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TOR SIGNALLING IN BUGS,
BRAIN AND BRAWN
Estela Jacinto and Michael N. Hall
TOR — a highly conserved atypical protein kinase and the ‘target of rapamycin’, an
immunosuppressant and anti-cancer drug — controls cell growth. TOR controls the growth of
proliferating yeast, fly and mammalian cells in response to nutrients. Recent findings, however,
indicate that TOR also controls the growth of non-proliferating cells, such as neurons and muscle
cells. Furthermore, TOR, by associating with regulatory proteins and inhibiting phosphatases,
controls the activity of multiphosphorylated effectors.
THE PHOSPHATIDYLINOSITOL
KINASE (PIK)-RELATED PROTEIN
KINASE FAMILY
(PIKK). A family of kinases that
share sequence homology with
lipid kinases but have a protein
kinase activity. They are
distinguished by the presence of
a unique carboxy-terminal
region (FATC) that is not
present in the PIK family.
Division of Biochemistry,
Biozentrum, University of
Basel, Klingelbergstrasse 70,
CH-4056 Basel,
Switzerland.
Correspondence to M.N.H.
e-mail [email protected]
doi:10.1038/nrm1018
The processes of cell growth and cell division are usually
coupled to give rise to cells of a specific size. There are
certain exceptions, however, in which one process
occurs independently of the other. For example, division without growth occurs during early development
when embryonic cells divide before commencing
growth. Growth without division occurs in predifferentiated cells, such as immature thymocytes, and in differentiated cells, such as neurons and muscle cells.
Why regulate cell growth? Organs and organisms
have characteristic sizes that are determined by the size
of their constituent cells. The regulation of cell growth
therefore ensures that there is an overall body plan with
properly proportioned organs. Defects in the regulation
of cell growth can result in cancer or a pathological
increase or decrease in organ size1–3.
What determines cell size? Environmental stimuli,
such as the presence of nutrients and growth factors,
and physical stimuli induce cell growth4. On reaching a
defined size, however, the exposure of cells to a saturating amount of any of these stimuli does not lead to further growth. A genetic programme, therefore, must also
govern cell growth and so ultimately control cell, organ
and organism size.
An increasing body of evidence points to the target of
rapamycin (TOR) signalling pathway as a central controller of cell growth5. Early studies in yeast and mammalian cells showed that TOR regulates growth in dividing cells by controlling several processes — including
transcription, translation and ribosome biogenesis —
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
that collectively determine the mass of the cell. Studies in
Drosophila melanogaster have shown recently that the
TOR signalling pathway, by controlling cell size, also
controls the overall body size. Finally, recent findings in
neurons and muscle cells indicate that TOR controls
growth of non-dividing cells, which provides further
evidence for a conserved and widespread role of TOR in
the regulation of cell growth.
TOR and its interactors
The discovery of the target of the antifungal activity of
rapamycin led to the identification of TOR. Mutations
in yeast that confer rapamycin resistance resulted in the
identification of three genes — FPR1, TOR1 and TOR2
(REF. 6). Rapamycin forms a complex with the FPR1 gene
product (FPR1), a peptidyl-prolyl isomerase that is
known as FK506-binding protein of 12 kDa (FKBP12)
in mammals, and this complex then binds and inhibits
TOR (BOX 1). This mode of action of rapamycin is conserved from yeast to humans.
TOR — which is found in yeast (TOR1 and TOR2 in
Saccharomyces cerevisiae and Schizosaccharomyces
pombe)7–9, fungi (TOR1 in Cryptococcus neoformans)10,
plants (AtTOR in Arabidopsis thaliana)11, worms
(CeTOR)12, flies (dTOR)13,14 and mammals (mTOR; also
known as FKBP12-rapamycin associated protein
(FRAP), rapamycin and FKBP12 target (RAFT) or
rapamycin target (RAPT)15–17) — is a member of the
PHOSPHATIDYLINOSITOL KINASE-RELATED PROTEIN KINASE (PIKK)
18
FAMILY
. The carboxy-terminal region of TOR shows high
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Box 1 | Immunosuppressants and their targets
The IMMUNOSUPPRESSANTS rapamycin, FK506 and cyclosporin A (CsA) are natural
bacterial or fungal secondary metabolites. Rapamycin (also known as sirolimus or
RAPA) is a lipophilic MACROLIDE produced by a bacterial strain, Streptomyces
hygroscopicus, that was originally isolated from a soil sample collected in Rapa-Nui
(Easter Island) — hence the name rapamycin. FK506 (tacrolimus), also a macrolide, is
produced by the soil bacterium Streptomyces tsukubaensis. CsA is a cyclic
UNDECAPEPTIDE that is produced by Tolypocladium inflatum. All three
immunosuppressants function by forming a complex with an IMMUNOPHILIN.
Rapamycin and FK506 form a complex with FK506-binding protein protein 12
(FKBP12), and CsA forms a complex with cyclophilin. The rapamycin–FKBP12,
FK506–FKBP12 and CsA–cyclophilin complexes elicit an immunosuppressive effect
by acting on targets in T-cell signalling pathways. Rapamycin–FKBP12 binds and
inhibits the target of rapamycin (TOR), which is a component of the T-cell
interleukin-2 (IL-2) signalling pathway. FK506–FKBP12 and CsA–cyclophilin bind
and inhibit calcineurin, a type 2B phosphatase in the T-cell receptor pathway.
Rapamycin also has several other clinical applications; it is showing promise in clinical
trials as an anti-cancer drug110, and has recently been used to coat cardiac STENTS as a
means to prevent proliferation of smooth muscle cells during RESTENOSIS111. Finally,
rapamycin has a potent antifungal activity.
IMMUNOSUPPRESSANT
A drug or compound that
inhibits an immune response
through inhibition of T-cell
growth and/or proliferation. It is
used mainly to prevent rejection
of organ grafts.
MACROLIDE
Any of several antibiotics that
contain a lactone ring and are
produced by Streptomyces sp.
UNDECAPEPTIDE
A peptide that is composed of a
chain of 11 amino-acid residues.
IMMUNOPHILIN
An intracellular protein that
binds immunosuppressive drugs.
