Amino Acid Regulation of TOR Complex 1
Joseph Avruch1, Xiaomeng Long, Sara Ortiz-Vega, Joseph Rapley,
Angela Papageorgiou, Ning Dai
Department of Molecular Biology and Diabetes Unit, Medical Services,
Massachusetts General Hospital and Department of Medicine, Harvard Medical
School, Boston, MA 02114, USA
1To whom correspondence should be addressed:
Diabetes Research Lab,
Department of Molecular Biology,
Massachusetts General Hospital,
Simches Research Center
185 Cambridge St,
Boston, MA 02114, USA.
Articles in PresS. Am J Physiol Endocrinol Metab (September 2, 2008). doi:10.1152/ajpendo.90645.2008
Copyright © 2008 by the American Physiological Society.
TOR complex 1 (TORC1), an oligomer of the mTOR (target of rapamycin)
protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GβL,
controls cell growth in all eukaryotes in response to nutrient availability, and in
metazoans, to insulin and growth factors, energy status and stress conditions. This review
focuses on the biochemical mechanisms that regulate mTORC1 kinase activity, with
special emphasis on mTORC1 regulation by amino acids. The dominant positive
regulator of mTORC1 is the GTP-charged form of the ras-like GTPase, Rheb. Insulin,
growth factors and a variety of cellular stressors regulate mTORC1 by controlling Rheb
GTP charging through modulating the activity of the Tuberous Sclerosis complex, the
Rheb GTPase activating protein. In contrast, amino acids, especially leucine, regulate
mTORC1 by controlling the ability of Rheb-GTP to activate mTORC1. Rheb binds
directly to mTOR, an interaction that appears to be essential for mTORC1 activation. In
addition, Rheb-GTP stimulates phospholipase-D1 to generate phosphatidic acid, a
positive effector of mTORC1 activation, and binds to the mTOR inhibitor FKBP38, to
displace it from mTOR. The contribution of Rheb’s regulation of PL-D1 and FKBP38 to
mTORC1 activation, relative to Rheb’s direct binding to mTOR, remains to be fully
defined. The rag GTPases, functioning as obligatory heterodimers, are also required for
amino acid regulation of mTORC1. As with amino acid deficiency however, the
inhibitory effect of rag depletion on mTORC1 can be overcome by Rheb overexpression,
whereas Rheb depletion obviates rag’s ability to activate mTORC1. The rag heterodimer
interacts directly with mTORC1 and may direct mTORC1 to the Rheb-containing
vesicular compartment in response to amino acid sufficiency, enabling Rheb-GTP
activation of mTORC1. The type III phosphatidyl inositol kinase also participates in
amino acid dependent mTORC1 activation, although the site of action of its product,
3’OH-phosphtidylinositol in this process is unclear.
Properties of TOR complex 1(TORC1):
TOR is the founding member of the PI-3 kinase-related protein (ser/thr) kinase
subfamily, identified in S. cerevisiae as the product of a mutant gene that confers a
dominant form of resistance to inhibition of growth/proliferation by the drug rapamycin
(43, 64). ScTOR is a critical regulator of the tightly coupled processes of cell growth (an
increase in cell mass/size) and proliferation, by virtue of its ability to control overall
mRNA translation in response to nutrient availability (6). The TOR polypeptides are
nearly 300kDa, and contain approximately 200kDa of aminoterminal noncatalytic
sequence, composed predominantly of HEAT repeats, and in the carboxyterminal third of
the protein a bi-lobed kinase domain whose amino acid sequence resembles that of the PI
lipid kinases more closely than that of the canonical protein kinases (58). Yeast contains
two TOR genes whereas only a single TOR gene is found in metazoans. Nevertheless, in
all eukaryotes, TOR is found in two independently regulated, functionally distinct hetero-
oligomeric complexes, TOR Complexes 1 and 2 (41, 59, 69). TOR Complex 1 (TORC1)
is the nutrient-responsive mediator of cell growth (i.e., cell mass/size) regulation, and is
composed of TOR and the WD-propeller domain containing proteins raptor and
Lst8/GβL (41, 59, 60, 69). Rapamycin, in a 1:1 complex with the polypeptide FKBP12,
binds directly to the TOR polypeptide in TOR complex 1 at a regulatory (FKBP12-
rapamycin binding/FRB) domain (mTOR AA 2014-2115) located slightly aminoterminal
to the mTOR catalytic domain (mTOR AA 2148-2430) and inhibits the TORC1 signaling
function (13, 14). The original rapamycin-insensitive ScTOR2 mutant (43, 64)contained
a single point mutation located in this segment (equivalent to Ser2035 in mTOR), that
eliminates the binding of the FKBP12/rapamycin complex. The mammalian TOR
Complex 2 (TORC2), in addition to TOR and Lst8, contains the essential polypeptides
rictor and sin1(54, 69, 96), and is concerned with the regulation of the actin cytoskeleton
and of certain AGC kinase subfamilies such as the Akt/PKBs (98) and novel PKCs (27,
48, 96). The TOR polypeptide in TORC2 is unavailable to the rapamycin/FKBP12
complex, and TORC2 is therefore not susceptible to direct inhibition by rapamycin,
although prolonged treatment with rapamycin can impair the assembly of TORC2 in
some cells (97). Lst8 is tightly bound to the mTOR catalytic domain in both complexes,
however its function in the mTORC1 complex is unclear, inasmuch as knockout of Lst8
is accompanied by a selective loss of mTORC2 function, whereas mTORC1 function
appears to be preserved (36). In contrast, raptor provides the substrate binding function to
TOR complex 1 and is thus essential; loss of raptor phenocopies loss of TORC1 (41, 73).
Surprisingly few direct substrates of the mammalian TORC1 complex are
known; best characterized are the translational regulatory proteins p70 S6 kinase/S6K1,
an AGC family kinase and 4E-BP, the inhibitor of the mRNA 5’ cap binding protein, eIF-
4E. S6K1 and 4E-BP bind directly to raptor through one or more short motifs; best
characterized is the TOS motif, which in S6K1 and 4E-BP has the form: Phe-Ac-φ-Ac-φ
(where Ac= Glu/Asp and φ-Leu/Ile/Met) (100); mutation of the Phe eliminates the ability
of these polypeptides to bind to raptor and to be phosphorylated by mTORC1 in cells (82,
100, 101). In the case of S6K1, if removal/inactivation of the TOS motif is accompanied
by deletion of the carboxyterminal noncatalytic tail, this doubly mutant S6K1, now
invisible in the cell to mTORC1, resembles Akt and so is instead phosphorylated at its
activating site (Thr389/412) by TORC2, in an insulin-responsive but rapamycin-resistant
manner (1, 11, 121). Although mTORC1 substrates other than S6K1 and 4E-BP are
known (e.g., STAT3, HIF1α, PRAS40, IRS1), the mTORC1-catalyzed phosphorylation
of S6K1 and 4E-BP has been shown to underlie, at least in part, the ability of mTORC1
to promote increased cell size (31). Rapamycin is perhaps the most selective protein
kinase inhibitor available, and is therefore a valuable probe of TORC1 function.
Rapamycin inhibits S6K1 activity by >98% in essentially all mammalian cells examined.
This inhibition of S6K1 activity (IC50 of ~2nM) is attributable to the ability of
rapamycin to prevent the TORC1-catalyzed phosphorylation of the S6K1 polypeptide at
Thr389/412, a regulatory site located in a hydrophobic segment carboxyterminal to the
S6K1 catalytic domain whose phosphorylation is indispensable for S6K1 activity (88).
Consequently, the extent of S6K1(Thr389/412) phosphorylation in cells is routinely used
as a reflection of TORC1 kinase activity (122). It should be emphasized however that
concentrations of rapamycin sufficient to fully inhibit this phosphorylation in intact cells
have little effect when added in vitro (as a complex with FKBP12) on the ability of
mTORC1 to directly phosphorylate S6K1(Thr389/412); fifty-fold or more higher
concentrations of rapamycin/FKBP12 are required for inhibition of TORC1 kinase in
vitro, and at these levels, the rapamycin/FKBP12 complex also promotes the dissociation
of raptor from mTOR (85). Thus the major mechanism by which rapamycin inhibits
mTORC1 signaling in vivo remains uncertain, but probably reflects a disruption of
raptor-mediated substrate presentation, instead of (or possibly, in addition to) an
inhibition of mTOR catalytic function. In contrast, the TOR inhibitors Ly294002
(IC50~5µM) and wortmannin (IC50~0.3µM) act at the kinase ATP binding site, the latter
through an irreversible covalent mechanism, and inhibit both TORC1 and TORC2 (7).
Regulation of TORC1: general features
Growth factors regulate TORC1 activity via either the PI3K/Akt pathway or the
Ras/MAPK pathway, which converge on the Tuberous Sclerosis heterodimeric complex
(TSC1-TSC2) to inhibit, by phosphorylation of TSC2, the GTPase activating function
(GAP activity) toward the small GTPase Rheb (ras homolog enriched in brain) (20, 49,
65). Rheb was identified as a gene whose expression is up regulated in rat brain by
seizure and stimuli that induce long-term potentiation (35, 124). Two Rhebs exist in
mammalian cells, Rheb1 and Rheb2, whereas a single Rheb gene is found in yeast and
Drosophila. Drosophila Rheb was identified as a gene whose loss of function led to a
decrease of cell size and whose gain of function led to cell-autonomous increase in cell
size (87, 99, 113). Genetic evidence in Drosophila places Rheb downstream of the TSC
complex, and biochemical studies established that the TSC complex functions as a
GTPase activating protein for Rheb (10, 33, 50, 115, 126). Elimination or inactivation of
either TSC1 or TSC2 results in an increase in the fractional GTP charging of Rheb to
well over 90% and in a constitutive activation of TORC1, which is not further augmented
by insulin or growth factors. Little is known concerning guanyl nucleotide exchange
factors acting on Rheb, although the Drosophila ortholog of the “translationally
controlled tumor protein” (TCTP) has been proposed to catalyze this reaction (46) .
