Molecular Biology Program and Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA
* Author for correspondence (e-mail: riddled{at}missouri.edu)
Accepted 6 May 2004
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SUMMARY |
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Key words: TOR, Raptor, daf-15, Dauer formation, Aging, Insulin
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Introduction |
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Recently, TOR was reported to interact with raptor (regulatory associated
protein of mTOR) to transduce nutrient signals to the downstream translation
machinery in mammals (Kim et al.,
2002; Hara et al.,
2002
). Raptor associates in a near stoichiometric ratio with mTOR
to form a complex that functions as the nutrient sensor
(Kim et al., 2002
). It was
proposed that raptor acts as a scaffold to bridge TOR with its putative
phosphorylation targets (Abraham,
2002
; Kim et al.,
2002
; Hara et al.,
2002
).
Insulin/IGF signaling is also essential for growth and body size
(Oldham and Hafen, 2003). Both
TOR and insulin/IGF signaling regulate a common set of effectors involved in
control of cell growth, including the translation initiation factor 4E-binding
protein (4EBP1) and the S6 ribosomal protein kinase
(Schmelzle and Hall, 2000
).
TOR is a potential downstream component of the insulin/IGF signaling
(Oldham and Hafen, 2003
), but
it is not clear how these two signaling pathways interact.
In C. elegans, insulin/IGF signaling regulates larval development
and adult life span (Kenyon et al.,
1993; Kimura et al.,
1997
). The first-stage (L1) larva responds to overcrowding and
limited food by arresting development as a long-lived dauer larva, an
alternative to the growing third-stage, L3
(Riddle and Albert, 1997
).
Pre-dauer L2 larvae accumulate fat in preparation for a prolonged period of
non-feeding. Dauer larvae have a constricted pharynx, shrunken intestinal
lumen, and a specialized cuticle. These traits are reversed when dauer larvae
resume development to the adult in response to food.
daf-15 mutants are Daf-c (dauer-formation constitutive). At the
second molt, they arrest development non-conditionally as dauer-like L3
larvae, but feeding is not completely suppressed. Electron microscopic
observation of daf-15(m81) showed that some tissues assume dauer
morphology and others do not (Albert and
Riddle, 1988). Head shape, cuticle and intestinal ultrastructure
are non-dauer, whereas sensory structure and excretory gland morphology are
intermediate between that of dauer and nondauer stages. daf-15 larvae
are neither able to complete dauer morphogenesis nor develop to the adult.
We report that daf-15 encodes the C. elegans ortholog of
raptor, and that a mutation in let-363, the gene encoding CeTOR
(Long et al., 2002), also
results in dauer-like larval arrest. Hence, raptor and TOR are required for
dauer morphogenesis and for maturation to the adult. daf-15 and
let-363 mutants shift metabolism to accumulate fat, as do pre-dauer
larvae. Life spans of daf-15 heterozygous adults are significantly
extended. We also show that daf-15 transcription is regulated by
daf-2 insulin/IGF signaling. Thus, DAF-15 is a point of integration
of insulin/IGF signaling and nutrient signaling pathways to regulate C.
elegans larval development, metabolism and longevity.
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Materials and methods |
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Life span
For heterozygous strains, wild-type L4 larvae were picked from populations
grown at 20°C and transferred to 25°C for survival tests. For some
controls (unc-63/+; + dpy-20/daf-15 +, daf-2; dpy-20/+ and
daf-16; dpy-20/+), the genotype of single animals grown at 25°C
was determined by progeny testing. Animals with the correct genotype were
pooled. The date when the animals were shifted to 25°C was designated as
day 1. SPSS Windows Version 11.5 was used for data analysis.
