cell size in
culture. The results of treating S2 cells and flies with rapamycin, an
inhibitor of TOR, suggest that the effects of dRheb are probably mediated by
dTOR.
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Materials and Methods |
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Mapping and lethal phase of dRhebAV4
Genomic DNA at the 3' end of the P{y+,5XUAS}
insert in dRhebAV4 was isolated by inverse PCR
(Sullivan et al., 2000).
Amplified product was cloned into the pCR4-TOPO vector (Invitrogen) and
sequenced. The insertion site was identified by FlyBLAST
(http://www.fruitfly.org/blast/).
For determination of lethal phase, eggs were collected from
dRhebAV4/TM6-GFP adults on yeasted apple juice agar plates
for 2 hours. After aging for various times, larvae were washed onto fresh
plates and scored under a fluorescence-equipped dissecting microscope for
genotype, based on the presence or absence of green fluorescent protein (GFP).
For effect of rapamycin on time of eclosion, 2-hour collections of 100 embryos
each were placed in multiple vials containing standard food with and without 1
µM rapamycin (Calbiochem).
Histology and phenotypic analysis
In situ hybridization to whole-mount embryos was performed as described
previously (Tautz and Pfeifle,
1989). Digoxigenin RNA probes (Roche Molecular Biochemicals) were
generated from cDNA templates of the dRheb open reading frame. BrdU
incorporation into embryos for 1 hour was carried out as described
(Smith and Orr-Weaver, 1991
)
and detected with anti-BrdU antibody (1:40, Becton Dickinson) using standard
techniques (Ashburner, 1989
).
Scanning electron microscopy and fixation, embedding and sectioning of adult
eyes were performed as described (Wolff
and Ready, 1991
; Sullivan et
al., 2000
). Light microscopy was performed with a Zeiss Axiophot
and images were acquired with a Sony DKC-500 digital camera and processed with
Adobe Photoshop. Cell size and number in the wing were determined as described
by Montagne et al. (Montagne et al.,
1999
). Wing areas were measured using Image J software on 10-12
wings for each genotype; anterior was defined by wing vein L2 and anterior
wing margin, posterior by vein L3 and posterior wing margin. Wing hair density
was determined by counting wing hairs in a 0.0275 mm2 area in the
anterior and in a 0.0544 mm2 area in the posterior of each wing.
Sample standard error was
5% of the mean for all measurements.
Drosophila cell culture, transfection, RNAi, cell cycle
analysis
S2 cells (1x106) were seeded and maintained in Schneider's
Drosophila medium (Gibco) supplemented with 10% fetal bovine serum
(Gibco) at 25°C. Transfection was carried out using Cellfectin
(Invitrogen) with 1 µg each of plasmids pPacFLAG-dRheb (expressing
FLAG-dRheb using the actin 5C promoter) and pRmHa-GFP (transfection
marker, expressing GFP using metallothionein promoter). For RNAi,
dRheb template containing T7 promoter sequence was generated by PCR
from the cDNA; double-stranded RNA (dsRNA) corresponding to the entire coding
sequence (546 bp) was synthesized using T7 Megascript kit (Ambion). S2 cells
were treated with 10 µg dRheb dsRNA with and without
pPacFLAG-dRheb according to Clemens et al.
(Clemens et al., 2000). For
cell cycle analysis by fluorescence-activated cell sorting (FACS), transfected
cells were fixed in 1% formalin/PBS and 70% ethanol, whereas RNAi-treated
cells were fixed in 70% ethanol alone. After fixing, cells were stained with
25 µg ml-1 propidium iodide and FACS was performed on a Becton
Dickinson FACScan. For immunoblotting, total cell extracts were prepared in
insect cell lysis buffer (BD Pharmingen) and electrophoresed on a 12% SDS
polyacrylamide gel; after blotting, proteins were immunodetected with
anti-FLAG antibody (Sigma).