Thus, Rheb-GTP is an activator of TORC1; as with rapamycin/FKBP12, Rheb does not
appear to regulate TORC2.
Cellular stresses inhibit TORC1 through a variety of mechanisms, which also
converge on the TSC complex; thus energy depletion or hypoxia, e.g., activate the AMP-
activated protein kinase, which (in collaboration with GSK3) phosphorylates TSC2 and
activates the TSC GAP function (51, 52). Glucocorticoids (118), hypoxia (111) and other
stresses induce expression of the REDD1 polypeptide, which binds 14-3-3 and thereby
relieves 14-3-3 inhibition of the TSC GAP function (21). Thus these negative regulatory
inputs to TORC1 appear to operate largely through up regulation of the GTPase
activating function of the TSC complex. AMPK can engender some inhibition of TORC1
in TSC null cells through the direct phosphorylation of raptor (38), although the potency
of this inhibition is far less than in the presence of an intact TSC. The aspect of TOR
regulation least well understood is the mechanism(s) by which amino acids control
TORC1 activity, the focus of the discussion to follow. One major distinction between
TORC1 regulation by amino acids as compared with growth factors or stress is that
amino acid regulation is only modestly altered in TSC null cells (89, 110), indicating that
the major site of amino acid regulation is downstream of, or on a pathway independent of
The concept of nutrient regulation of TOR emerged from studies in
Saccharomyces cerevisiae, where treatment with rapamycin or inactivation of both TOR
genes elicits a profound (90%) decrease in mRNA translation resulting in a proliferative
arrest very early in the cell cycle, as well as an inhibition of several other anabolic
programs and concomitant activation of catabolic programs such as autophagy (6). This
arrest phenotype strongly resembles Go, the state seen with nutrient deprivation or
growth on very poor nitrogen or carbon sources. Thus, as a dominant regulator of cell
growth and proliferation, ScTOR was proposed to be part of a nutrient responsive
pathway and perhaps was itself regulated by nutrient signals. Later studies suggested that
ScTORC1 is most responsive to glutamine sufficiency, inasmuch as both glutamine
depletion and rapamycin result in nuclear localization and activation of several
transcription factors that mediate glutamine synthesis (19); nevertheless, the molecular
components of a putative glutamine regulatory mechanism impinging on ScTOR remain
undefined. The first evidence for a role of TOR in the nutrient regulation of cell growth
in an intact metazoan was provided by the finding that loss of function of Drosophila
TOR arrests larval development and yields cellular phenotypes that mimic those caused
by amino acid deprivation, i.e., a significant reduction in nucleolar size, substantial lipid
vesicle aggregation in the larval fat body, and a cell-type specific pattern of cell cycle
arrest (84, 125).
Evidence for a relatively direct regulation of TORC1 by amino acids in
mammalian cells was first provided by the finding that a 1-2 hour removal of amino acids
from tissue culture medium results in the selective inhibition of S6K1 and
dephosphorylation of 4E-BP, rendering these targets unresponsive to insulin; readdition
of amino acids to basal levels, in the absence of serum or insulin, restores 4E-BP
phosphorylation, S6K1 activity and their responsiveness to insulin, whereas raising the
amino acid concentrations further can fully activate p70 S6 kinase such that insulin elicits
no further activation (42). Amino acid activation of p70 S6 kinase is inhibited by
rapamycin and is mediated by phosphorylation of the same array of sites as occurs with
insulin, thus reflecting the activity of TORC1. Importantly, the doubly-mutant,
rapamycin-resistant variant of S6K1 described above was found to also be resistant to
inhibition by amino acid withdrawal, establishing that amino acid regulation of S6K1
occurs either at mTORC1 or upstream. Withdrawal of amino acids does not interfere with
insulin’s ability to activate Akt, an output that requires activation of the type 1A PI-3
kinase and TORC2 (98); moreover, the ability of amino acid withdrawal to inhibit S6K1
is only modestly delayed in TSC null cells, and is effected without alteration in Rheb
GTP charging (89, 110). Thus, the pathway through which amino acids regulate
mTORC1 is largely independent of the type1A PI-3 kinase and does not involve
regulation of Rheb GTP charging. Notably however, overexpression of recombinant
Rheb to very high levels, e.g., 10-100 fold greater than endogenous Rheb, is able to
overcome fully the inhibition of mTORC1 signaling engendered by amino acid
withdrawal (70-72) In vivo, overexpression of Rheb in Drosophila is sufficient to
counteract the effect of amino acid starvation in tissues such as the fat body and the
salivary gland (113). Removal/readdition of amino acids does not alter the kinase activity
assayed in vitro of the TOR polypeptides extracted from cells exposed to these treatments
(37, 42, 70) (although see (37) ) suggesting that the amino acid regulation of TOR may
not involve a stable modification of TORC1 components, but more likely a reversible
inhibition of the ability of endogenous Rheb-GTP to activate mTORC1.
Regulation of TORC1: the primacy of leucine
Withdrawal of most amino acids singly for 1-2 hours inactivates TORC1
signaling to differing extents, however withdrawal of leucine or arginine is each nearly as
effective in down regulating TORC1 signaling as withdrawal of all amino acids (42), and
the preeminent effect of leucine withdrawal has been consistently observed in a variety of
cell types. Some cell types, e.g. hepatoma lines, are quite resistant to amino acid
withdrawal, perhaps because of high rates of endogenous autophagy (107). The unique
signaling function of leucine in the regulation of metabolism, in part through the
regulation of mTOR signaling, has been extensively supported by studies in vivo,
primarily in rodents (62, 112), but in humans as well (16, 22). Thus it is clear that leucine
regulates protein synthesis in skeletal muscle, as well as protein degradation in skeletal
muscle and liver, through TORC1-dependent and independent mechanisms. Skeletal
muscle protein synthesis is stimulated by leucine uniquely among the branched chain
amino acids, and the ability of leucine to augment the increase in protein synthesis
elicited by resistance training in man, and to restore this response in older individuals has
been well documented. A substantial component of the effect of leucine is independent of
insulin (which stimulates leucine uptake), however leucine also promotes insulin
secretion in vivo and together leucine and insulin stimulate muscle protein synthesis
synergistically (reviewed in (62)). Leucine stimulation of muscle protein synthesis is
inhibited by rapamycin, as is the ability of leucine to selectively promote S6K1 and 4E-
BP phosphorylation and the assembly of the eIF-4F complex, the primary basis for
leucine-stimulated translational initiation. Leucine also acts on the central nervous system
to control overall food intake through TORC1(and food selection, probably through
GCN2) (40, 77). Direct administration of L-leucine (but not L-valine) in the vicinity of
the arcuate nuclei region in rat hypothalamus stimulates hypothalamic TOR signaling,
and results in decreased food intake (anorexia) and significant weight loss (18). Leucine-
induced anorexia is inhibited by rapamycin, demonstrating that TORC1 signaling is
required. TORC1 activation is also required for the ability of intracerebroventricular
leptin and ciliary neurotrophic factor (CNTF) to reduce food intake in mice. Both
polypeptides stimulate hypothalamic S6K1/S6 phosphorylation and the anorectic
response to these agents is largely eliminated in S6K1 null mice (17). Mice fed a high fat
diet (HFD) develop resistance to the ability of leptin to reduce food intake, whereas
CNTF retains its potency; similarly, a HFD eliminates the ability of leptin to promote
hypothalamic S6K1/S6 phosphorylation whereas the response to CNTF persists (17).
Thus, leucine in concert with insulin stimulates TORC1 signaling in skeletal muscle to
promote the translation of mRNAs that underlie cell enlargement. Leucine also promotes
Leptin synthesis in the adipocyte (76, 91), and both leucine and leptin action on leptin-
sensitive neurons, signals that reflect immediate and long-term adequate nutrition,
activate TORC1 to suppress further food intake.
The stimulation of TORC1 by leucine appears to be initiated at an intracellular
site. In Xenopus oocytes, extracellular leucine is unable to promote S6K1
phosphorylation, but expression of a recombinant system L transporter confers
responsiveness to extracellular leucine and direct intracytoplasmic injection of L-leucine
(or trp, phe, arg, lys and gly but not D-leu, ala, pro, glu or gln) stimulates S6K1-P in a
rapamycin-sensitive manner (15). This contrasts with S. cerevisiae, where the dominant
receptor for amino acids is the plasma membrane protein Ssy1p, a nontransporting
member of the amino acid permease family (123), as well as with the leucine regulation
of autophagy (56, 57, 79). In rat hepatocytes, a polymer consisting of 5-8 leucines
attached by amide linkage to the alpha and epsilon amino groups of a branched lysine
polymer, Mr~1900, inhibits macroautophagy without evident cellular entry and at a
molar concentration comparable to free leucine, whereas a similar polymer of isoleucine
is ineffective. Moreover although insulin inhibition of macroautophagy in hepatocytes is
reversed by rapamycin, leucine inhibition is not sensitive to rapamycin (57, 79). Thus the
hepatic leu-sensitive pathway to macroautophagy and the Ssy1p pathway are TORC1-
independent, as is the leucine activated GCN2 output.