STENT
A small, mesh-like tube made
from stainless steel that is placed
permanently inside an artery to
hold it open to improve the flow
of blood.
RESTENOSIS
A re-narrowing or blockage of
an artery at the same site at
which treatment, such as an
angioplasty or stent procedure,
has already taken place.
118
homology to lipid kinases, but evidence supports a role
for TOR as a serine/threonine protein kinase19–21.
TOR has several known or putative protein–protein interaction domains (FIG.1). The amino-terminal region contains ~20 tandem HEAT REPEATS22. An
internal region of TOR contains a so-called FAT DOMAIN23.
The carboxy-terminal region of TOR contains the FRB
domain, which is the FKBP12/FPR1–rapamycin binding site. This is followed by the catalytic domain that
shows homology to phosphatidylinositol kinases,
and finally the FATC sequence. Consistent with the
presence of many protein–protein interaction
domains in TOR, TOR is part of multiprotein
complexes.
TOR complexes in yeast. TOR is a high-molecularweight protein (~280 kDa) that forms a complex
with several proteins. In yeast, two functionally distinct TOR complexes have been identified 24. TOR
complex 1 (TORC1) contains TOR1 or TOR2 and
the evolutionarily conserved proteins kontroller of
growth 1 (KOG1) and LST8. KOG1, which contains
four internal HEAT motifs and seven WD40 REPEATS,
might function as a scaffold protein to couple TOR to
its targets24 (FIG.1). LST8 has seven WD40 repeats, and
functions in nutrient-sensitive permease sorting and
regulation of the retrograde signalling protein
(RTG)-1–RTG3 transcription factor (see below)25,26.
TOR complex 2 (TORC2) contains TOR2, adheres
voraciously to TOR2 (AVO)-1, AVO2, AVO3 and
LST8. AVO1 has a domain that is weakly similar to
the Ras-binding domain (RBD) of Ras target proteins. AVO3 contains a RasGEFN (guaninenucleotide exchange factor) domain that is found in
exchange factors and activating proteins for Ras-like
small GTPases. AVO2 contains five ankyrin repeats
and is the only non-essential protein among the
TORC1 and TORC2 subunits24.
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TOR complexes in mammalian cells. In mammalian
cells, mTOR forms a complex with raptor and
mLST8, which are the mammalian orthologues of
KOG1 and LST8 (REFS 24,27,28), respectively. This
complex, which is the mammalian equivalent of
TORC1, has been named the ‘nutrient-sensitive complex’ 28. Recently, tuberous sclerosis 1 (TSC1; also
known as hamartin) and TSC2 (tuberin), the products of two tumour suppressor genes, have also been
shown to bind mTOR29. TSC1 and TSC2 have a negative effect on TOR signalling, whereas all of the
other TOR-binding proteins that have been
described above seem to have a positive effect (see
below)29–31.
Regulators of TOR in yeast
In yeast, nutrients such as carbon and nitrogen stimulate cell growth (BOX 2;TABLE 1). Studies in yeast initially showed that the TOR signalling pathway
responds to nutrients32. Mutants that are defective in
TOR function (that is, they lack both TOR1 and
TOR2), or cells that have been treated with
rapamycin, phenotypically resemble cells that have
been starved or are in G0. This phenotype includes a
downregulation of protein synthesis and ribosome
biogenesis, an upregulation of autophagy and ubiquitindependent protein degradation, specific changes in
transcription and an increase in messenger RNA
turnover. As disruption of TORC1 mimics
rapamycin treatment, TORC1 seems to mediate the
signalling pathway or pathways that control these
rapamycin-sensitive, growth-related processes in
response to nutrients 24. So, TORC1 mediates the
temporal control of cell growth (FIG.2). The disruption of TOR2 alone causes a defect in the cell-cycledependent polarization of the actin cytoskeleton33. A
polarized actin cytoskeleton orientates the secretory
pathway and, consequently, the delivery of newly
made proteins and lipids to the growth site, or bud,
on the yeast cell surface. This function, which is specific to TOR2, is rapamycin-insensitive. The disruption of TORC2 mimics TOR2 depletion, and
TORC2, unlike TORC1, is rapamycin-insensitive24.
So, TORC2 mediates the signalling pathway that
controls polarization of the actin cytoskeleton,
thereby mediating the spatial control of cell growth.
Furthermore, the two structurally and functionally
distinct TOR complexes provide a molecular basis
for the diversity, specificity and selective rapamycin
sensitivity of TOR signalling in yeast.
The effects of TOR in yeast
Protein synthesis. Yeast TOR1 and TOR2 (as part of
TORC1) control protein synthesis probably by activating
the translation initiation factor eIF4E. Downregulation
of cap-dependent TRANSLATION INITIATION is one of the earliest effects that is observed after rapamycin treatment32.
TOR also controls the transcription of ribosomal proteins and the synthesis and processing of ribosomal RNA
and transfer RNA, which emphasizes the role of TOR in
controlling translation34–37.
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HEAT
FAT
HEAT
FRB
Kinase
FATC
TOR
RNC
HEAT
WD40
KOG1
(raptor)
WD40
LST8
(mLST8)
RBD
AVO1
Ankyrin
AVO2
RasGEFN
AVO3
Figure 1 | Structural domains of TOR and its binding partners. The structural domains of the
target of rapamycin (TOR) are evolutionarily conserved. The KOG1/raptor domain structure is also
conserved. The RNC (raptor N-terminal conserved) domain consists of highly conserved residues
that are found in all KOG1/raptor orthologues. Regions in AVO1 and AVO3 have homology to the
Ras-binding domain (RBD) and RasGEFN domain, respectively. The structural domain of LST8 is
also highly conserved, consisting of seven WD40 repeats. AVO2 contains five ankyrin repeats.
AVO, adheres voraciously to TOR2; FAT; a domain that was found in the FRAP (mTOR), ATM and
TRRAP proteins; FRB, the binding site for FKBP12–rapamycin; HEAT, an amino-acid sequence
motif that was first identified in huntingtin, elongation factor 3, regulatory A subunit of PP2A and
TOR; KOG1, kontroller of growth 1.