Sudan black staining
Heterozygous mutant strains were grown at 20°C and dauer-like
segregants (daf-15 and let-363 homozygotes or daf-15;
daf-d double mutants) were hand-picked 1 day after dauer-like arrest.
daf-2(e1370) dauer larvae grown at 25°C were harvested one day
after dauer arrest. Control animals [N2 and daf-16(mgDf47)] were
grown at 20°C on ample food. Staining was performed as described by Kimura
et al. (Kimura et al.,
1997).
daf-15 cloning
SNP mapping (Wicks et al.,
2001) was used to localize daf-15. Cosmid rescue was done
as described previously (Jia et al.,
2002
). To test three candidate genes carried by cosmid C10C5 with
dsRNA treatment, part of each gene was amplified by PCR using the following
primers (underlined): C10C5.1, 5' CGT GCG TCG TCA AAT TGC TG
3', 5' CAC TCC ATG TCT CAG TGG TG 3'; C10C5.2,
5' GGA TTC GGT GGA CTC GGT CA 3', 5' GTT GGC AAG GAG TCT CAC
TCT 3'; C10C5.6, 5' TGA CTT CGA ACA TGT GCT GAC
3', 5' TCT CGC AGT ATC ATC GAC CAT 3'. The PCR fragments
were cloned into the pGEMT vector (Promega), and the Riboprobe Combination
System-SP6/T7 (Promega) was used to transcribe RNA in vitro according to the
manufacturer's protocol. Double-stranded RNA was synthesized and injected as
described by Fire et al. (Fire et al.,
1998
).
To confirm the exon-intron junctions predicted by genefinder, 5' and 3' regions of the daf-15 cDNA were isolated by RT-PCR of total RNA from mixed-stage N2 animals. The cDNA fragments were cloned into pGEM-T (Promega) and sequenced. To identify lesions in m81 and m634, DNA from homozygous mutant animals [segregants from unc-24(e138) daf-15(m81)/nT1 or unc-24(e138) daf-15(m634)/nT1, respectively] was amplified using gene-specific primers. Resulting PCR fragments were cloned into pGEM-T and sequenced.
daf-15::gfp (green fluorescent protein)
Gene-specific primers were used to amplify 1.8 kb of daf-15
promoter plus DNA from the ATG start codon to 67 bp inside exon 3 (5'
CCC AAG CTT GGA ATT TCC AAA ACG GTC GAG 3' and 5' ACG CGT CGA CCT
ACT TCC TGC GAT ATC TTC GAC 3'). The PCR fragment was digested and
cloned into the HindIII/SalI sites of gfp vector
pPD95.75. The daf-15::gfp plasmid DNA(100 ng/µl) and the pRF4
rol-6(su1006) marker plasmid (100 ng/µl) were microinjected into
the ovaries of N2 adults to generate transgenic lines. Images are from
mEx157 [rol-6(su1006) daf-15p::Exon1-3::gfp]. Use of GFP as a
reporter was described by Chalfie et al.
(Chalfie et al., 1994).
daf-16 RNAi treatment was performed by feeding according to Kamath
et al. (Kamath et al., 2000)
to examine whether the expression of daf-15::gfp is regulated by
DAF-16. L4 transgenic daf-2 animals carrying daf-15::gfp and
rol-6 were grown on food carrying vector only, or daf-16
RNAi food, for 24 hours at 20°C. The adult animals were transferred to
fresh plates and shifted to 25°C. GFP expression was examined in L2-stage
transgenic progeny.
To construct the daf-16 RNAi plasmid, 1.1 kb of the daf-16-coding region was amplified from C. elegans genomic DNA using primers 5'-CGG GAT CCG TCA CAA TCT GTC TC-3' and 5'-CCC AAG CTT GAA GTT AGT GCT TGG C-3'. The PCR product was cloned into the L4440 vector between the BamHI and HindIII sites, then transformed into HT115. This RNAi construct effectively suppressed the Daf-c phenotype of daf-2(e1370) mutants.
Semi-quantitative RT-PCR
daf-2(e1370) and daf-16(mgDf47); daf-2(e1370) mutant
animals were prepared as described by Lee et al.