Although leucine action on TORC1 is initiated subsequent to leucine entry,
whether this is mediated by leucine itself or by some covalently liganded or metabolically
transformed product is not known. Regarding the features of the leucine molecule
required for TORC1 activation, modification of the alpha amino group (acetylation,
methylation) eliminates the ability of leucine to promote S6K1-P in H4-EII hepatoma
cells, however these derivatives are inhibitory, with an IC50 at ten fold excess over
leucine (108). In contrast, leucinamide is quite active both on H4-EII (108) and when
injected into Xenopus oocytes (15) but Lynch observed considerable conversion of
leucinamide to leucine by adipocytes (75). Leucinol, where an alcohol replaces the
leucine carboxyl, is an inhibitor of leucyl-tRNA synthetase; microinjection of leucinol or
tryptophanol into Xenopus oocytes generates a weak stimulation of S6K1-P compared
with the same amount of leucine, and this response is increased further with higher doses
of the alcohols (15). In Jurkat cells however, L-Leucinol is moderately inhibitory to
S6K1 (other amino alcohols, e.g., histidinol, are much more inhibitory) (47), whereas it is
without effect on basal or leucine-stimulated 4E-BP phosphorylation in adipocytes (74,
75). The basis for this variability in response to leucinol is unknown, and leaves open the
possibility that the mechanism of leucine action may vary with the cell background.
Specifically, it raises the question of whether cross regulation of the TORC1 pathway
consequent to GCN2 activation occurs in a cell- or time- or otherwise conditional
GCN2 is activated when the level of any amino acid diminishes sufficiently to
cause the accumulation of uncharged tRNAs, which are direct activators of the GCN2
kinase (44). GCN2 phosphorylates eIF2A at Ser51, resulting in the sequestration of the
eIF2A-GEF, eIF-2B; this greatly reduces the rate of general translational initiation, while
up regulating the translation of a subset of mRNA, e.g., the transcription factor ATF4,
which promotes expression of an array of genes including those coding for amino acid
biosynthetic proteins. Mice fed a leucine-deficient diet (or any diet deficient in an
essential amino acid) consume much less food (34) and exhibit semi-starvation, including
a suppression of protein synthesis in skeletal muscle and liver (2); GCN2 null mice fed
this diet also eat less but fail to suppress hepatic protein synthesis or undergo the
dephosphorylation of hepatic S6K1 and 4E-BP that occurs in wildtype mice pair-fed this
deficient diet, indicating that GCN2 is required for the down regulation of hepatic
mTORC1 signaling, at least in this circumstance. In contrast to the sustained liver weight
in the GCN2 null mice fed the leucine-deficient diet, muscle wasting in these mice is
profound, indicating major tissue-specific differences in the regulation of protein balance
and perhaps TORC1 signaling (2). As to possible mechanisms by which activation of
GCN2 might regulate mTORC1, one of the genes unregulated in response to the
increased abundance of ATF4 is GADD34, a protein phosphatase 1 regulatory protein
(83). GADD34 binds to both TSC1 and TSC2 (78, 120), and has been observed to
promote the dephosphorylation of TSC2 (Thr1462), a major Akt phosphorylation site,
which is claimed to disinhibit the TSC GAP function, reduce Rheb-GTP charging and
thus inhibit mTORC1. The likelihood that coordinate, reciprocal regulation of the GCN2
and TORC1 signaling occurs in response to amino acid deficiency is logical, and further
examination of the tissue-specific interactions of the GCN2 and mTORC1 pathways is
Activation of GCN2 occurs in response to deficiency of any essential amino acid,
however regulation of mTORC1 is most responsive to specific individual amino acids.
The nearly comparable inhibitory effect of leucine and arginine (42) withdrawal is
especially puzzling; apart from their participation in polypeptide chain elongation
through the mediation of six tRNAs, these amino acids share no commonalities in their
transport or metabolic fates. The possibility e.g., that arginine affects mTORC1 activity
through the mediation of NO is unexplored. Finally, it should be mentioned that
alterations in the extracellular concentration of amino acids whose transport is Na+-
linked, e.g., glutamine (but not leucine), will lead to parallel alterations in cell hydration;
cell swelling induced by any mechanism is accompanied by activation of several anabolic
pathways including mTORC1, whereas cell dehydration is inhibitory (63,102).
Mechanisms of TORC1 regulation: Rheb
As regards the biochemical mechanism by which Rheb activates mTORC1, Rheb
does not achieve this effect through modulating amino acid uptake, as overexpressing
Rheb (or TSC1/2) has no effect on the steady state concentration of individual amino
acids within the cells, including the concentrations of branched chain amino acids and
arginine. In addition, although removing extracellular amino acids lowers the total
intracellular amino acid level, overexpressing Rheb does not protect against the decline
nor does overexpression of TSC1/2 increase the decline (81). Similarly, in Drosophila S2
cells Rheb overexpression activates TORC1 signaling but does not promote the import of
glucose, bulk amino acids, or arginine, (39). Therefore, Rheb does not activate TORC1
signaling by modulating the intracellular amino acid levels.
Considerable evidence supports the view that Rheb activates TORC1 through a
direct interaction with mTOR. Rheb binds directly to the upper, small lobe of the TOR
kinase domain (mTOR AA 2148-2300) in TORC1 (71). The ability of Rheb to bind
directly to the mTOR catalytic domain is consistent with the occurrence of such an
interaction in the normal process by which Rheb-GTP activates TORC1, however the
affinity of Rheb for TOR is rather weak and an interaction of endogenous Rheb with
endogenous mTOR has not been identified, even in TSC null cells. Nevertheless, other
physiologically important interactions between a small GTPase and its effector, e.g., ras-
GTP and type 1A PI-3 kinases, exhibit comparably low affinity (86), and this may be
mitigated somewhat by the substantial colocalization of Rheb and mTORC1 on
overlapping endomembrane compartments. A more unusual feature of the Rheb-mTOR
interaction, and a significant caveat to its physiologic significance is that the ability of
recombinant Rheb to bind mTOR, in vitro and in cells, is not dependent upon, or
stimulated by Rheb GTP charging; nucleotide-deficient, inactive Rheb mutants actually
bind mTOR more tightly than does wildtype Rheb. Nevertheless when mTOR is
coexpressed with such nucleotide-deficient Rheb mutants, the mTOR polypeptides
retrieved with these Rheb mutants are essentially devoid of kinase activity when assayed
in vitro (71). This finding indicates that the interaction of mTOR with a native,
presumably GTP charged Rheb is essential for the ability of mTOR (at least in TOR
complex 1) to achieve catalytic competence. The regions of Rheb that mediate its
interaction with mTOR is not known; Rheb mutagenesis indicates that mutation of both
the Rheb switch1 domain (71), whose configuration is highly GTP dependent, and the
switch 2 domain(70), which is hardly altered by GTP, both disable the ability of
overexpressed Rheb to stimulate mTORC1 signaling in amino acid deprived cells.
Further evidence for the importance of a direct Rheb/TOR interaction in the activation of
TOR is provided by the findings of Tamanoi and colleagues who selected hyperactive
mutants of S. pombe Rheb; in vitro, these mutants exhibit a marked decrease in affinity
for GDP without altered GTP binding (117). Although wildtype S. pombe Rheb cannot
be coprecipitated with S. pombe TOR and does not comigrate with SpTOR on sucrose
density gradients, such hyperactive S. pombe Rheb mutants, expressed at wildtype levels,
do coprecipitate and comigrate with endogenous S. pombe TOR. The concomitant
enhancement in signaling efficacy and affinity for SpTOR exhibited by the SpTOR
mutants implies that the two phenotypes are causally related, however this remains
inferential. Finally, Sancak et al. (95) reported that the direct addition of Rheb-GTP to
mTORC1 in vitro results in activation of the TORC1 kinase activity; this result, if
confirmed and defined in molecular terms, would provide incontrovertible support for the
role of a direct Rheb/mTOR interaction in TORC1 regulation.
A ready potential explanation for the ability of amino acid withdrawal to inhibit
mTORC1 signaling is provided by the finding that withdrawal of amino acids (or of just
leucine or arginine) diminishes the ability of recombinant Rheb to bind to endogenous or
coexpressed recombinant mTOR (72). This effect of amino acid withdrawal may also
explain why substantial Rheb overexpression is needed, generating Rheb-GTP levels well
above those found in TSC null cells, to overcome the inhibition of mTORC1 engendered
by amino acid withdrawal. The biochemical mechanism responsible for the ability of
amino acid withdrawal to inhibit the Rheb/mTOR interaction is not known, however
amino acid regulation of the Rheb-mTOR interaction is directed entirely through mTOR
and is mediated through the lower lobe of the TOR kinase domain; deletion of the lower
lobe (mTOR AA 2301-2430) eliminates the ability of amino acid withdrawal to inhibit
Rheb binding to the upper lobe (mTOR AA 2148-2300) without significantly altering
Rheb binding per se. Thus the mechanism by which amino acid sufficiency regulates
mTORC1 appears to be intimately related to the mechanism by which Rheb activates
mTORC1; unfortunately however, the mechanism(s) by which Rheb regulates mTOR are
not yet unclear.