HEAT REPEATS
An amino-acid sequence motif
that was first identified in
huntingtin, elongation factor 3,
regulatory A subunit of PP2A
and TOR. Each repeat varies in
length between 37 and 43 amino
acids,occurs as anti-parallel
α-helices,and is repeated tandemly
at least three times in every protein.
Most of the proteins that contain
this motif are large,are known to
be part of a complex and function
in transport processes.
FAT DOMAIN
(FRAP, ATM, TRRAP). A
domain spanning ~500 amino
acids that is found in the PIKK
and TRRAP protein families .
This domain is found aminoterminal to the kinase domain,
and in combination with the
FATC domain, which is found at
the extreme carboxyl terminus.
FAT and FATC domains are
speculated to function in
protein–protein interactions.
WD40 REPEAT
A repeat of ~40 amino acids
with a characteristic central
Trp–Asp motif.
Protein stability. In the presence of nutrients, TOR controls protein stability by inhibiting both autophagy and
ubiquitin-dependent protein degradation. Autophagy,
which is the vacuolar targeting and degradation of bulk
cytoplasm, is triggered by the dephosphorylation and
activation of the APG1–APG13 (autophagy 1; 13)
kinase complex in response to starvation. TOR inhibits
autophagy by maintaining APG1 in a phosphorylated
state38,39. Similarly, TOR prevents ubiquitylation, vacuolar
targeting and degradation of the tryptophan transporter
TAT2 by maintaining the serine/threonine kinase NPR1
in a phosphorylated — and thereby inactive — state40,41.
NPR1 might phosphorylate TAT2 directly to trigger its
ubiquitylation and internalization. Interestingly, mTOR
also inhibits autophagy and nutrient transporter
turnover in mammalian cells, but the mechanism of
this inhibition is unknown42–44.
Transcription. In yeast, TOR negatively controls the
transcription of starvation-specific genes, by regulating
the nuclear localization of several nutrient-responsive
transcription factors34,35,45. TOR maintains the GATA-TYPE
TRANSCRIPTION FACTOR GLN3 in a phosphorylated state
and thereby tethered to the cytoplasmic protein URE2
(REF. 45). When TOR is inactivated — by nitrogen starvation or rapamycin treatment — GLN3 is dephosphorylated, released from URE2 and translocated into the
nucleus to induce the transcription of genes that are
required for the use of secondary nitrogen sources.
URE2 is also phosphorylated in a TOR-dependent
manner34,35.
TOR negatively controls the transcription of stressresponsive genes by sequestering the general stress
transcription factors MSN2 and MSN4 in the
cytoplasm45. TOR sequesters the MSNs possibly by promoting the association of MSN2 and MSN4 with the
cytoplasmic 14-3-3 proteins BMH1 and BMH2 (REF. 45).
Finally, TOR negatively regulates the heterodimeric
transcription factor that is composed of RTG1 and
RTG3 (REFS 46,47). The mechanism by which TOR
inhibits RTG1–RTG3 is unknown, but epistasis analysis
indicates that this inhibition might occur through
RTG2 and MKS1 (REFS 48–50). TOR seems to antagonize
RTG2, which is a negative regulator of MKS1 (REF. 49),
and MKS1 inhibits RTG1–RTG3 (REFS 49,50). So, a
model is that TOR inhibits RTG1–RTG3 through a cascade of three negative regulatory steps. In the absence
of glutamine, RTG1–RTG3 moves into the nucleus and
induces the transcription of genes that encode enzymes
Box 2 | Nutrient signalling in yeast
Yeast cells grow and proliferate in response to ambient nutrients such as nitrogen and carbon sources. The quantity and
quality of nutrient sources are detected by a diverse set of sensing and regulatory systems. In the presence of good
nitrogen sources, such as ammonium or glutamine, the transcription of genes that are involved in the use of a poorer
nitrogen source is repressed by the TOR signalling pathway, a phenomenon that is known as ‘nitrogen catabolite
repression’112. All nitrogen sources are eventually converted to ammonia and glutamate, which are then converted to
glutamine — the preferred nitrogen source and a key intermediate in yeast nitrogen metabolism. In the presence of a
poor nitrogen source, such as proline or urea, genes that are required to scavenge and metabolize these secondary
nitrogen sources are upregulated as a result of inactivation of the TOR pathway. The TOR pathway, which also responds
to nutrients other than the nitrogen source, controls several growth-related processes, including ribosome biogenesis
and protein synthesis. SSY1, an AMINO-ACID PERMEASE-like protein at the plasma membrane, is required for sensing
extracellular amino acids and inducing genes that encode other amino-acid permeases113. The GCN system (general
control non-derepressible) senses intracellular amino acids and induces synthesis of amino-acid biosynthetic genes in
response to amino-acid deprivation. Glucose, which is the preferred carbon source of yeast, is detected by RGT2
(restores glucose transport 2) and SNF3 (sucrose non-fermenting 3), two plasma-membrane proteins that resemble
glucose transporters, and by the G-protein-coupled receptor GPR1 (REF. 114). A high ambient concentration of glucose
causes the repression of genes that are involved in respiration, GLUCONEOGENESIS and metabolism of alternative sugar
sources. RGT2 and SNF3 signal through the AMP-activated protein kinase SNF1. GPR1 signals through the cyclic
AMP–protein kinase A pathway.
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In mammalian cells, growth is stimulated by a combination of nutrients and growth factors (TABLE 1).
Accumulating evidence indicates that mTOR might
mediate signalling in response to both stimuli (FIG.3).
of mTOR blocks signalling through the PI3K pathway51,52. Reduction of neoplastic proliferation and
tumour size by inhibition of mTOR in PTEN-deficient
cells is due largely to the inhibition of RIBOSOMAL S6 PROTEIN
KINASE (S6K, also known as p70 S6 kinase).
The targets of mTOR, S6K and eukaryotic INITIATION
FACTOR 4E-BINDING PROTEIN (4E-BP; also known as phosphorylated heat and acid-stable protein regulated by insulin,
PHAS-I), are also components of the insulin–PI3K–PDK1
–Akt/PKB (protein kinase B) pathway53.