Expression and purification of dRheb and biochemical analyses
dRheb coding sequence, amplified from a plasmid cDNA library
(provided by F. Laski, University of California, Los Angeles) by PCR, was
inserted into vector pET28a(+) (Novagen). Total cell lysates of bacteria
expressing His-tagged dRheb, induced by IPTG, were prepared in lysis buffer
(20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 0.5 mM MgCl2, 10 µM
GTP) using a French press. Protein was purified using Probond resin
(Invitrogen) and stored in 50% glycerol at -20°C. GTP binding was
determined by nitrocellulose filtration assay
(Finlin et al., 2001).
His-dRheb (2 µg) was incubated with [
-35S]GTP (1250 Ci
mmol-1, Amersham) in binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 1 mM DTT, 1 mM EDTA, 40 µg ml-1 bovine serum albumin) in
the presence of 1 mM MgCl2 at 37°C. At various time points,
samples were diluted into ice-cold wash buffer (20 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 10 mM MgCl2, 1 mM DTT) and nitrocellulose-bound radioactivity
was determined by scintillation counting. Specificity of nucleotide binding
was assessed with [
-35S]GTP in the presence of a 20-fold
excess of unlabeled competing nucleotide for 30 minutes, normalized to binding
in the absence of competitor. GTPase activity
(Tanaka et al., 1991
) was
assayed by measuring loss of 32P from His-dRheb preloaded with
[
-32P]GTP (3000 Ci mmol-1) in binding buffer
containing 0.1% Triton X-100, 4 µM GTP and 1 mM MgCl2 at
37°C for 30 minutes. Unlabeled ATP (5 mM) was included to inhibit
nonspecific phosphatase activity. Hydrolysis was initiated by adjusting the
MgCl2 concentration to 10 mM. Reactions were terminated at the
indicated time points and bound radioactivity was assessed by nitrocellulose
binding. All biochemical assays described here were carried out in
triplicate.
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Results |
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We screened approximately 5000 P{y+,5XUAS} lines for
lethality in combination with bynGAL4, which drives expression
specifically in the developing posterior gut
(Iwaki and Lengyel, 2002), to
identify genes affecting hindgut morphogenesis. Among the roughly 7% of these
lines that were lethal with bynGAL4, we identified one (designated
AV4) that gave a dramatically enlarged (both in width and length) hindgut in
the first instar larva (Fig.
1B,D). As determined by inverse PCR, this chromosome carries an
insertion of the P-element in the 5' UTR of dRheb; this allele
is designated dRhebAV4
(Fig. 1A). In situ
hybridization confirms that, as expected from the orientation of the inserted
5XUAS, combining dRhebAV4 with
bynGAL4 causes overexpression of dRheb mRNA in the hindgut
(Fig. 1C). When the P-element
insert of dRhebAV4 is mobilized by transposase
and precisely excised (confirmed by PCR), combination of the excision
chromosome with bynGAL4 has no effect on hindgut size (data not
shown), again confirming that the effects observed in combination with the
bynGAL4 driver are due to the P{y+,5XUAS}
insertion in dRheb.
|
dRheb is an active GTPase
The dRheb gene is predicted to encode a 182 amino acid protein
that contains G-boxes characteristic of the Ras superfamily of G-proteins.
Fig. 2A shows the predicted
amino acid sequence of dRheb, together with those of the yeast and human
Rhebs. Yeast and fly each have a single Rheb gene; the most similar
human gene is hRheb1 (Gromov et
al., 1995; Mizuki et al.,
1996
), which maps to chromosome 7
(Mizuki et al., 1996
). We have
identified an additional, more divergent human Rheb gene, designated
hRheb2, which maps to chromosome 12. The overall sequence identity
between Drosophila and S. pombe Rheb is 51%, and that
between Drosophila and human Rheb1 is 63%
(Fig. 2A). Consistent with this
high sequence conservation, dRheb, hRheb1 and hRheb2 can
replace the function of S. pombe Rheb (data not shown).
|
Recombinant dRheb binds [-35S]GTP (a non-hydrolysable GTP
analog) in a manner that is both time and nucleotide-concentration dependent
(Fig. 2B, data not shown). The
binding of GTP to dRheb is enhanced by addition of Mg2+ (data not
shown) and is specific for guanine nucleotides, with a preference for the
binding of GTP over GDP (Fig.