Mechanisms of TORC1 regulation: Phospholipase D and phosphatidic acid
Two mechanisms have been proposed whereby Rheb can activate TORC1
indirectly, without the requirement for a direct Rheb/mTOR interaction. One such
pathway occurs through the generation of phosphatidic acid (PA), produced by
Phospholipase-D catalyzed hydrolysis of phosphatidylcholine (PC). Inhibition of PA
accumulation using 1-butanol (but not 2-butanol) (29) or RNAi-induced depletion of PL-
D1(28) reduces serum-stimulated S6K1 and 4E-BP phosphorylation, providing strong
support to the view that PL-D1, through the generation of PA, provides a positive input to
mTORC1. The site of PA action appears to be mTOR itself; salt-sensitive binding of PA-
containing PC vesicles to mTOR occurs through a site within the mTOR FRB domain,
mediated by several basic residues, especially Arg2109. Addition of an
FKBP12/rapamycin complex displaces the PA vesicles from an isolated FRB domain,
whereas the rapamycin-insensitive FRB mutant, Ser2035Ile, binds the PA vesicles in a
rapamycin-insensitive manner. The mTOR(Arg2109Ala) mutant exhibits unaltered
kinase activity in vitro but only ~ 60% the efficacy of mTOR-WT in activating
coexpressed S6K1(29). Recently Sun et.al., (114) reported that Rheb, added directly in
vitro, binds to and stimulates the activity of PL-D1 in a GTP dependent manner. Rheb is
thus added to the extensive array of upstream regulators of PL-D, which includes protein
kinase C isoforms, other small GTPases of the ARF, Rho and Ras families and,
particularly, the phosphoinositide, phosphatidylinositol 4,5-bisphosphate. In this
pathway, Rheb-GTP stimulation of PL-D1 generates PA, which through its direct binding
to mTOR, contributes to TORC1 activation in vivo; stimulation of TORC1 kinase
activity in vitro by addition of PA has not been observed. PA may act by directing the
localization of mTORC1, enabling its proximity to Rheb or other elements, rather than by
direct modulation of its catalytic function; a similar role for PA has been suggested in the
regulation of the Raf kinase. Alternatively, PA may participate in mTOR regulation by
FKBP38, discussed next.
Mechanisms of TORC1 regulation: FKBP38
A second mechanism for Rheb activation of TOR that does not require a direct
Rheb-TOR interaction involves the rapamycin-insensitive peptidyl prolyl cis-trans
isomerase (PPI) FKBP38. Bai et.al., (5) retrieved FKBP38 in a two-hybrid screen with a
Rheb bait and demonstrated coprecipitation of the endogenous polypeptides from 293
cells. FKBP38 encompasses an FKBP12-like PPI domain, the site of Rheb binding,
followed by three TPR repeats, a canonical Ca++/calmodulin(CM) binding domain, and
near its carboxyterminus, a transmembrane domain (67, 109). The FKBP38 polypeptide
is bound to endomembranes and mitochondrial outer membrane via a carboxyterminal
transmembrane domain, so that the bulk of the polypeptide faces into the cytoplasm.
Previous work had shown that FKBP38 catalytic function is activated by a calcium-
calmodulin complex (EC50~290nM) binding to the Ca++/CM binding domain,
displacing it from the PPI domain (25, 26). Thereby freed, the PPI domain is able to bind
and inhibit Bcl2 in a Ca++ dependent manner, thereby contributing to Ca++-induced
apoptosis in a variety of cells. Ca++/CM binding to FKBP38 also enables the three
tetratricopeptide (TPR) repeats to bind HSP90, itself a Ca++/CM binding protein. The
ternary complex of FKBP38/Ca++CM/HSP90 is enzymatically inactive and unable to
bind Bcl2; thus HSP90 availability controls the ability of Ca++ to make available the
FKBP38 PPI domain to its partners (24). FKBP38 had also been identified as one of the
transcripts significantly unregulated in HeLa cells overexpressing either TSC1 or TSC2,
and RNAi-induced depletion of FKBP38 reversed the (10-14%) reduction in HeLa cells
size (FSC) caused by overexpression of TSC1 or TSC2 (92). Bai et.al., (5) demonstrated
that overexpression of FKBP38 inhibits the phosphorylation of 4E-BP caused by amino
acid readdition or Rheb overexpression, whereas RNAi-induced depletion of endogenous
FKBP38 up regulates basal S6K1(Thr389/412) and 4E-BP phosphorylation and
ameliorates the inhibition of these phosphorylations that occurs with serum or amino acid
withdrawal. Moreover, addition of FKBP38 in vitro inhibited the mTORC1 kinase
activity. Thus FKBP38 appears to be an endogenous inhibitor of mTORC1. Importantly,
FKBP38 binds directly to Rheb-GTP in preference to Rheb-GDP and also binds, in a
rapamycin-insensitive manner, to an mTOR segment (1967-2191) that encompasses the
FRB domain (AA 2015-2114). The binding of FKBP38 to mTOR(1967-2191), a segment
that does not bind Rheb, is nevertheless inhibited by addition of Rheb-GTP (but not
Rheb-GDP), presumably because the binding to Rheb-GTP to FKBP38 interferes with
the ability of FKBP38 to bind mTOR. FKBP38 binds through its FKBP12-like domain to
mTOR; the ability of Rheb-GTP to displace FKBP38 from mTOR is consistent with the
ability of Rheb-GTP to also bind to this FKBP12-like segment. The regions of Rheb that
mediate its binding to mTOR and FKBP38 are not defined, however the nucleotide
deficient RhebD60K mutant, which binds both mTOR and FKBP38 more strongly than
does wildtype Rheb, does not displace FKBP38 from mTOR(1967-2191) suggesting that
Rheb may use different domains for the binding of these two partners. Serum addition,
which promotes Rheb GTP charging, enhances the binding of Rheb to FKBP38 and
reduces FKBP38 binding to mTOR, presumably reflecting the ability of Rheb-GTP to
displace FKBP38 from mTOR. Amino acid withdrawal decreases the binding of Rheb to
FKBP38 while increasing FKBP38 binding to mTOR; inasmuch as amino acid
withdrawal does not alter Rheb-GTP charging, an explanation for this behavior is not
Caveats and questions: PL-D/PA and FKBP38
Altogether, the data point to a significant role for FKBP38 in the regulation of
mTORC1; nevertheless, questions remain regarding both the PL-D1/PA and FKBP38
mechanisms of TORC1 regulation. First, there is thus far a lack of supporting genetic
evidence for either mechanism. PL-D null flies exhibit reduced viability during
cellularization but PL-D deficient adults are overtly normal (66), inconsistent with a
significant role for PL-D in Drosophila TOR regulation. Drosophila encodes an FKBP38
homolog (CG5482) however the cell/organ screens that have revealed such elements as
Rheb, TSC, REDD, etc, have not (as yet) identified CG5482 as a regulator of cell size.
More definitively, SpRheb is a dominant regulator of TORC1 activity (3) despite the
absence of an FKBP38 homolog in S. pombe. Conceivably, the participation of PL-D
and/or FKBP38 in TORC1 regulation may be limited to higher metazoans, e.g.,
vertebrates. Rheb is certainly the dominant proximate regulator of mammalian TORC1
for all inputs, however the relative contributions of Rheb’s interaction with mTOR, PL-
D1 or FKBP38 to the activation of TORC1 remains to be established, and several
mechanisms may be operating simultaneously, in a parallel or interdependent way.
Inasmuch as PA binding to mTOR is competitive with the rapamycin/FKBP12 complex
(29), PA may also participate in the relief of mTOR inhibition by FKBP38. Important
modulators of FKBP38 action include the level of intracellular calcium and/or HSP90.
The ability of amino acid withdrawal/readdition to enhance/reduce the FKBP38/mTOR
interaction while reduce/enhance the FKBP38/Rheb interaction is unexplained. Perhaps,
as with the effect of amino acid withdrawal on the Rheb/mTOR interaction (72), amino
acid withdrawal may act through TOR to enhance FKBP38 binding near the FRB
domain, thereby diminishing FKBP38 availability to Rheb. Placing the site of action of
amino acid regulation of TORC1 at TOR itself, rather than at Rheb, provides a
framework for explaining TORC1 regulation in S. cerevisiae, where FKBP38 homologs
do not exist and Rheb does not appear to regulate TORC1. Although it is likely that a
variety of mechanisms (Fig.1), including Rheb-independent inputs, exist for the
regulation of TORC1, a more complete understanding of the mechanism by which Rheb
regulates mTORC1 will be required before the mechanism of amino acid regulation is
Mechanisms of TORC1 regulation: The Rag GTPases
The Rag subfamily of GTPases, Rag-A-D have recently emerged as components
of the pathway by which amino acids regulate TORC1 signaling. The rags are small GTP
binding proteins that encode the GX4GKS/T and DXXG motifs; rag A and B, but not C
and D, contain an appropriately situated NXXD motif, whereas all four contain an
HKM/VD that might instead participate in guanosine binding. No candidate S/CA motifs
are evident and farnesylation or myristoylation motifs are lacking (103). Human rags
A(313AA) and B(346AA) are over 98% identical, differing only by an aminoterminal
extension in rag B of 33 amino acids; rag B also has a longer isoform with a 28AA insert
after AA76, into the center of what is likely the switch 1 loop. Rag C (399AA) and D
(400AA) are 77% identical overall, differing primarily over their aminoterminal 60 and
carboxyterminal 30 amino acids. Rag A/B are only ~20% identical to rag C/D, and each
is about 17-18% identical to Ha-ras, with long carboxyterminal extensions beyond the
Ha-ras sequence; the rag A/B polypeptides associate as heterodimers with rag C/D
through these carboxyterminal segments (103) and function as obligatory heterodimers.