What is the nature of the link between mTOR and
the insulin signalling pathway in the control of S6K
and 4E-BP? mTOR and the insulin signalling pathway
converge on S6K and 4E-BP to mediate the complex,
hierarchical phosphorylation of these common downstream targets54–56. However, recent evidence indicates
that the insulin pathway might also impinge on mTOR
signalling upstream of mTOR. Genetic evidence in
flies and biochemical evidence in mammalian cells
indicate that the TSC1–TSC2 complex binds and
inhibits TOR29,30,57. The TSC1–TSC2 complex is inactivated in response to insulin by Akt-mediated phosphorylation of three sites in TSC2 (REF. 31). Akt also
phosphorylates mTOR directly, on Ser2448 (where Ser
is serine), but the significance of this phosphorylation
is uncertain, as a substitution of Ser2448 with alanine
does not affect mTOR signalling58,59. Furthermore,
mTOR seems to respond positively to phosphatidic
acid and ATP, both of which are produced in response
to growth factors60,61. So, mTOR signalling is controlled
by growth-factor inputs upstream and downstream of
mTOR.
Several studies have placed the TSC1–TSC2 complex
upstream of TOR29,30,57. However, there is one conflicting report that suggests that the TSC complex regulates
S6K1 independently of mTOR62*. It is difficult to reconcile this discrepancy as the conflicting reports30,57,62
describe essentially the same experiment but with
opposite results. It is also worth noting that the binding of TSC1 and TSC2 to TOR was shown using overexpressed, recombinant TOR29.
As TOR controls cell growth in yeast and plants,
which lack a PI3K signalling pathway, the PI3K pathway
seems to have been grafted onto the TOR pathway late
in evolution. The joint control by the mTOR and PI3K
pathways meets the need of multicellular animals to
coordinate cell-autonomous growth with overall body
growth. Multicellularity evolved independently in plants
and animals, and plants must use another strategy to
coordinate growth. Interestingly, worms have both a
TOR and a PI3K pathway, but, in this case, the PI3K
pathway does not seem to be involved in controlling cell
growth12.
Growth factors. The mTOR pathway mediates growth
factor signalling through the phosphatidylinositol 3kinase (PI3K) pathway. Studies using phosphatase and
tensin homolog (PTEN)-deficient cancer cells, in which
the PI3K pathway is activated owing to the upregulation
of the lipid second messenger phosphatidylinositol-3,4,5trisphosphate (PtdIns(3,4,5)P3), indicate that inhibition
Nutrients. Similar to yeast TOR, mTOR also responds
to the availability of nutrients. Branched-chain amino
acids, particularly leucine, activate the mTOR signalling
pathway63–66. A high level of ambient amino acids, even
in the absence of insulin, promotes phosphorylation
and activation of S6K, without activating Akt.
Withdrawal of amino acids, which mimics rapamycin
Table 1 | Nutrient and/or growth-factor signalling proteins
Yeast
Human
Function*
TOR1/2
mTOR
Protein kinase, nutrient signalling
KOG1
raptor
Scaffold protein
LST8
mLST8
Permease sorting,
negatively regulates RTGs
AVO1
hSIN1(?)
Actin cytoskeleton, Ras signalling
AVO2
–
Actin cytoskeleton
AVO3
–
Actin cytoskeleton, sphingolipid
metabolism
TAP42
α4
Phosphatase interactor/regulator
TIP41
mTIP41
Binds and regulates TAP42 in yeast
PPH21/22
PP2A ctalytic
Phosphatase catalytic subunit
TPD3/CDC55
A/B regulatory
PP2A regulatory subunit
SIT4
PP2A catalytic
Phosphatase catalytic subunit
SAPs
–
SIT4 associated proteins
NPR1
–
Ser/Thr kinase, permease regulation
GLN3
–
GATA transcription factor, nitrogen regulation
RTG1/3
–
Transcription factor, TCA cycle
–
PI3K
PI3 kinase, mitogen signalling
–
Akt/PKB
Protein kinase, binds PtdInsP3
–
PDK1
Protein kinase, binds PtdInsP3
–
PTEN
Phospholipid phosphatase
–
TSC1 (hamartin)
Negatively regulates mTOR
–
TSC2 (tuberin)
Negatively regulates mTOR
–
S6K
Protein kinase, translation activator
–
4E-BP
eIF4E binding protein, translation inhibitor
*Characterized or putative functions are listed. 4E-BP, eukaryotic initiation factor 4E-binding protein;
α4, mammalian homologue of TAP42; AVO, adheres voraciously to TOR2; CDC55, cell-divisioncycle mutant 55; eIF4E, eukaryotic initiation factor 4E; GLN3, glutamine; KOG1, kontroller of growth
1: LST8, lethal with sec-thirteen 8 ; NPR1, nitrogen permease reactivator 1; PDK1,
3-phosphoinositide dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PKB, protein
kinase B; PP2A, protein phosphatase 2A; PPH, protein phosphatase; PTEN, phosphatase and
tensin homologue; RTG, retrograde signalling protein; S6K, ribosomal S6 protein kinase; SAP, SIT4associated protein; SIT4, suppressors of initiation of transcription; SIN1, sty1 interactor; TAP42, type
2A-phosphatase associated protein, 42 kDa; TCA, tricarboxylic acid; TIP41, TAP42-interacting
protein, 41 kDa; TOR, target of rapamycin; TPD3, tRNA processing deficient 3; TSC, tuberous
sclerosis complex.
TRANSLATION INITIATION
The first step in protein synthesis,
wherein the initiating ribosome
scans along the messenger RNA
and identifies the initiator codon
to begin translation in the proper
reading frame.
GATA-TYPE TRANSCRIPTION
FACTORS
A family of transcription factors
that contain a zinc-finger motif
that was first identified in the
vertebrate GATA-1 protein.
These transcription factors bind
the consensus sequence GATA in
the 5′ non-coding regions of
constitutive and inducible genes.
120
of the tricarboxylic acid (TCA) cycle. As GLN3 and
RTG1–RTG3 respond to intracellular levels of glutamine, the TOR pathway seems to sense glutamine,
among other unknown nutrient compounds47. So, TOR
broadly controls growth-related metabolism by sequestering several transcription factors in the cytoplasm.
Regulators of mammalian TOR
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REVIEWS
Nutrients
Cell membrane
?