2C). dRheb also exhibits intrinsic GTPase activity, as shown by
the slow but significant loss of radioactivity from dRheb preloaded with
[
-32P]GTP (Fig.
2D). Hydrolysis of GTP to GDP is confirmed by the appearance of
32P in GDP (revealed by thin layer chromatography, data not shown)
after incubation of dRheb with [
-32P]GTP. Our detection of
Rheb GTPase activity shows that the presence of arginine at the position
corresponding to amino acid 12 of Ras [at which most Ras-family members have
glycine (Bourne et al., 1991
)]
does not eliminate GTP-hydrolysing activity of the protein.
Overexpression of dRheb promotes tissue and cellular growth
and progression into S-phase
Overexpression of dRheb leads to enlargement of the larval
hindgut, so we tested the generality of this effect by driving dRheb
expression in other tissues. In the larval salivary glands, dRheb
overexpression also results in significant enlargement (data not shown). When
dRheb is overexpressed in the developing eye imaginal disc, using the
GMR-GAL4 driver, dramatically enlarged eyes are seen in the adult
(Fig. 3A-F). Similarly,
overexpression of dRheb in clones of cells in the eye and antennal
discs, using the 'flip-out' method
(Pignoni and Zipursky, 1997)
and eyFLP, results in an enlarged eye, antenna and head
(Fig. 3G-J). When compared with
control eyes (Fig. 3K), the
ommatidia of these eyes are much larger than normal, are not organized into
the normal hexagonal array and are frequently flanked by extra bristles
(Fig. 3L). The larger overall
eye and ommatidial size, and the extra bristles further support the notion
that dRheb overexpression promotes growth. The fact that bristles in
the dRheb-overexpressing eyes are larger (thicker) than normal
(Fig. 3K,L), together with the
fact that each bristle is derived from a single cell
(Wolff and Ready, 1993
),
suggests that dRheb might promote growth at the level of the
individual cell.
|
Overexpression of dRheb also promotes growth in the wing, an organ
consisting of two opposed, flat, single-layered epithelial sheets.
Overexpression of dRheb in the dorsal epithelial compartment causes
the wing to curve downwards (Fig.
4C,D), presumably owing to an increase in area of the dorsal
compartment. When dRheb is overexpressed in the posterior compartment
of the wing, the posterior area is increased by 36%
(Fig. 4A,B), further supporting
a role for dRheb in growth. To distinguish between effects on cell
number versus cell size, we counted wing hairs (each of which is produced by a
single cell) in normal and dRheb-overexpressing compartments
(Fig. 4E,F). Because the cell
density in the dRheb-overexpressing compartment is only 67% of that
in the wild-type compartment, we conclude that dRheb-overexpressing
cells are significantly larger (i.e. each cell occupies more area) than
wild-type cells. Wing hair density, combined with compartment area
measurements, shows that total cell number in the posterior compartment is the
same whether or not dRheb is being overexpressed. Thus, in the wing,
dRheb overexpression results in an increase in cell size but not cell
number. This is similar to what has been described for overexpression of
activated Ras1 and Myc; in Drosophila, these genes
are believed to affect cell cycle progression via their primary function as
regulators of cell growth (Johnston et
al., 1999; Prober and Edgar,
2000
; Prober and Edgar,
2001
).
|
In the developing embryo, dRheb expression correlates with DNA
replication. Immediately after fertilization, as rapid, syncytial nuclear
divisions take place (Foe et al.,
1993), dRheb mRNA is present at a high, uniform level.
This level decreases as rates of nuclear division slow, begins increasing
again during stage 11 and becomes significantly higher in tissues undergoing
endocycles (i.e. midgut) and mitoses (i.e. central nervous system). Later in
embryogenesis (stages 13-16), as revealed by in situ hybridization and BrdU
incorporation, the same regions that show strong dRheb expression are
also carrying out DNA synthesis (Fig.