In the most active form, the rag A/B is GTP charged, whereas the rag C/D is GDP
charged. Although evidence exists that ras, e.g., may also function as dimers (53), the
formation of stable dimers, and especially, the discordant phosphorylation state of the
guanyl nucleotide in each half of the active form of the rag dimer are entirely unique
features of these small GTPases. The rag proteins are homologous to the Gtr1p and Gtr2p
proteins of S. cerevisiae; human rag A[Q66L] will rescue a Gtr1 LOF mutant (45). Gtr1
was originally retrieved in mutant form in screens for suppressors of a RCC1 (Ran-GEF)
mutant and wildtype Gtr1 was shown to negatively regulate Gsp1/Ran through Gtr2 (80,
103). Recombinant rag A-GTP is predominantly cytoplasmic, whereas rag-A-GDP is
seen in a speckled pattern in the nucleus and rag C/D, when coexpressed, follows rag A
(103). The reported association of Gtr1/2 or rag with a variety of nuclear proteins (104,
116, 119) supports the likelihood that these GTPases cycle through the nucleus. Gao and
Kaiser (32) however identified a primarily endosomal localization for Gtr1/2; they
retrieved Gtr1 and Gtr2 in a screen for genes required for the function of the general
amino acid transporter GAP1, which is translocated to the cell surface in response to
amino acid deficiency (12, 93). Pull-down studies with the Gtr1/2 polypeptides recovered
the proteins EGO1/GSE3, EGO3/GSE2 and Ltv1, all of which were required for GAP1
function. A constitutive complex of Gtr1, Gtr2, EGO1 and EGO3 is tethered to
endosomes through EGO3; EGO1/3 are small (184/162AA) polypeptides without
metazoan homologues. The ability of the complex to enable translocation of GAP1 to the
PM depends on a direct interaction between Gtr2p and GAP1, GTP charging of Gtr1p
and secondarily, GDP charging of Gtr2p (32). The EGO/Gtr complex, which remains on
the endsome, probably promotes the loading of GAP1 onto a cycling vesicle for
translocation to the PM. The EGO/Gtr complex is relatively specific for GAP1 among
AA permeases, although Gtr1 was reported earlier to be important for functional
expression of the phosphate transporter, Pho84 (8).
The first evidence linking these small GTPases with TORC1 came from screens
in S. cerevisiae. Dubouloz et.al., (23) identified strains mutant for EGO1/3 and Gtr2 as
deficient in their recovery from rapamycin inhibition. The EGO/Gtr complex was
observed to associate with the vacuolar membrane; in response to rapamycin the vacuole
enlarges greatly in size, due to fusion with autophagic vesicles. During recovery from
rapamycin, the excess vacuolar membrane is endocytosed into the vacuole, a process
called microautophagy; this does not occur in the EGO/Gtr mutants. Retrograde traffic of
vacuolar membrane to other compartments is not impaired in the EGO mutants,
indicating that this process is not sufficient to restore TORC1 activity. In contrast,
mutations that promote the synthesis or inhibit the degradation of glutamate/glutamine
suppress the effects of EGO LOF on recovery from rapamycin. More recently, a screen in
S. pombe (127) seeking genes that enhance the growth defect of a partial TOR1
(predominantly TORC1) LOF mutation retrieved all four components of the EGO/Gtr
complex, as well as vps34/vps15, many vps class C genes that are required for vesicle
trafficking, along with numerous genes encoding ribosomal and mitochondrial
components. As to the mechanism by which impairment of vesicle traffic synergizes with
TORC1 LOF, temperature sensitive vps11 or vps18 class C mutants shifted to the
nonpermissive temperature exhibited very pronounced (50-90%) and relatively selective
decreases in the intracellular levels of glutamate and/or glutamine. Taken together, these
data suggest that the EGO/Gtr complex, among its various actions, controls membrane
traffic to promote adequate intracellular amino acid (especially Glu/Gln) levels.
The existence of a direct physical and functional interaction between the rag
GTPases and TORC1 was demonstrated by the finding that immunoprecipitates of
TORC1 from mammalian cells coprecipitate rag C (94). The rags were shown to bind as
heterodimers to endogenous and coexpressed TORC1, apparently through a direct
association with raptor. Binding is strongly dependent upon rag A/B in the GTP form is
and enhanced if rag C/D is GDP charged. Independently, Kim et.al., (61) generated
shRNA against all 132 Drosophila small GTPases and found that depletion of the
Drosophila rag (A/B) and (C/D) homologs, as well as of DRheb selectively reduced
DS6K1[Thr398-P]. A constitutively activated mutant of rag B, expressed together with
rag C is able to restore S6K1-P in amino acid deprived cells with a potency similar to
Rheb, whereas stressors that inhibit TORC1 at the level of the TSC complex continue to
inhibit mTORC1 despite expression of a constitutively activated rag B+C complex. Thus,
depletion of endogenous Rheb-GTP greatly diminishes the stimulatory effect of
recombinant rag B+C and sufficient recombinant Rheb-GTP can activate TORC1
independently of rag. Dominant inhibitory Rag A/B variants suppress the response to
both amino acids and to insulin (61, 94). Although endogenous Rheb-GTP requires active
rag to promote TORC1 signaling, the ability of recombinant, overexpressed Rheb to
activate mTORC1 is largely unaffected if rags are depleted with RNAi. Conversely, the
ability of rags, even if overexpressed in active form, to stimulate TORC1 requires Rheb-
GTP. Evidence for a specific role of rags in the nutrient-dependent regulation of TORC1
comes from Drosophila; overexpression of activated variants of the rag heterodimer
greatly increase cell size under starvation conditions, with little impact in the fed state.
Conversely, dominant inhibitory rag mutants decrease cell size in the fed, but not the
fasted state (61).
As to the mechanism of rag action in the regulation of TORC1, Sancak et.al. (94)
report that readdition of amino acids to deprived cells increases GTP charging of
endogenous rag from 44 to 63%, and increases the association of rag with TORC1, the
latter demonstrable only by addition of cross-linker to the cells prior to lysis.
Immunocytochemistry shows mTOR to be diffusely cytoplasmic in amino acid deprived
cells but to aggregate onto a rab7-rich vesicular compartment upon readdition of amino
acids. Rag B behaves similarly, but the constitutively active rag B[Q99L] is constitutively
associated with this rab7 compartment, as is Rheb. Depletion of rag A/B, rag C/D or
raptor with shRNAs prevents the recruitment of mTOR to these rab7 vesicles by amino
acids. Sancak et.al., (94) propose that amino acids, by promoting rag A/B GTP charging,
promote rag association with raptor and the recruitment of mTORC1 to the (rab7-
containing) membrane compartment that contains the mTORC1 proximal activator, Rheb
Caveats and questions: the rag GTPases
The evidence indicates that rags are critical components of the pathway by which
amino acids regulate TORC1 activity. The major question is whether these GTPases are
situated upstream or downstream of the amino acid signal, or perhaps both. The
properties of the yeast EGO/Gtr complexes described above (23, 32, 127) indicates that
they function by a variety of mechanisms to promote the accumulation of intracellular
amino acids. Although there is no evidence for a structural or functional equivalent of an
EGO/Gtr complex in mammalian cells, it will be important to determine whether the
activated mammalian rag heterodimer increases intracellular amino acid levels in
deprived cells, and if so, whether its ability to activate mTORC1 is dependent on such an
increase. If rag activation of TORC1 is mediated primarily by an increase in intracellular
amino acids, the site of amino acid action would remain to be defined.
Several subcellular localizations and itineraries for the rag heterodimers have
been described (23, 32, 94, 103); this may reflect the existence of compositionally and
functionally distinct rag-containing complexes, whose function is to enable the
movement of polypeptides among membrane compartments or, for TORC1, perhaps from
cytosol to a membrane compartment, in response to stimuli that alter their GTP charging.
The ability of amino acids to regulate rag GTP charging, if confirmed, along with ability
of the GTP-activated rag heterodimer to bind raptor (23, 32, 94, 103) provide the
strongest evidence for an action of the rags as mediators of amino acid signaling to
mTORC1. If the function of the rag A/B GTP charging is to promote the binding of the
rag heterodimer to raptor, what enables the transfer of the complex to the rab7
compartment in response to amino acid sufficiency? In any case, the mechanism by
which amino acids regulate rag GTP charging is of considerable interest.
Clearly, the rag heterodimer may plausibly function both upstream and
downstream of TORC1, promoting sufficient intracellular amino acids through TORC1-
independent control of vesicle trafficking of relevant polypeptides, while simultaneously
acting directly on mTORC1 to enable its optimal activation by Rheb-GTP.