KOG1
LST8
TOR1/2
LST8
Rapamycin AVO1
TOR2
AVO2
AVO3
TAP42
P
TIP41
RTG2
TIP41
MKS1
SIT4
PPH
GLN3
?
Actin organization
Translation
TOR as a protein-phosphatase regulator
NPR1
MSN2/4
RTG1/3
Permease turnover
Transcription
Figure 2 | Two functionally distinct TOR complexes in yeast. Target of rapamycin (TOR)
complex 1 (TORC1) contains either TOR1 or TOR2, together with KOG1 and LST8. TORC1
mediates the rapamycin-sensitive function of TOR, which leads to the activation of translation,
inhibition of protein turnover and inhibition of transcription of starvation-specific genes. TOR
controls several of these processes by inhibiting type 2A phosphatases. TOR complex 2
(TORC2) consists of TOR2, LST8, AVO1, AVO2, and AVO3. The TORC2 complex mediates actin
cytoskeleton organization and is insensitive to rapamycin. AVO, adheres voraciously to TOR2;
GLN3, glutamine; KOG1, kontroller of growth 1; MSN, multicopy suppressors of snf1 mutation;
NPR1, nitrogen permease reactivator 1; PPH, protein phosphatase; RTG, retrograde signalling
protein; SIT4, suppressors of initiation of transcription 4; TAP42, type 2A-phosphatase
associated protein 42 kDa; TIP41, TAP42-interacting protein 41 kDa.
AMINO-ACID PERMEASE
A protein that transports amino
acids from the outside to the
inside of the cell. In yeast, these
proteins contain 12 membranespanning segments, and are
either broadly specific for a
group of structurally related
amino acids or highly specific
for individual amino acids.
GLUCONEOGENESIS
The metabolic formation of
carbohydrates from noncarbohydrate organic
precursors.
RIBOSOMAL S6 PROTEIN KINASE
(S6K). A protein kinase that
phosphorylates the ribosomal
protein S6. S6 is involved in the
translation of messenger RNA
transcripts that contain a
polypyrimidine tract at their
transcriptional start site.
through raptor28, although the effect of nutrients on
the mTOR–raptor association is debated24,27,67. The precise roles of raptor, TSC1–TSC2 and possibly mLST8 in
controlling mTOR activity in response to amino acids
remain to be determined. However, as S6K and 4E-BP
phosphorylation in response to both insulin and
amino acids is rapamycin sensitive, mTOR integrates
nutrient and insulin signals to control cell growth.
Interestingly, as the production of insulin as well as the
response to insulin is sensitive to nutrients, mTOR as a
nutrient sensor might also be involved in regulating
insulin production68.
treatment, leads to rapid dephosphorylation of S6K
and 4E-BP. Furthermore, rapamycin treatment renders
the phosphorylation of S6K refractory to amino-acid
stimulation63. Although amino acids seem to regulate
the mTOR pathway, the effect of amino acids on
mTOR kinase activity is unclear. Dennis et al. reported
that amino acids have no effect on mTOR kinase activity towards S6K, as assayed with mTOR that was
immunopurified from amino acid stimulated cells60.
However, Kim et al. have carried out similar experiments, but with conditions that favour the inclusion of
raptor, the newly identified mTOR interactor protein.
In the presence of raptor, mTOR kinase activity is
increased when cells are stimulated with leucine28.
Consistent with the above observations, raptor has
been proposed to function as a scaffold protein that
links mTOR to S6K and 4E-BP27. Finally, mTOR is also
thought to respond to a mitochondrial signal, but this
signal could simply be amino acids that are synthesized
in the mitochondria47.
How might mTOR kinase activity be regulated by
amino acids? The recent observations that loss of the
TSC1–TSC2 complex results in an increase in S6K
activity and renders cells resistant to amino-acid starvation imply that amino acids, as well as growth factors,
signal to TOR through inhibition of the TSC COMPLEX30.
The finding that nutrient deprivation stabilizes the
mTOR–raptor association and inhibits mTOR kinase
activity indicates that nutrients might regulate mTOR
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Regulation of type 2A protein phosphatases (PP2As) is
an important mechanism of TOR signalling. Several
downstream effectors of TOR, such as NPR1 and GLN3
in yeast, and S6K and 4E-BP in mammals, are multiphosphorylated proteins that are dephosphorylated
rapidly after rapamycin treatment. The regulation of
phosphatases, in addition to kinases, by TOR ensures a
rapid and coordinated response to nutrient deprivation.
PP2A. In yeast and mammals, the PP2A catalytic (C)
subunits are broadly active; target specificity is determined by the associated regulatory (A or B) subunits69. The regulatory subunits of phosphatases are
diverse and, as well as controlling the substrate specificity, they also determine the subcellular localization
of the phosphatase complex. The PPH21 or PPH22
catalytic subunit of yeast PP2A associates with the
regulatory A subunit, TPD3, and one of two B regulatory subunits, CDC55 and RTS1. Mammalian PP2A
consists of a core dimer of a catalytic subunit that is
associated with a scaffolding A subunit. This core
dimer associates with one of various regulatory B
subunit isoforms or splice variants.
SIT4. SIT4 is a yeast PP2A-related catalytic subunit that
associates with one of four regulatory proteins that are
known as SAPs (SIT4-associated proteins) — SAP4,
SAP155, SAP185, and SAP190 (REFS 70,71). Deletion of
three of the SAP genes (SAP155, SAP185, and SAP190)
phenocopies a SIT4 deletion, which indicates that the
SAPs regulate SIT4 positively.
SIT4, PP2A and TAP42. In yeast, SIT4 and PP2A also
interact with the essential protein TAP42 (type 2A
associated protein-42kDa)72. TAP42 binds the catalytic
subunits of PP2A and SIT4 in a nutrient-dependent,
rapamycin-sensitive manner, and a TAP42 mutation
confers rapamycin resistance, which indicates that
TAP42 is a component of the TOR pathway. TAP42
seems to compete with other regulatory subunits for
binding to the phosphatase catalytic subunit73. TOR
promotes the binding of TAP42 to the phosphatase
catalytic subunit and thereby prevents the binding of
other regulatory subunits. What is the consequence of
TOR-stimulated binding of TAP42 to SIT4 and PP2A?