5A, Flybase). Thus, embryonic mRNA expression patterns and levels
are consistent with a role for dRheb in promoting S-phase.
|
To characterize the effect of dRheb further at the cellular level, Drosophila S2 cells were co-transfected with vectors expressing dRheb and GFP (a marker for transfection). Examination of the cell cycle profile of transfected (GFP plus dRheb-overexpressing) cells by FACS reveals that increased levels of dRheb result in an increase in the proportion of cells in S-phase (Fig. 5B). In addition, analysis of cell size by forward scatter analysis reveals that S2 cells overexpressing dRheb are slightly larger than control (GFP alone) cells (Fig. 5C).
The overexpression studies described above show that dRheb can promote two processes: increase in cell size and cell cycle progression. The correlation between dRheb expression and DNA synthesis in the embryo, and the effect of dRheb overexpression in cultured cells both suggest that dRheb promotes progression into S-phase. The increased size of cultured cells, eye bristles and wing cells overexpressing dRheb suggests that dRheb promotes increase in mass of individual cells (cell growth). These two distinct effects of dRheb raise the question of whether dRheb affects the cell cycle and cell growth independently or via a pathway that coordinates both of these processes.
Loss of dRheb function results in reduced cell and tissue
size
Although the results of overexpression studies described above suggest a
role for dRheb in regulation of cell growth and cell cycle
progression, removal of gene activity is necessary unequivocally to establish
required function. Of great utility is the fact that the
dRhebAV4 allele, in addition to allowing ectopic
expression when combined with a GAL4 driver, is also homozygous lethal.
Precise excision of the P-element in the dRhebAV4
chromosome results in a reversion to viability, confirming that the
dRhebAV4 lethal phenotype is due to disruption of
dRheb activity resulting from the P-element insertion. To determine
the lethal phase of dRhebAV4 homozygotes, we
collected eggs for 2 hours from heterozygous parents and determined the number
of surviving homozygous mutant larvae relative to heterozygous sibs as a
function of time (Fig. 6A). Embryos homozygous for dRhebAV4 hatch into first
instar larvae that grow very slowly, move lethargically, and die by 72 hours
without molting into second instar larvae
(Fig. 6B,C). dRheb is
thus required for growth of the whole organism.
|
To assess the requirement for dRheb in individual cells, we made
clones of cells lacking dRheb in the eye. For this purpose, we
recombined FRT(82B) onto the dRhebAV4
chromosome; combination of this chromosome with eyFLP should, in
principle, allow generation of w;dRheb-/- clones in the
background of a w+;dRheb+/- eye. In this
background, however, dRheb-/- clones were not detected,
similar to what has been reported for clones lacking Ras1 function
(Prober and Edgar, 2000). In
both cases, the absence of detectable w clones is probably due to
cell competition, a process in which faster-growing cells out-compete slowly
growing cells, which are then eliminated by apoptosis
(Morata and Ripoll, 1975
). To
give dRheb-/- clones a growth advantage, we generated them
in a Minute (M+/-) background (Minute
genes regulate protein synthesis; thus M+/+ cells grow more rapidly
than surrounding M+/- cells)
(Andersson et al., 1994
). Heads
and eyes containing multiple dRheb-/-
M+/+ clones are dramatically smaller than those containing
dRheb+/+clones (Fig.
7H,I). Sections of these eyes show that the
dRheb-/- ommatidia are smaller overall because they are
composed of dramatically smaller cells
(Fig. 7J cf. C).
|
Two important inferences can be drawn from analysis of these dRheb loss-of-function clones. First, because eyes bearing multiple dRheb-/- clones are smaller than wild-type, dRheb must play a required role in tissue growth. Second, the smaller size of individual dRheb-/- M+/+ cells (Fig. 7J) indicates that dRheb is required in a cell autonomous manner for cell growth (increase in mass).