(Lee et al., 2003). Total RNA
was extracted with an acid guanidinium thiocyanate-phenol-chloroform mixture
(Chomczynski and Sacchi, 1987
).
mRNA was purified using a PolyATract mRNA Isolation System (Promega). All
RT-PCR was performed with SuperScript One-Step RT-PCR for Long Template
(Invitrogen). The primers used for daf-15 and rpl-21 are:
daf-15, 5' TGA CTT CGA ACA TGT GCT GAC 3' and 5'
TCT CGC AGT ATC ATC GAC CAT 3'; rpl-21, 5' ATG ACT AAC
TCC AAG GGT C 3' and 5' TCA CGC AAC AAT CTC GAA AC 3'.
Electrophoretic mobility shift assays (EMSA)
To perform EMSA, we first amplified a daf-16 cDNA using gene
specific primers (5' CGG GAT CCA TGA ACG ACT CAA TAG ACG AC 3',
and 5' CCC AAG CTT CAA ATC AAA ATG AAT ATG CTG C 3') from total
RNA of mixed-stage N2. The PCR fragment was digested and cloned into the
BamHI/HindIII sites of protein expression vector pET-28a
(Novagen) with a His tag at the C terminus. The construct was confirmed by DNA
sequencing.
His-tagged DAF-16 was expressed in E. coli strain BL21 (DE3) after induction by 0.25 mM isopropyl-thio-ß-D-galactopyranoside at 20°C for 2 hours. The fusion protein was purified using Ni-NTA agarose (Qiagen) according to the manufacturer's protocol. The DNA-binding abilities of purified His-tagged DAF-16 were tested by EMSA. The sequence of the daf-15 probe (with the IRS in bold) was 5'- TTT TGC ACG AAA TAT TTT TTC TTA AAC TCG -3', and the sequence of the mutated probe (with base-pair changes underlined) was 5'- TTT TGC ACG AAA GAG GGT TTC TTA AAC TCG -3'. Oligonucleotides for the sense and antisense strand of each probe were annealed, and the double-stranded probes were end-labeled using T4 polynucleotide kinase (Fermentas) following the manufacturer's protocol. 10 nM 32P-labeled probe, 200 ng His-tagged DAF-16 protein and 10 ng salmon sperm DNA (as a non-specific competitor) were used for each reaction. For competition experiments, a fivefold or a tenfold excess of either wild-type or mutant probe was added to each reaction. The DNA and protein were mixed and incubated at room temperature for 15 minutes, and the products separated on 5% PAGE gels. Gels were dried prior to autoradiography.
Mutagenesis of IRS elements in the promoter and first intron of daf-15
Mutations were introduced with the QuickChange site-directed mutagenesis
kit (Stratagene) following the manufacturer's instructions. Primers (5'-
GAC TCG AAT AAA TAA AGA GGG TTT TAA ATT AAG ATA TTC G
-3' and 5'- GGA ATA TCT TAA TTT AAA ACC CTC
TTT ATT TAT TCG AGT C -3') were used to mutate the IRS that is 237 bp
upstream of the ATG. Primers (5'- GCA ATT TTG CAC GAA AGA
GGG TTT CTT AAA CTC GGT TTC C -3' and 5'- GGA AAC CGA
GTT TAA GAA ACC CTC TTT CGT GCA AAA TTG C -3')
were used to mutate the IRS in the first intron at +127. The mutated
nucleotides are underlined in the primer sequences. The mutations were
confirmed by sequencing.
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Results |
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daf-15 encodes the C. elegans ortholog of raptor and
exhibits sequence conservation with protein family members from mammals,
Drosophila, Arabidopsis and yeast
(Kim et al., 2002;
Hara et al., 2002
;
Wedaman et al., 2003
). The
gene spans
8.3 kb from the predicted ATG initiator site to the TAA
terminator. All predicted intron-exon junctions were confirmed by RT-PCR. The
5.4 kb cDNA (GenBank Accession Number AY396716) encodes a predicted 205 kDa
protein.