NPR1 and GLN3 are dephosphorylated rapidly in a
SIT4-dependent manner after rapamycin treatment,
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Growth factors
Nutrients
Cell membrane
?
Raptor
mLST8
mTOR
Rapamycin
TSC1
TSC2
PtdInsP3
PDK1
PtdInsP2
Akt
PI3K
PTEN
PP2A
4E-BP
S6K
elF4E
S6
Translation
Transcription
Transcription
Figure 3 | mTOR integrates signals from nutrients and growth factors leading to cell
growth. Growth-factor stimulation activates phosphatidylinositol 3-kinase (PI3K) which
phosphorylates phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at position 3 to generate
PtdIns(3,4,5)P3. The protein kinases Akt and 3-phosphoinositide-dependent protein kinase-1
(PDK1) are recruited to the membrane and bind PtdIns(3,4,5)P3through their pleckstrin homology
(PH) domains. Membrane-bound Akt is activated, leading to the phosphorylation of
TSC2/tuberin. This renders the TSC complex unstable and inactive, and thereby relieves the
inhibitory constraint on the mammalian target of rapamycin (mTOR). Nutrients, by an unknown
mechanism, activate the mTOR complex, which consists of mTOR bound to raptor and mLST8.
mTOR promotes the hierarchical phosphorylation of the translation activator S6K and the
translation inhibitor 4E-BP either directly or indirectly by the inhibition of protein phosphatase 2A
(PP2A). PDK1 and an unidentified growth factor-responsive kinase also phosphorylate S6K and
4E-BP, respectively. Phosphorylation of S6K and 4E-BP promotes translation, via the ribosomal
protein S6 and the eukaryotic initiation factor 4E (eIF4E), respectively. mTOR also controls gene
transcription in response to nutrients and in cooperation with growth factor stimulation5. 4E-BP,
eukaryotic initiation factor 4E-binding protein; S6K, ribosomal S6 protein kinase; TSC2, tuberous
sclerosis complex 2.
INITIATION FACTOR 4E-BINDING
PROTEIN
(4E-BP; PHAS-I). When
dephosphorylated, 4E-BP
negatively regulates capdependent translation by
binding and inhibiting the
eukaryotic initiation factor 4E
(eIF4E).
TSC COMPLEX
This consists of TSC1, a protein
that is predicted to form coiledcoil structures and contains a
putative transmembrane
domain, and TSC2, a protein
that contains a coiled-coil
domain and a Rap GTPaseactivating protein (GAP)
domain. Mutations in either
TSC1 or TSC2 are responsible
for tuberous sclerosis, a genetic
disorder that is characterized by
hamartomas in various organs.
122
which implies that TAP42 inhibits SIT4 and possibly
PP2A40,45,74. Interestingly, TAP42 binds only a small
fraction of the more abundant catalytic subunits,
which indicates that TAP42 might only inhibit a specific pool of PP2A or SIT4 (REF. 72). TAP42 might also
activate PP2A or SIT4 towards specific targets, but
such targets are unknown.
TOR regulation of TAP42. TOR might regulate
TAP42 both directly and indirectly. TOR phosphorylates TAP42 in vitro, and TAP42 is dephosphorylated
in a PP2A-dependent manner in rapamycin-treated
cells73. This implies that TOR phosphorylates TAP42
and thereby directly controls the binding of TAP42
to PP2A. By contrast, TOR seems to control the
binding of TAP42 to SIT4 indirectly, as indicated by
the observation that TAP42 dephosphorylation after
rapamycin treatment is significantly slower than
SIT4–TAP42 dissociation and SIT4 activation40,45,72–74.
TOR controls the binding of TAP42 to SIT4 through
the TAP42-interacting protein TIP41 (REF. 74). TOR, by
maintaining TIP41 phosphorylation, promotes the
| FEBRUARY 2003 | VOLUME 4
binding of TAP42 to SIT4. After TOR inactivation,
TIP41 is dephosphorylated, binds TAP42 and thereby
induces the release and activation of SIT4. SIT4, in turn,
dephosphorylates more TIP41. So, TIP41 is part of a
feedback loop, the purpose of which is to rapidly amplify
phosphatase activity in response to TOR-inactivating
conditions.
Mammalian TOR and phosphatases. TOR also controls
phosphatase activity in mammalian cells. mTOR phosphorylates PP2A in vitro, and rapamycin activates
phosphatase activity in vivo75. mTOR might therefore
regulate PP2A through direct phosphorylation.
Furthermore, PP2A binds and inactivates S6K75. Finally,
a mammalian orthologue of TAP42, α4, associates with
mammalian PP2A catalytic subunits76,77. Although the
rapamycin sensitivity of this association is controversial, S6K activation is defective in α4-disrupted cells,
which suggests that α4 might function in the mTOR
pathway77. α4 also associates with the microtubuleassociated and OPITZ SYNDROME protein Midline-1 (MID1).
MID1, a UBIQUITIN LIGASE that is involved in normal midline development, targets PP2A for degradation78.
Interestingly, overexpression of α4 promotes the
dephosphorylation of MID1, which indicates that
MID1 might be a target of α4–PP2A79. In support of
a phosphatase activity for this complex, elongation
factor-2 is also dephosphorylated when α4 is overexpressed80. The role of α4 in mTOR signalling remains
to be determined.
TOR in Drosophila growth control
Similar to the situation in mammals, the TOR and insulin
signalling pathways in Drosophila converge to coordinately regulate cell size81. Loss-of-function mutations in
the genes that encode the positive elements of these pathways, such as dTOR, dIRS (insulin receptor substrate; also
known as chico), dPI3K, dPDK1, dAkt and dS6K result in
smaller cells and, in some cases, smaller but correctly
proportioned flies (FIG. 4)13,14,82–86. Conversely, loss-offunction mutations in the genes that encode the negative
elements of these pathways, such as dPTEN, dTSC1, and
dTSC2, lead to an increased cell size87–89. In addition,
expression of a highly active d4E-BP1, also a negative regulator, decreases cell size90. dTOR regulates dS6K and
probably also d4E-BP, which indicates that dTOR might
control cell size through protein synthesis.