Mechanisms of TORC1 regulation: Type III Phosphatidylinositol kinase/vps34
Several reports propose a role for the type III PI-3 kinase, the homolog of the S.
cerevisiae vps34, as a signaling intermediate in the amino acid regulation of TORC1 (9,
81). Type III PI-3 kinase vps34, together with its kinase-like partner, vps15,
phosphorylates PI exclusively at the 3’ OH group, and is the only PI kinase in S.
cerevisiae. In mammalian cells, the hvps34/hvps15 heterodimer is best recognized for its
many roles in endosomal trafficking and sorting, and for its requirement in the initiation
of autophagy (4, 68). PI3P is found primarily on early endosomes and the internal
vesicles of multivesicular endosomes; PI-3P-rich microdomains serve as platforms to
build trafficking complexes through the interaction of PI-3P with proteins that contain
FYVE and PX domains. The important evidence linking type III PI-3 kinase to mTORC1
includes the finding that extracellular amino acids are capable of regulating hvps34 lipid
kinase activity (9), perhaps through a Ca++ dependent mechanism (37); withdrawal of
extracellular amino acid reduces immunoprecipitable hvps34 activity by ~50% (9). In
addition, depletion of vps34 or vps15 in mammalian cells inhibits amino acid-stimulated
S6K1 phosphorylation, and overexpression of recombinant vps34 and vps15 increase
S6K1 phosphorylation, especially if amino acids are present. Overexpression of a dimeric
FYVE domain sequesters PI-3P itself and also inhibits S6K1 phosphorylation (81).
Moreover, some coprecipitation of hvps34 with endogenous mTOR is detectable (37).
Taken together, these findings are supportive of the conclusion that hvps34, through the
generation of PI-3P, participates in the activation of mTORC1. It should be noted
however that genetic elimination of the type III PI 3 kinase in Drosophila has no impact
on DTORC1 signaling (55), and we observe that RNAi depletion of vps34 or vps 15 in
C.elegans recapitulates none of the phenotypes of CeTOR or CeRaptor deficiency ((90);
XL and JA, unpublished). It remains possible however that vps34, through the generation
of PI-3P, participates in the amino acid signaling to TORC1 only in vertebrate or
mammalian cells. Such participation might be as a direct regulator of the mTORC1
kinase (not evident in vitro) or by contributing to the generation of a membrane
compartment necessary for optimal mTORC1 activation. Alternatively however, it is also
possible that the action of PI-3 kinase is much more indirect, and is mediated by its
contributions to vesicle trafficking e.g., to the lysosome and thus to the provision of
intracellular amino acids.
Mechanisms of TORC1 regulation: MAP4K3/Germinal center kinase-related kinase
Findlay et al., (30) carried out an RNAi screen for Drosophila protein kinases
necessary for the hyperphosphorylation of DS6K1, and identified a Ste20 family member
(MAP4K3) whose depletion inhibited S6K1 and 4E-BP phosphorylation, and whose
overexpression increased S6K1-P in an PI-3 kinase independent but rapamycin sensitive
manner. The activity of the recombinant MAP4K3 was reduced by amino acid
withdrawal and restored by amino acid readdition, but was insensitive to insulin or
rapamycin. MAP4K3/ Germinal center kinase-related protein kinase has been previously
implicated in the TNFa and Wnt3a regulation of JNK activation in lymphocytes (105,
106). The site of action of this protein kinase and its physiologic role in the regulation of
TORC1 signaling remain to be defined.
Acknowledgements: We thank Jeanette Prendable for assistance in preparation of the
manuscript. The work of the au cited herein was supported by NIH grants DK17776 and
mTOR or rictor-mTOR phosphorylates its hydrophobic motif site. J Biol Chem 280:
2. Anthony TG, McDaniel BJ, Byerley RL, McGrath BC, Cavener DR,
McNurlan MA, and Wek RC. Preservation of liver protein synthesis during dietary
leucine deprivation occurs at the expense of skeletal muscle mass in mice deleted for
eIF2 kinase GCN2. J Biol Chem 279: 36553-36561, 2004.
3. Aspuria PJ, Sato T, and Tamanoi F. The TSC/Rheb/TOR signaling pathway in
fission yeast and mammalian cells: temperature sensitive and constitutive active mutants
of TOR. Cell Cycle 6: 1692-1695, 2007.
4. Backer JM. The regulation and function of Class III PI3Ks: novel roles for
Vps34. Biochem J 410: 1-17, 2008.
5. Bai X, Ma D, Liu A, Shen X, Wang QJ, Liu Y, and Jiang Y. Rheb activates
mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318: 977-980,
Ali SM and Sabatini DM. Structure of S6 kinase 1 determines whether raptor-
TOR controls translation initiation and early G1 progression in yeast. Mol Biol Cell 7:
7. Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC, Jr., and
Abraham RT. Direct inhibition of the signaling functions of the mammalian target of
rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.
Embo J 15: 5256-5267, 1996.
8. Bun-Ya M, Harashima S, and Oshima Y. Putative GTP-binding protein, Gtr1,
associated with the function of the Pho84 inorganic phosphate transporter in
Saccharomyces cerevisiae. Mol Cell Biol 12: 2958-2966, 1992.
9. Byfield MP, Murray JT, and Backer JM. hVps34 is a nutrient-regulated lipid
kinase required for activation of p70 S6 kinase. J Biol Chem 280: 33076-33082, 2005.
10. Castro AF, Rebhun JF, Clark GJ, and Quilliam LA. Rheb binds tuberous
sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and
farnesylation-dependent manner. J Biol Chem 278: 32493-32496, 2003.
11. Cheatham L, Monfar M, Chou MM, and Blenis J. Structural and functional
analysis of pp70S6k. Proc Natl Acad Sci U S A 92: 11696-11700, 1995.
12. Chen EJ and Kaiser CA. Amino acids regulate the intracellular trafficking of the
general amino acid permease of Saccharomycescerevisiae. Proc Natl Acad Sci U S A 99:
13. Chen J, Zheng XF, Brown EJ, and Schreiber SL. Identification of an 11-kDa
FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated
protein and characterization of a critical serine residue. Proc Natl Acad Sci U S A 92:
14. Choi J, Chen J, Schreiber SL, and Clardy J. Structure of the FKBP12-
rapamycin complex interacting with the binding domain of human FRAP. Science 273:
15. Christie GR, Hajduch E, Hundal HS, Proud CG, and Taylor PM.
Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase
in a target of rapamycin-dependent manner. J Biol Chem 277: 9952-9957, 2002.
16. Coffey VG and Hawley JA. The molecular bases of training adaptation. Sports
Med 37: 737-763, 2007.
17. Cota D, Matter EK, Woods SC, and Seeley RJ. The role of hypothalamic
mammalian target of rapamycin complex 1 signaling in diet-induced obesity. J Neurosci
28: 7202-7208, 2008.
18. Cota D, Proulx K, Smith KA, Kozma SC, Thomas G, Woods SC, and Seeley
RJ. Hypothalamic mTOR signaling regulates food intake. Science 312: 927-930, 2006.
19. Crespo JL, Powers T, Fowler B, and Hall MN. The TOR-controlled
transcription activators GLN3, RTG1, and RTG3 are regulated in response to
intracellular levels of glutamine. Proc Natl Acad Sci U S A 99: 6784-6789, 2002.
20. Crino PB, Nathanson KL, and Henske EP. The tuberous sclerosis complex. N
Engl J Med 355: 1345-1356, 2006.
21. DeYoung MP, Horak P, Sofer A, Sgroi D, and Ellisen LW. Hypoxia regulates
TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3
shuttling. Genes Dev 22: 239-251, 2008.
Barbet NC, Schneider U, Helliwell SB, Stansfield I, Tuite MF, and Hall MN.
regulation of mammalian target of rapamycin signalling and human skeletal muscle
protein synthesis. Curr Opin Clin Nutr Metab Care 11: 222-226, 2008.
23. Dubouloz F, Deloche O, Wanke V, Cameroni E, and De Virgilio C. The TOR
and EGO protein complexes orchestrate microautophagy in yeast. Mol Cell 19: 15-26,
24. Edlich F, Erdmann F, Jarczowski F, Moutty MC, Weiwad M, and Fischer G.
The Bcl-2 regulator FKBP38-calmodulin-Ca2+ is inhibited by Hsp90. J Biol Chem 282:
25. Edlich F, Maestre-Martinez M, Jarczowski F, Weiwad M, Moutty MC,
Malesevic M, Jahreis G, Fischer G, and Lucke C. A novel calmodulin-Ca2+ target
recognition activates the Bcl-2 regulator FKBP38. J Biol Chem 282: 36496-36504, 2007.
26. Edlich F, Weiwad M, Erdmann F, Fanghanel J, Jarczowski F, Rahfeld JU,
and Fischer G. Bcl-2 regulator FKBP38 is activated by Ca2+/calmodulin. Embo J 24:
27. Facchinetti V, Ouyang W, Wei H, Soto N, Lazorchak A, Gould C, Lowry C,
Newton AC, Mao Y, Miao RQ, Sessa WC, Qin J, Zhang P, Su B, and Jacinto E. The
mammalian target of rapamycin complex 2 controls folding and stability of Akt and
protein kinase C. Embo J 27: 1932-1943, 2008.
28. Fang Y, Park IH, Wu AL, Du G, Huang P, Frohman MA, Walker SJ, Brown
HA, and Chen J. PLD1 regulates mTOR signaling and mediates Cdc42 activation of
S6K1. Curr Biol 13: 2037-2044, 2003.