The N-terminal RNC (raptor N-terminal conserved) domain consists of three
highly conserved blocks (Fig.
1B,C). All of the raptor family members, including DAF-15, share
34-52% amino acid identity (67-79% similarity) in this domain
(Kim et al., 2002)
(Fig. 1C). An ethylmethane
sulfonate-induced GC-to-AT mutation in exon 5 was identified in
daf-15(m81). It changes an invariant amino acid (G194S) in the first
block of the RNC domain (Fig.
1B,C). G194 is the second glycine in a sequence of ten amino acids
(LFHYNGHGVP) that is invariant in all raptor family members
(Fig. 1C), indicating that this
domain is functionally important. It might be a key region in mediating
raptor's interaction with TOR or other proteins. The daf-15(m634)
allele carries a nonsense mutation in exon 8 that changes TGG (W440) to a TGA
stop, resulting in a truncated protein retaining only the first two blocks of
the RNC domain (Fig. 1B,C). In
addition to the RNC domain, all raptor orthologs have three HEAT repeats
followed by seven WD40 repeats in the C-terminal third of the protein
(Fig. 1B). Both HEAT and WD40
domains mediate protein-protein interactions, implying that raptor could be a
scaffold for the TOR kinase to interact with regulatory proteins
(Kim et al., 2002
;
Hara et al., 2002
;
Wedaman et al., 2003
).
LET-363 and DAF-15 comprise the TOR/raptor complex in C. elegans
Having implicated loss of raptor function in dauer-like larval arrest, we
next examined TOR, which is encoded by let-363. The let-363
mutants were reported to arrest development at the L3 stage, exhibiting a
phenotype thought to result from global inhibition of mRNA translation
(Long et al., 2002). As the
let-363 mutation was originally isolated on a chromosome marked with
dpy-5(e61), we separated it from the closely linked dpy
marker in order to observe the mutant morphology in a wild-type genetic
background. The let-363 larvae are similar to daf-15
(dauer-like both in morphology and movement), consistent with a similarity in
gene function. Blast searches of the C. elegans genome revealed only
one raptor (Kim et al., 2002
;
Hara et al., 2002
) and one TOR
(Long et al., 2002
), as
previously reported. We propose that LET-363 and DAF-15 comprise the
TOR/raptor complex in C. elegans.
let-363 is expressed in most, if not all, cells from the early
embryo to adulthood (Long et al.,
2002). We constructed a daf-15::gfp reporter that fuses
the gfp sequence in frame with the first 71 amino acids of DAF-15.
This reporter was expressed in many cells and tissues, including the nervous
system, the intestine, gonadal distal tip cells, the excretory cell,
hypodermal cells, and pharyngeal and body wall muscles
(Fig. 2). The expression was
observed in all stages, including starvation-induced dauer larvae. An overall
expression pattern similar to that of let-363 is consistent with the
idea that DAF-15 and LET-363 interact.
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|
A heterozygous daf-15 mutation extends both mean and maximum adult life span. The mean life span of daf-15(m81) + /+ dpy-20(e1282) adults was increased by 30% compared with dpy-20/+ controls, and maximum life span increased by 4±0 days (mean±s.d.), or 19% (Table 1; Fig. 4A). When dpy-20 was replaced with another balancing marker, unc-24(e138), mean life span was extended 13% by daf-15(m81) (Table 1; Fig. 4B). Mean life span of unc-24(e138) daf-15(m634)/nT1 was 33% longer than that of the control strain unc-24(e138)/nT1 (Table 1; Fig. 4C).
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Genetic interaction between DAF-2/insulin and CeTOR signaling pathways
Insulin signaling can modulate the TOR pathway in mammals
(Schmelzle and Hall, 2000),
and in C. elegans the DAF-2 insulin/IGF pathway functions to inhibit
its major target, DAF-16, a FOXO family transcription factor
(Lin et al., 1997
;
Ogg et al., 1997
). We examined
the effect of the daf-16(mgDf47) null mutation
(Ogg et al., 1997
) on the
daf-15 Daf-c phenotype, and found daf-15 to be epistatic.