To investigate whether these inferred roles of dRheb can be
demonstrated at the cellular level, we used dsRNA
(Clemens et al., 2000) to
inhibit Rheb function in Drosophila S2 cells. Addition of
dsRNA corresponding to the entire coding sequence of dRheb to the
culture medium almost completely inhibits expression of FLAG-tagged dRheb
(Fig. 8A). Characterization of
cell cycle profiles by FACS showed a dramatic increase in the proportion of
cells in G1-phase by day four to five in dRheb dsRNA-treated cells;
this effect persisted to at least day 8
(Fig. 8B; notice the increased
proportion of cells in G2-phase in controls). In addition to having an effect
on the cell cycle, inhibition of dRheb also has a significant effect
on cell growth. Forward scatter analysis reveals a dramatic reduction in cell
size after the addition of dRheb dsRNA
(Fig. 8C). Both the diminution
in cell size and the accumulation of cells in G1-phase after dRheb
inhibition follow roughly the same time course (i.e. both are maximal by day
five) (Fig. 8B,C).
|
From the loss-of-function studies in the whole organism and in cultured cells described above, we conclude that dRheb affects both cell growth (mass increase) and cell cycle progression promoting transition of cells from G1 to S phase.
dTOR is required for dRheb-mediated cell
growth
Control of cell growth and cell cycle progression can, in principle, be
regulated by parallel independent pathways or through a signal that
coordinates both (Coelho and Leevers,
2000). In both yeast and Drosophila, mutations in
cell-cycle-specific genes (such as cyclin E) result in cell cycle
arrest with an associated increase in cell size owing to continued cell growth
(Johnston et al., 1977
;
Neufeld et al., 1998
),
although overexpression of these genes results in smaller cells. Because loss
of dRheb function in both cultured cells and in the whole organism
results in reduced cell size, it is likely that dRheb coordinates
cell cycle and cell growth. We therefore considered the possibility that
dRheb might impinge on the insulin and TOR signaling pathways, which
are major contributors to the regulation of cell growth in both
Drosophila and mammalian cells
(Kozma and Thomas, 2002
;
Oldham and Hafen, 2003
).
Because dRheb larvae exhibit a growth arrest similar to dTOR
mutants and larvae starved for amino acids, we used rapamycin treatment to
investigate whether dRheb interacts, either in the whole organism or
in cultured cells, with dTOR. Under normal growth conditions, flies
with either one or two copies of dRheb eclose (emerge from the pupal
case) at the same time; in the presence of rapamycin, however, larvae with
only one wild-type copy of dRheb grow more slowly, eclosing two days
later than their wild-type sibs (Fig.
9A). Reduced dRheb thus sensitizes the organism to the
growth-inhibiting effect of rapamycin. We also examined possible involvement
of dTOR in dRheb function in S2 cells, in which, as
expected, treatment with rapamycin causes cells to decrease in size
(Fig. 9B). Significantly, the
cell-growth-promoting effect of overexpressing dRheb is blocked by
rapamycin (Fig. 9C). This
latter result indicates that the effect of dRheb overexpression
depends on the functional activity of dTOR; in other words, dTOR is
epistatic to (downstream of) dRheb.
|
![]() |
Discussion |
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An important question is whether Rheb affects downstream effectors of TOR,
most particularly S6K, which modulates growth by controlling protein
synthesis. Recent studies have demonstrated a link between dRheb and the
dTOR/dS6K pathway (Saucedo et al.,
2003; Stocker et al.,
2003
). Transient expression of dRheb in
Drosophila S2 cells was found to lead to the activation of p70S6K,
and inhibition of dRheb expression by small interfering RNA blocked
amino-acid-induced activation of p70S6K
(Saucedo et al., 2003
). In
addition, in Drosophila dRheb mutants, p70S6K activity was reported
to be decreased (Stocker et al.,
2003
). We have recently observed that transient expression of
human Rheb in mammalian cells (HEK293) induces activation (phosphorylation) of
S6K (C.L.G. and F.T., unpublished). Although these results might be taken to
suggest that Rheb acts solely through TOR and S6K, it is important to realize
that dS6K-/- flies develop into adults with reduced body
size (Montagne et al., 1999
),
whereas, as shown here, dRheb-/- flies do not survive.