29. Fang Y, Vilella-Bach M, Bachmann R, Flanigan A, and Chen J. Phosphatidic
acid-mediated mitogenic activation of mTOR signaling. Science 294: 1942-1945, 2001.
30. Findlay GM, Yan L, Procter J, Mieulet V, and Lamb RF. A MAP4 kinase
related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem J 403: 13-
31. Fingar DC, Salama S, Tsou C, Harlow E, and Blenis J. Mammalian cell size is
controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev 16:
32. Gao M and Kaiser CA. A conserved GTPase-containing complex is required for
intracellular sorting of the general amino-acid permease in yeast. Nat Cell Biol 8: 657-
33. Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, Stocker H,
Kozma SC, Hafen E, Bos JL, and Thomas G. Insulin activation of Rheb, a mediator of
mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell 11: 1457-1466, 2003.
34. Gietzen DW, Hao S, and Anthony TG. Mechanisms of food intake repression in
indispensable amino acid deficiency. Annu Rev Nutr 27: 63-78, 2007.
35. Gromov PS, Madsen P, Tomerup N, and Celis JE. A novel approach for
expression cloning of small GTPases: identification, tissue distribution and chromosome
mapping of the human homolog of rheb. FEBS Lett 377: 221-226, 1995.
36. Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J,
Brown M, Fitzgerald KJ, and Sabatini DM. Ablation in mice of the mTORC
components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to
Akt-FOXO and PKCalpha, but not S6K1. Dev Cell 11: 859-871, 2006.
Drummond MJ and Rasmussen BB. Leucine-enriched nutrients and the
SC, Thomas AP, and Thomas G. Amino acids activate mTOR complex 1 via
Ca2+/CaM signaling to hVps34. Cell Metab 7: 456-465, 2008.
38. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez
DS, Turk BE, and Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic
checkpoint. Mol Cell 30: 214-226, 2008.
39. Hall DJ, Grewal SS, de la Cruz AF, and Edgar BA. Rheb-TOR signaling
promotes protein synthesis, but not glucose or amino acid import, in Drosophila. BMC
Biol 5: 10, 2007.
40. Hao S, Sharp JW, Ross-Inta CM, McDaniel BJ, Anthony TG, Wek RC,
Cavener DR, McGrath BC, Rudell JB, Koehnle TJ, and Gietzen DW. Uncharged
tRNA and sensing of amino acid deficiency in mammalian piriform cortex. Science 307:
41. Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, Tokunaga C,
Avruch J, and Yonezawa K. Raptor, a binding partner of target of rapamycin (TOR),
mediates TOR action. Cell 110: 177-189, 2002.
42. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, and Avruch J.
Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a
common effector mechanism. J Biol Chem 273: 14484-14494, 1998.
43. Heitman J, Movva NR, and Hall MN. Targets for cell cycle arrest by the
immunosuppressant rapamycin in yeast. Science 253: 905-909, 1991.
44. Hinnebusch AG. Translational regulation of GCN4 and the general amino acid
control of yeast. Annu Rev Microbiol 59: 407-450, 2005.
45. Hirose E, Nakashima N, Sekiguchi T, and Nishimoto T. RagA is a functional
homologue of S. cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway. J Cell Sci
111 ( Pt 1): 11-21, 1998.
46. Hsu YC, Chern JJ, Cai Y, Liu M, and Choi KW. Drosophila TCTP is essential
for growth and proliferation through regulation of dRheb GTPase. Nature 445: 785-788,
47. Iiboshi Y, Papst PJ, Kawasome H, Hosoi H, Abraham RT, Houghton PJ, and
Terada N. Amino acid-dependent control of p70(s6k). Involvement of tRNA
aminoacylation in the regulation. J Biol Chem 274: 1092-1099, 1999.
48. Ikenoue T, Inoki K, Yang Q, Zhou X, and Guan KL. Essential function of
TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. Embo J
27: 1919-1931, 2008.
49. Inoki K, Corradetti MN, and Guan KL. Dysregulation of the TSC-mTOR
pathway in human disease. Nat Genet 37: 19-24, 2005.
50. Inoki K, Li Y, Xu T, and Guan KL. Rheb GTPase is a direct target of TSC2
GAP activity and regulates mTOR signaling. Genes Dev 17: 1829-1834, 2003.
51. Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X, Yang Q, Bennett
C, Harada Y, Stankunas K, Wang CY, He X, MacDougald OA, You M, Williams
BO, and Guan KL. TSC2 integrates Wnt and energy signals via a coordinated
phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126: 955-968, 2006.
52. Inoki K, Zhu T, and Guan KL. TSC2 mediates cellular energy response to
control cell growth and survival. Cell 115: 577-590, 2003.
Gulati P, Gaspers LD, Dann SG, Joaquin M, Nobukuni T, Natt F, Kozma
essential for Raf-1 activation. J Biol Chem 275: 3737-3740, 2000.
54. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, and Hall MN.
Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.
Nat Cell Biol 6: 1122-1128, 2004.
55. Juhasz G, Hill JH, Yan Y, Sass M, Baehrecke EH, Backer JM, and Neufeld
TP. The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR
signaling in Drosophila. J Cell Biol 181: 655-666, 2008.
56. Kadowaki M, Karim MR, Carpi A, and Miotto G. Nutrient control of
macroautophagy in mammalian cells. Mol Aspects Med 27: 426-443, 2006.
57. Kanazawa T, Taneike I, Akaishi R, Yoshizawa F, Furuya N, Fujimura S, and
Kadowaki M. Amino acids and insulin control autophagic proteolysis through different
signaling pathways in relation to mTOR in isolated rat hepatocytes. J Biol Chem 279:
58. Keith CT and Schreiber SL. PIK-related kinases: DNA repair, recombination,
and cell cycle checkpoints. Science 270: 50-51, 1995.
59. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage
H, Tempst P, and Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive
complex that signals to the cell growth machinery. Cell 110: 163-175, 2002.
60. Kim DH, Sarbassov DD, Ali SM, Latek RR, Guntur KV, Erdjument-
Bromage H, Tempst P, and Sabatini DM. GbetaL, a positive regulator of the
rapamycin-sensitive pathway required for the nutrient-sensitive interaction between
raptor and mTOR. Mol Cell 11: 895-904, 2003.
61. Kim E, Goraksha-Hicks P, Li L, Neufeld TP, and Guan KL. Regulation of
TORC1 by Rag GTPases in nutrient response. Nat Cell Biol, 2008.
62. Kimball SR and Jefferson LS. New functions for amino acids: effects on gene
transcription and translation. Am J Clin Nutr 83: 500S-507S, 2006.
63. Krause U, Bertrand L, Maisin L, Rosa M, and Hue L. Signalling pathways and
combinatory effects of insulin and amino acids in isolated rat hepatocytes. Eur J Biochem
269: 3742-3750, 2002.
64. Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, and
Hall MN. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol
kinase homolog required for G1 progression. Cell 73: 585-596, 1993.
65. Kwiatkowski DJ and Manning BD. Tuberous sclerosis: a GAP at the crossroads
of multiple signaling pathways. Hum Mol Genet 14 Spec No. 2: R251-258, 2005.
66. LaLonde M, Janssens H, Yun S, Crosby J, Redina O, Olive V, Altshuller
YM, Choi SY, Du G, Gergen JP, and Frohman MA. A role for Phospholipase D in
Drosophila embryonic cellularization. BMC Dev Biol 6: 60, 2006.
67. Lam E, Martin M, and Wiederrecht G. Isolation of a cDNA encoding a novel
human FK506-binding protein homolog containing leucine zipper and tetratricopeptide
repeat motifs. Gene 160: 297-302, 1995.
68. Lindmo K and Stenmark H. Regulation of membrane traffic by
phosphoinositide 3-kinases. J Cell Sci 119: 605-614, 2006.
69. Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, Bonenfant D,
Oppliger W, Jenoe P, and Hall MN. Two TOR complexes, only one of which is
Inouye K, Mizutani S, Koide H, and Kaziro Y. Formation of the Ras dimer is
rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 10: 457-468,
70. Long X, Lin Y, Ortiz-Vega S, Busch S, and Avruch J. The Rheb switch 2
segment is critical for signaling to target of rapamycin complex 1. J Biol Chem 282:
71. Long X, Lin Y, Ortiz-Vega S, Yonezawa K, and Avruch J. Rheb binds and
regulates the mTOR kinase. Curr Biol 15: 702-713, 2005.
72. Long X, Ortiz-Vega S, Lin Y, and Avruch J. Rheb binding to mammalian
target of rapamycin (mTOR) is regulated by amino acid sufficiency. J Biol Chem 280:
73. Long X, Spycher C, Han ZS, Rose AM, Muller F, and Avruch J. TOR
deficiency in C. elegans causes developmental arrest and intestinal atrophy by inhibition
of mRNA translation. Curr Biol 12: 1448-1461, 2002.
74. Lynch CJ. Role of leucine in the regulation of mTOR by amino acids: revelations
from structure-activity studies. J Nutr 131: 861S-865S, 2001.
75. Lynch CJ, Fox HL, Vary TC, Jefferson LS, and Kimball SR. Regulation of
amino acid-sensitive TOR signaling by leucine analogues in adipocytes. J Cell Biochem
77: 234-251, 2000.