That is, the daf-16; daf-15 double mutants exhibit the
daf-15 dauer-like phenotype, confirming previous work using the
daf-16(m26) allele (Albert and
Riddle, 1988
). daf-16 also failed to suppress
daf-15 fat accumulation (Fig.
3). Compared with the single daf-16(mgDf47) mutant (which
is similar to wild-type L3 larvae) daf-16(mgDf47); daf-15(m81) double
mutants accumulate fat (Fig.
3E,F). Taken together with data for let-363, the
let-363/daf-15 pathway could be downstream of, and regulated
by, the daf-2 pathway. Alternatively, it may be a new pathway that
functions in parallel with daf-2 signaling to regulate C.
elegans larval development and metabolism.
We also examined the genetic interactions between daf-15 and daf-2 with respect to life span. The mean life span of daf-2(e1370); + dpy-20/daf-15(m81) + was increased by 12% relative to the daf-2(e1370); dpy-20(e1282)/+ controls (P<0.01, log-rank test), and the maximum life span was increased by 12±5 days (mean±s.d.) (Table 1; Fig. 4D). These results suggest that daf-15 may act in parallel to daf-2, but the interpretation must be guarded, because we used daf-15/+ strains to test the genetic interactions. Reduction, but not elimination, of daf-15 function leads to increased life span.
daf-16 activity, which is required for daf-2 life span
extension (Kenyon, 1997), is
also required for increased daf-15/+ longevity. As daf-16
life span is shorter than that of N2
(Larsen et al., 1995
;
Gems et al., 1998
), we were
not surprised that the mean life span of daf-16(mgDf47); dpy-20/+
decreased by 13% relative to dpy-20/+
(Table 1). The mean life span
of daf-16(mgDf47); + dpy-20/daf-15(m81) + was 39%
shorter than that of + dpy-20/daf-15(m81) +, and was similar to that
of daf-16(mgDf47); dpy-20/+ (Table
1; Fig. 4E). Hence,
loss of daf-16 function suppressed the effect of daf-15 on
longevity, but did not suppress the daf-15 Daf-c or fat deposition
phenotypes. The latter results indicate that daf-15 acts either
downstream or in parallel with daf-16, raising questions about the
mechanism by which daf-16 suppresses daf-15/+ longevity.
DAF-16 negatively regulates daf-15 transcription
Loss of DAF-16 transcription factor activity may suppress daf-15/+
mutant life span by enhancing transcription of the wild-type allele. We
performed semi-quantitative RT-PCR to compare levels of daf-15 mRNA
in daf-2(e1370) (in which DAF-16 is activated) and
daf-16(mgDf47); daf-2(e1370), in which there is no DAF-16 activity.
The daf-15 mRNA level was reduced in daf-2
(Fig. 5A, lane 1) compared with
daf-16; daf-2 (Fig.
5A, lane 2), indicating that DAF-16 negatively regulates the
expression of daf-15.
|
DAF-16 can bind specifically to the daf-15 IRS in vitro. We performed electrophoretic mobility shift assays (EMSAs) as shown in Fig. 5B. His-tagged DAF-16 protein expressed in bacteria binds directly to the daf-15 IRS (lane 2), but not to the mutant probe with four consensus T residues changed to G (lane 5). The binding was effectively self-competed with fivefold and tenfold excesses of unlabeled daf-15 IRS probes (lanes 3 and 4). As expected, the formation of protein-DNA complex was not competed by the corresponding concentrations of unlabeled mutant probes (lanes 6 and 7).