Regulation of dTOR. How is dTOR regulated? A
reduction in the dosage of the dTOR gene suppresses
the lethality and the cell-size defect that is caused by a
dTsc mutation29. Furthermore, in cells with decreased
dTSC expression, dS6K phosphorylation is resistant to
amino-acid starvation, but remains rapamycin sensitive. Finally, loss of both the dTSC complex and dS6K,
like the loss of dS6K alone, results in decreased cell
growth (that is, ds6k is epistatic to dTsc29). These results
indicate that dTOR is regulated negatively by dTSC, and
this inhibition of dTOR by dTSC is relieved by amino
acids. But how is dTSC regulated? Similar to mammalian
TSC, dTSC2 is phosphorylated and inactivated by dAkt.
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Growth in non-proliferating cells
+/+
I-1/I-1
Figure 4 | A TOR-signalling defect in Drosophila causes smaller cells and a smaller
animal. Shown are wild type (+/+) and ds6k (l-1/l-1) mutant flies. The ds6k allele is a partial loss-offunction mutation. dTOR and ds6k-null mutations are lethal. S6K, ribosomal S6 protein kinase;
TOR; target of rapamycin. Reproduced with permission from REF. 86 © (1999) American
Association for the Advancement of Science.
Indeed, most dAkt-mediated growth signalling is
transduced by phosphorylation of two highly conserved sites in dTSC231. Mutation of these two sites in
dTSC2, such that they can no longer be phosphorylated, stabilizes the dTSC complex and inhibits cell
growth. Furthermore, destabilization of the complex
by mutation of dTsc1 promotes cell growth that is not
further enhanced by dAkt overexpression. Although
these observations indicate that dTSC is controlled by
dAkt, they do not account for the amino-acid-mediated inactivation of dTSC, as dAkt is not controlled by
amino acids. So, in both flies and mammals, it remains
to be determined how TSC responds to amino acids.
Nevertheless, the above studies indicate that in higher
eukaryotes, both the nutrient-induced TOR pathway
and the insulin pathway determine cell size.
TOR in muscle cells. An increased workload on a
muscle causes an increase in muscle mass. This
increase in mass, or hypertrophy, is due to an increase
in the size of individual muscle cells (BOX 3). A reduced
workload, ageing or myopathies lead to atrophy,
which is a loss of skeletal-muscle mass due to shrinkage of individual muscle cells. How a mechanical
stimulus is propagated as a chemical signal, and the
molecular mechanisms that underlie skeletal-muscle
hypertrophy are poorly understood. However, muscle
hypertrophy requires an increase in the rate of protein
synthesis and signalling by the mTOR and the
insulin-like growth factor (IGF1) pathways91–94.
Treatment with rapamycin prevents IGF1-induced
hypertrophy, whereas expression of activated Akt promotes hypertrophy91,92,95.Also, increased phosphorylation
of mTOR at Ser2448, a site that is phosphorylated by
Akt in vitro, is associated with muscle hypertrophy96.
The mTOR effector S6K shows a rapamycin-sensitive
increase in phosphorylation in response to muscle
load92,97.
The role of mTOR and the Akt pathways in musclecell growth is supported by the finding that transgenic mice that overexpress activated Akt specifically
in the heart have enlarged cardiomyocytes, which
results in a larger heart 98. The Akt-induced size
increase is rapamycin-sensitive, indicating that the
effect is mediated by mTOR. The observed increase
in myocyte size is independent of cell division, as an
increase in heart size occurs when myocytes are in a
postmitotic state98. Taken together, the above studies
on muscle cells show how both cell and organ size
are controlled by mTOR, and provide an example of
how mTOR can control growth in non-proliferating
cells.
Box 3 | Skeletal-muscle hypertrophy
OPITZ SYNDROME
Opitz G/BBB syndrome is a
congenital disorder that arises
from defects in ventral midline
development. Manifestations of
this disorder include, among
others, mental retardation, cleft
lip and palate, and genitourinary
defects.
UBIQUITIN LIGASE
An enzyme that couples the
small protein ubiquitin to lysine
residues on a target protein; it
marks the target protein for
destruction by the proteasome.
SATELLITE CELLS
Myogenic stem cells that are able
to proliferate and form new
myofibres.
In response to an increased workload, skeletal muscles
IGF1
Mechanical load
Nutrients
undergo hypertrophic growth. Load-induced hypertrophy
occurs as a result of protein synthesis that is induced by
Akt
the insulin-growth factor 1 (IGF1) and
Ca2+/calmodulin
calcineurin–nuclear factor of activated T cells (NFAT)
Rapamycin
mTOR
Calcineurin
signalling pathways. However, recent studies challenge the
prominent role given to the calcineurin–NFAT pathway,
and argue for an important role for Akt and mTOR in
NFAT
4E-BP
S6K
muscle hypertrophy91,92. Activation of Akt after IGF1
stimulation promotes translation by the phosphorylation
Transcription
of translational regulators and mTOR targets S6K and 4ETranslation
BP. Calcium could couple a mechanical stimulus to the
IGF1 pathway and lead to increased gene transcription by
Nucleus
activating the calcium-dependent phosphatase
calcineurin, which dephosphorylates and activates
transcription factors such as NFAT115.
Skeletal-muscle hypertrophy also occurs during muscle regeneration. As adult skeletal muscles are terminally
differentiated, muscle regeneration involves activation of SATELLITE CELLS that are located at the periphery of myofibres116.
Activated satellite cells, which are also known as myoblasts, proliferate and express myogenic markers. The myoblasts
then migrate and fuse to existing muscle fibres (hypertrophy) or fuse together to form new myofibres (hyperplasia).
Myoblast proliferation and fusion are stimulated by growth factors such as IGF1.