Thus, dS6K might not be the only target of dRheb.
As shown here, dRheb exhibits intrinsic GTPase activity despite having
arginine at the position corresponding to glycine-12 of Ras, a residue known
to be crucial for GTPase activity. Our results are in agreement with the
reported intrinsic GTPase activity of mammalian Rheb by Im et al.
(Im et al., 2002), who also
described that the ratio of GTP to GDP bound to Rheb in mammalian cells is
higher than that bound to Ras. The possibility that dRheb might also have high
GTP:GDP ratio could explain why overexpression of wild-type dRheb has
significant growth promoting effects, as shown here. In our experiments, we
did not use dRhebQ63L, a mutant analogous to the constitutively active Ras,
because it has previously been shown that expression of S. pombe
RhebQ64L or RhebS20N (analogous to a dominant negative Ras) in S.
pombe cells has no detectable effect
(Mach et al., 2000
). Similar
observations were made with mammalian Rheb
(Clark et al., 1997
).
Our finding of GTPase activity for dRheb raises the possibility that there
is a dRheb GTPase-activating protein (GAP). A candidate for such an activity
is the TSC1/TSC2 complex; TSC1 and TSC2 are tumor suppressors that act to
regulate TOR negatively (Gao and Pan,
2001; Potter et al.,
2001
; Tapon et al.,
2001
; Radimerski et al.,
2002
). Interestingly, the C-terminal portion of TSC2 contains a
GAP domain that is reported to function as a GAP for Rap1 and Rab5
(Wienecke et al., 1995
;
Xiao et al., 1997
). Our
results linking dRheb to TOR raise the possibility, currently under
investigation, that TSC2 is a GAP for Rheb.
We have previously reported that, in S. pombe, Rheb inhibition
leads to cell cycle arrest at G0/G1 phase
(Yang et al., 2001). Because
this growth arrest can be rescued by expression of dRheb, there is
conservation of function between Rhebs in two evolutionarily very distant
organisms. It is therefore significant that TOR and TSC homologues exist in
S. pombe (Kawai et al.,
2001
; Weisman and Choder,
2001
; Matsumoto et al.,
2002
). Notably, disruption of tor2+ in S.
pombe leads to growth arrest, whereas disruption of
tsc2+ leads to amino acid uptake defect
(Weisman and Choder, 2001
;
Matsumoto et al., 2002
); both
of these phenotypes are reminiscent of those seen for yeast rheb
mutants (Urano et al., 2000
;
Yang et al., 2001
). Further
experiments are needed to examine possible genetic interactions between the
tor, tsc and rhb1 genes in S. pombe. As for
mammalian Rheb, it has been reported that Rheb inhibits activation of Raf
kinase and that overexpression of Rheb in NIH3T3 cells antagonizes Ras
transformation (Clark et al.,
1997
; Im et al.,
2002
). It will be important to examine whether mammalian Rheb also
functions in the insulin/mTOR signaling pathway.
Our observations suggesting that Rheb is involved in the TOR pathway could
have implications for cancer therapy, because TOR activity appears to be
upregulated in several human cancers
(Hidalgo and Rowinsky, 2000).
Because of its interaction with TOR, Rheb might be an important player in
tumorigenesis. As previously reported, farnesylation is required for Rheb
activity as well as its cellular localization
(Clark et al., 1997
;
Urano et al., 2000
; Yang et
al., 2000); farnesyltransferase inhibitors (FTIs), anticancer drugs currently
being evaluated in clinical trials, might block Rheb function. It is
interesting to note that FTI has been shown to inhibit p70S6K activation in
mammalian cells (Law et al.,
2000
). TOR function is blocked by rapamycin, initially used as an
immunosuppressant but currently under investigation as an anticancer drug
(Crespo and Hall, 2002
).
Therefore, both Rheb and TOR can be targets for anticancer drug therapy.
Further investigations are expected to provide insights that will be relevant
for the design of future drug therapies.
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Acknowledgments |
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![]() |
Footnotes |
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![]() |
References |
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