To determine whether DAF-16 binds IRS elements in vivo, we mutated the two
IRS elements most proximal to the ATG start codon in the daf-15p::gfp
reporter construct. One is 237 bp upstream of the ATG, and the other is in the
first intron at +127. The wild-type and the mutant gfp reporter
constructs were introduced into daf-2(e1370) mutants at a
concentration of 1 ng/µl. The GFP expression was observed only in the
excretory cell in some transgenic animals. We reduced daf-16 activity
with RNAi (Kamath et al.,
2000) to compare the expression pattern of daf-15p::gfp
(wild-type and mutant IRS) in L2 larvae
(Table 2). We compared
daf-2(e1370) grown on control vector food versus
daf-2(e1370) grown on daf-16 RNAi food. The RNAi treatment
was judged to be effective because it suppressed the Daf-c phenotype.
|
Taken together, our data indicate that DAF-16 negatively regulates daf-15 transcription. Hence, the daf-16 mutation may suppress daf-15/+ life span extension by derepressing transcription of the wild-type gene. In homozygous mutants, however, daf-16 cannot suppress the Daf-c and metabolic defect of daf-15 because there is no wild-type DAF-15 produced. The homozygotes form dauer-like larvae and accumulate fat independently of DAF-16.
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Discussion |
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In Drosophila, Tor is also required for normal growth.
Tor homozygotes arrested development as larvae
(Zhang et al., 2000;
Oldham et al., 2000
). Loss of
Tor resulted in lipid vesicle aggregation in the larval fat body
(Zhang et al., 2000
), a fat
accumulation phenotype similar to that observed in let-363 and
daf-15 mutants. Thus, the function of TOR signaling is conserved
between C. elegans and Drosophila.
We have shown that raptor modulates C. elegans adult life span.
Similar results have been reported recently
(Vellai et al., 2003) using
the let-363(h111) dpy-5(e61) mutant, but these results are difficult
to interpret because the mutant larvae do not mature to the adult. Hence, the
relationship of their survival to adult aging is not clear. Nevertheless,
Vellai et al. (Vellai et al.,
2003
) did report that the let-363 arrested larvae
accumulate fat, which is consistent with our results. Extension of adult life
span in daf-15/+ heterozygotes clearly established a role for
TOR/raptor signaling in the normal modulation of longevity. Reduction of TOR
signaling extends longevity, but loss of function is lethal. Study of other
dauer-like mutants may reveal additional components of the TOR/raptor pathway,
and should contribute to the understanding of how nutrient availability
influences life span in other organisms.
Insulin/IGF and nutrient signaling converge at DAF-15 (raptor)
Insulin/IGF signaling controls cellular and organismal growth. The TOR
pathway also regulates a variety of processes contributing to cell growth.
Disruption of either pathway in C. elegans results in larval arrest.
Previous work has shown that in the daf-2(e1370); daf-15(m81) double
mutant, the daf-15 mutation was epistatic to daf-2
(Albert and Riddle, 1988).
Here, we show that daf-15 is also epistatic to daf-16 for
both Daf-c and metabolic phenotypes. Therefore, the TOR pathway is required
for insulin/IGF signaling to control C. elegans development and
metabolism. These results are consistent with those in Drosophila
that show a TOR mutation to be epistatic to PTEN
(Zhang et al., 2000
;
Oldham et al., 2000
), which
acts downstream of the insulin receptor.
Our study reveals a possible mechanism by which insulin signaling regulates
the TOR pathway. DAF-16, the major target of insulin signaling, can negatively
regulate daf-15 transcription, either directly by binding PEPCK-like
IRS in the regulatory region of daf-15 or indirectly through other
unknown transcription factors. Mammalian TOR controls the translation
machinery via activation of the p70S6K protein kinase and via
inhibition of the translation inhibitor 4E-BP
(Schmelzle and Hall, 2000).