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Box 4 | Synaptic plasticity in neurons
Synaptic plasticity refers to
Presynapse
the ability of synapses to
undergo modification in
response to a stimulus. The
Nutrients
morphological and
? ?
biochemical changes that
Postsynapse
Ca2+/
Rapamycin
accompany changes in
mTOR
calmodulin
synaptic strength are the
Nucleus
Translation PKA
basis for learning and
117
ERK/MAPK
memory . In the model
Transcription
organism Aplysia, shortterm memory entails the
transient stimulation of
synapses by
neurotransmitters, which
leads to the presynaptic production of the second messenger cyclic AMP and the subsequent activation of protein kinase A
(PKA). PKA phosphorylates substrates such as ion channels and proteins of the exocytosis machinery in the pre-synapse
and consequently enhances the availability and release of transmitters. Short-term synaptic changes therefore involve
modification of pre-existing proteins, which leads to modification of pre-existing synaptic connections. The conversion of
short-term memory to long-term memory requires new protein synthesis immediately after re-stimulation of a synapse. In
the hippocampus of mammalian brain, both presynaptic and postsynaptic changes have been proposed to mediate longterm potentiation (LTP). Ca2+-dependent signalling pathways mediate synaptic changes, for example by enhancing
neurotransmitter release in the presynapse and increasing responsiveness of glutamate receptors in the postsynapse. As a
neuron makes several synaptic connections, an important issue is whether LTP is cell-wide or synapse-specific. Although
nuclear events are required, long-term changes are specific to the stimulated synapse and require local protein synthesis.
messenger RNAs that are stored in dendrites are activated after synaptic stimulation. Translation of these mRNAs, which is
mediated by the mTOR pathway, facilitates LTP. The mechanism by which mTOR is activated in neurons, possibly by
neurotransmitters, neurotrophic factors, or nutrients, is unknown. ERK/MAPK, extracellular-signal-regulated
kinase/mitogen-activated protein kinase.
LONG-TERM POTENTIATION
(LTP). LTP is a specific example
of coincidence detection,
whereby the high-frequency
stimulation of a neuron
increases the magnitude of
subsequent responses, an effect
that can last for days. LTP is
believed to underlie some kinds
of learning and memory.
SYNAPSE
The point of contact and
transfer of information from
one neuron to another.
RNA INTERFERENCE
(RNAi). The process by which an
introduced double-stranded
RNA specifically silences the
expression of genes through
degradation of their cognate
messenger RNAs.
124
TOR in neurons. Memory formation is achieved by
changes in synaptic strength or plasticity, a process
which involves LONG-TERM POTENTIATION (LTP) or, in the
snail Aplysia, long-term facilitation (LTF) (BOX 4).
Studies in Aplysia have shown that, despite nuclear
events, long-term changes in synaptic function and
structure are confined to a stimulated SYNAPSE and
require local protein synthesis from pre-existing
mRNAs. So proteins are synthesized and deposited
specifically at a stimulated synapse, which in turn leads
to the synapse-specific growth that is necessary to
encode memory99,100.
As the control of protein synthesis is an important
aspect of LTP/LTF, it is perhaps not surprising that
mTOR is involved in LTP/LTF100,101. The induction of
LTF in invertebrates and LTP in hippocampal slices by
electrical stimulation or by brain-derived neurotrophicfactor stimulation is inhibited by rapamycin101,102.
Postsynaptic protein synthesis and S6K activity in
response to a stimulus are also inhibited by
rapamycin103,104. Finally, components of the mTOR and
insulin pathways are enriched at postsynaptic sites101,105.
These findings show that mTOR controls synaptic protein synthesis and so also memory formation in
response to a stimulus. Furthermore, this is an interesting example of mTOR controlling cell growth in a localized manner and, again, in non-proliferating cells.
mTOR might also control overall neuronal growth, as
the deletion of PTEN in the brain results in enlargement
of neuronal cells106.
| FEBRUARY 2003 | VOLUME 4
Does mTOR control the size of proliferating cells?
Cultured mammalian cells treated with rapamycin
have a reduced proliferative rate and are smaller at all
stages of the cell cycle107. RNA INTERFERENCE (RNAi)-mediated inhibition of mTOR or raptor also leads to
decreased size in proliferating cells28. Finally, S6K1deficient mice have smaller pancreatic β-cells as a
result of a growth defect during embryogenesis108. So,
mTOR also seems to control cell size in proliferating
cells, which is consistent with previous findings for
dTOR and yeast TOR.
Conclusion and perspective
Studies over the past seven years have shown that TOR
controls the growth of proliferating yeast, Drosophila
and mammalian cells. In addition, TOR also controls
the growth of non-proliferating neurons and muscle
cells. An important mechanism of TOR signalling is the
phosphorylation of multi-phosphorylated effectors, by
activating protein kinases and inhibiting protein phosphatases. An unanswered question is how TOR senses
and is activated by the presence of nutrients. The further
characterization of several recently identified regulatory
proteins that associate with TOR should provide clues as
to how TOR carries out its many functions in controlling cell growth.
The importance of TOR in regulating cell growth is
underscored by the finding that rapamycin is not only
an immunosuppressant but also an effective anti-cancer
drug. The effect of rapamycin on tumour growth seems
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to be twofold. In addition to blocking the growth of
tumour cells directly, it also has an indirect effect by preventing the growth of new blood vessels (angiogenesis)
that supply oxygen and nutrients to the tumour cells109.
Furthermore, understanding the TOR signalling pathway
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Acknowledgments
We thank M. Ruegg, T. Schmelzle, and K. Tatchell for their comments on the manuscript. This work was supported by grants from
the Cancer Research Institute (E.J.), the Swiss National Science
Foundation and the Canton of Basel (M.N.H.).
Online links
DATABASES
The following terms in this article are linked online to:
Saccharomyces Genome Database:
http://genome-www.stanford.edu/Saccharomyces/
AVO1 | AVO2 | AVO3 | KOG1 | LST8 | PPH21 | PPH22 | SIT4 |
TIP41
Swiss-Prot: http://www.expasy.ch/
4E-BP | CDC55 | eIF4E | FKBP12 | FRAP | GLN3 | FRAP | NPR1 |
PDK1 | PTEN | raptor | RTG1 | RTG3 | TAP42 | TSC1 | TSC2 |
TOR1 | TOR2 | TPD3
FURTHER INFORMATION
Michael N. Hall’s laboratory:
http://www.biozentrum.unibas.ch/Research/Biochemistry/Hall/
hall.html
Access to this interactive links box is free online.
*Please note that this sentence has been corrected and differs from the print version. On
page 120, right column of the print version,
the sentence reads ‘However, there is one
conflicting report which suggests that mTOR
regulates S6K1and 4E-BP independently of
mTOR62.’
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