However, this mechanism apparently is not used in C. elegans, because
Cep70 RNAi did not phenocopy loss of CeTOR function and search of the C.
elegans genome failed to detect a 4E-BP ortholog
(Long et al., 2002
). Recently,
FOXO, the Drosophila DAF-16 ortholog, was reported to positively
regulate the transcription of 4E-BP (Puig
et al., 2003
). Repression of daf-15 transcription in
C. elegans by DAF-16 may result in inhibition of translation, as does
regulation of 4E-BP transcription in Drosophila.
TOR is a nutrient checkpoint in yeast, Drosophila and mammalian
cells. In yeast, rapamycin treatment mimics the effects of starvation. In
Drosophila, amino acid deprivation phenocopies the Tor
mutant phenotype. CeTOR-deficient C. elegans larvae also share some
features with starved L3 larvae (Long et
al., 2002). We propose that LET-363 and DAF-15 couple the DAF-2
signal and nutrient availability to regulate C. elegans development
and longevity. When nutrients are abundant, LET-363/DAF-15 relays a nutrient
sufficiency signal to downstream effectors and daf-2 signaling
enhances transcription of daf-15, which further stimulates TOR
signaling to respond to the elevated nutrients
(Fig. 6A). However, when
nutrients are limited, reduction of TOR activity and DAF-16-mediated
repression of DAF-15 transcription activate genes for dauer arrest
(Fig. 6B). It is notable that
the TOR pathway is required both for growth and dauer formation.
let-363 and daf-15 developmental arrest is accompanied by
fat accumulation and a dauer-like morphology, but neither mutant is able to
complete dauer morphogenesis.
|
Caloric restriction extends life span in C. elegans
(Klass, 1977;
Lakowski and Hekimi, 1998
) and
in a wide spectrum of other organisms. As the TOR pathway primarily responds
to nutrient availability, caloric restriction may extend life span by
decreasing TOR activity. Interestingly, it has been reported that caloric
restriction and reduced insulin signaling may exhibit their aging effects at
least partly by their common stimulatory action on autophagy
(Bergamini et al., 2003
). If
insulin/IGF and nutrient signaling converge at raptor, TOR signaling could be
a central pathway mediating caloric restriction.
Raptor is a potential therapeutic target for PTEN-deficient tumors
Given that the TOR and insulin pathways are highly conserved among
divergent species, crosstalk between these pathways via a DAF-16 ortholog may
exist in humans. In C. elegans, DAF-16 functions downstream of the
AGE-1 phosphoinositide 3-kinase (PI3K), which mediates daf-2
signaling (Morris et al.,
1996). The DAF-18 PTEN phosphatase
(Ogg and Ruvkun, 1998
), a
tumor suppressor in humans, indirectly activates DAF-16 by dephosphorylating
PIP3. As DAF-16 inhibits raptor transcription, a PTEN null mutation would be
expected to increase TOR activity and promote cell growth, potentially
contributing to the growth of PTEN-deficient tumors in humans. Thus, TOR
inhibitors such as rapamycin could inhibit growth of these tumors. Indeed,
PTEN-null cells are sensitive to inhibition of TOR by a rapamycin derivative
(CCI-779) (Neshat et al.,
2001
; Podsypanina et al.,
2001
), which is currently in phase II clinical trials as an
anticancer agent (Huang and Houghton,
2003
). Therefore, further work in C. elegans may help
elucidate the molecular mechanism of the anticancer effect of agents like
CCI-779. DAF-16 repression of DAF-15 transcription also suggests that agonists
of FOXO transcription factors in humans are potential anticancer agents. A
conditional daf-15 mutant could be used to screen for such candidate
agents predicted to enhance the Daf-c phenotype of the mutant.
Conclusion
We found that daf-15 encodes the C. elegans raptor, and
we established roles for TOR/raptor in controlling C. elegans
development, metabolism and life span. Additionally, we identified a mechanism
by which insulin/IGF signaling regulates the TOR pathway via DAF-16. The role
of the TOR pathway in aging may contribute to the understanding of how
insulin/IGF signaling and nutrient availability influence life span.
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ACKNOWLEDGMENTS |
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