76. Lynch CJ, Gern B, Lloyd C, Hutson SM, Eicher R, and Vary TC. Leucine in
food mediates some of the postprandial rise in plasma leptin concentrations. Am J Physiol
Endocrinol Metab 291: E621-630, 2006.
77. Maurin AC, Jousse C, Averous J, Parry L, Bruhat A, Cherasse Y, Zeng H,
Zhang Y, Harding HP, Ron D, and Fafournoux P. The GCN2 kinase biases feeding
behavior to maintain amino acid homeostasis in omnivores. Cell Metab 1: 273-277, 2005.
78. Minami K, Tambe Y, Watanabe R, Isono T, Haneda M, Isobe K, Kobayashi
T, Hino O, Okabe H, Chano T, and Inoue H. Suppression of viral replication by stress-
inducible GADD34 protein via the mammalian serine/threonine protein kinase mTOR
pathway. J Virol 81: 11106-11115, 2007.
79. Mordier S, Deval C, Bechet D, Tassa A, and Ferrara M. Leucine limitation
induces autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes
through a mammalian target of rapamycin-independent signaling pathway. J Biol Chem
275: 29900-29906, 2000.
80. Nakashima N, Noguchi E, and Nishimoto T. Saccharomyces cerevisiae putative
G protein, Gtr1p, which forms complexes with itself and a novel protein designated as
Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p. Genetics 152:
81. Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P, Byfield MP,
Backer JM, Natt F, Bos JL, Zwartkruis FJ, and Thomas G. Amino acids mediate
mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase.
Proc Natl Acad Sci U S A 102: 14238-14243, 2005.
82. Nojima H, Tokunaga C, Eguchi S, Oshiro N, Hidayat S, Yoshino K, Hara K,
Tanaka N, Avruch J, and Yonezawa K. The mammalian target of rapamycin (mTOR)
partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR
signaling (TOS) motif. J Biol Chem 278: 15461-15464, 2003.
protein response by GADD34-mediated dephosphorylation of eIF2alpha. J Cell Biol 153:
84. Oldham S, Montagne J, Radimerski T, Thomas G, and Hafen E. Genetic and
biochemical characterization of dTOR, the Drosophila homolog of the target of
rapamycin. Genes Dev 14: 2689-2694, 2000.
85. Oshiro N, Yoshino K, Hidayat S, Tokunaga C, Hara K, Eguchi S, Avruch J,
and Yonezawa K. Dissociation of raptor from mTOR is a mechanism of rapamycin-
induced inhibition of mTOR function. Genes Cells 9: 359-366, 2004.
86. Pacold ME, Suire S, Perisic O, Lara-Gonzalez S, Davis CT, Walker EH,
Hawkins PT, Stephens L, Eccleston JF, and Williams RL. Crystal structure and
functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell
103: 931-943, 2000.
87. Patel PH, Thapar N, Guo L, Martinez M, Maris J, Gau CL, Lengyel JA, and
Tamanoi F. Drosophila Rheb GTPase is required for cell cycle progression and cell
growth. J Cell Sci 116: 3601-3610, 2003.
88. Pearson RB, Dennis PB, Han JW, Williamson NA, Kozma SC, Wettenhall
RE, and Thomas G. The principal target of rapamycin-induced p70s6k inactivation is a
novel phosphorylation site within a conserved hydrophobic domain. Embo J 14: 5279-
89. Roccio M, Bos JL, and Zwartkruis FJ. Regulation of the small GTPase Rheb
by amino acids. Oncogene 25: 657-664, 2006.
90. Roggo L, Bernard V, Kovacs AL, Rose AM, Savoy F, Zetka M, Wymann
MP, and Muller F. Membrane transport in Caenorhabditis elegans: an essential role for
VPS34 at the nuclear membrane. Embo J 21: 1673-1683, 2002.
91. Roh C, Han J, Tzatsos A, and Kandror KV. Nutrient-sensing mTOR-mediated
pathway regulates leptin production in isolated rat adipocytes. Am J Physiol Endocrinol
Metab 284: E322-330, 2003.
92. Rosner M, Hofer K, Kubista M, and Hengstschlager M. Cell size regulation by
the human TSC tumor suppressor proteins depends on PI3K and FKBP38. Oncogene 22:
93. Rubio-Texeira M and Kaiser CA. Amino acids regulate retrieval of the yeast
general amino acid permease from the vacuolar targeting pathway. Mol Biol Cell 17:
94. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L,
and Sabatini DM. The Rag GTPases bind raptor and mediate amino acid signaling to
mTORC1. Science 320: 1496-1501, 2008.
95. Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E,
Carr SA, and Sabatini DM. PRAS40 is an insulin-regulated inhibitor of the mTORC1
protein kinase. Mol Cell 25: 903-915, 2007.
96. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-
Bromage H, Tempst P, and Sabatini DM. Rictor, a novel binding partner of mTOR,
defines a rapamycin-insensitive and raptor-independent pathway that regulates the
cytoskeleton. Curr Biol 14: 1296-1302, 2004.
Novoa I, Zeng H, Harding HP, and Ron D. Feedback inhibition of the unfolded
Markhard AL, and Sabatini DM. Prolonged rapamycin treatment inhibits mTORC2
assembly and Akt/PKB. Mol Cell 22: 159-168, 2006.
98. Sarbassov DD, Guertin DA, Ali SM, and Sabatini DM. Phosphorylation and
regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101, 2005.
99. Saucedo LJ, Gao X, Chiarelli DA, Li L, Pan D, and Edgar BA. Rheb promotes
cell growth as a component of the insulin/TOR signalling network. Nat Cell Biol 5: 566-
100. Schalm SS and Blenis J. Identification of a conserved motif required for mTOR
signaling. Curr Biol 12: 632-639, 2002.
101. Schalm SS, Fingar DC, Sabatini DM, and Blenis J. TOS motif-mediated raptor
binding regulates 4E-BP1 multisite phosphorylation and function. Curr Biol 13: 797-806,
102. Schliess F, Richter L, vom Dahl S, and Haussinger D. Cell hydration and
mTOR-dependent signalling. Acta Physiol (Oxf) 187: 223-229, 2006.
103. Sekiguchi T, Hirose E, Nakashima N, Ii M, and Nishimoto T. Novel G
proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B. J Biol
Chem 276: 7246-7257, 2001.
104. Sekiguchi T, Todaka Y, Wang Y, Hirose E, Nakashima N, and Nishimoto T.
A novel human nucleolar protein, Nop132, binds to the G proteins, RRAG A/C/D. J Biol
Chem 279: 8343-8350, 2004.
105. Shi CS, Huang NN, Harrison K, Han SB, and Kehrl JH. The mitogen-
activated protein kinase kinase kinase kinase GCKR positively regulates canonical and
noncanonical Wnt signaling in B lymphocytes. Mol Cell Biol 26: 6511-6521, 2006.
106. Shi CS, Leonardi A, Kyriakis J, Siebenlist U, and Kehrl JH. TNF-mediated
activation of the stress-activated protein kinase pathway: TNF receptor-associated factor
2 recruits and activates germinal center kinase related. J Immunol 163: 3279-3285, 1999.
107. Shigemitsu K, Tsujishita Y, Hara K, Nanahoshi M, Avruch J, and Yonezawa
K. Regulation of translational effectors by amino acid and mammalian target of
rapamycin signaling pathways. Possible involvement of autophagy in cultured hepatoma
cells. J Biol Chem 274: 1058-1065, 1999.
108. Shigemitsu K, Tsujishita Y, Miyake H, Hidayat S, Tanaka N, Hara K, and
Yonezawa K. Structural requirement of leucine for activation of p70 S6 kinase. FEBS
Lett 447: 303-306, 1999.
109. Shirane M and Nakayama KI. Inherent calcineurin inhibitor FKBP38 targets
Bcl-2 to mitochondria and inhibits apoptosis. Nat Cell Biol 5: 28-37, 2003.
110. Smith EM, Finn SG, Tee AR, Browne GJ, and Proud CG. The tuberous
sclerosis protein TSC2 is not required for the regulation of the mammalian target of
rapamycin by amino acids and certain cellular stresses. J Biol Chem 280: 18717-18727,
111. Sofer A, Lei K, Johannessen CM, and Ellisen LW. Regulation of mTOR and
cell growth in response to energy stress by REDD1. Mol Cell Biol 25: 5834-5845, 2005.
112. Stipanuk MH. Leucine and protein synthesis: mTOR and beyond. Nutr Rev 65:
Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF,
Breuer S, Thomas G, and Hafen E. Rheb is an essential regulator of S6K in controlling
cell growth in Drosophila. Nat Cell Biol 5: 559-565, 2003.
114. Sun Y, Fang Y, Yoon MS, Zhang C, Roccio M, Zwartkruis FJ, Armstrong
M, Brown HA, and Chen J. Phospholipase D1 is an effector of Rheb in the mTOR
pathway. Proc Natl Acad Sci U S A 105: 8286-8291, 2008.
115. Tee AR, Manning BD, Roux PP, Cantley LC, and Blenis J. Tuberous sclerosis
complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a
GTPase-activating protein complex toward Rheb. Curr Biol 13: 1259-1268, 2003.
116. Todaka Y, Wang Y, Tashiro K, Nakashima N, Nishimoto T, and Sekiguchi T.
Association of the GTP-binding protein Gtr1p with Rpc19p, a shared subunit of RNA
polymerase I and III in yeast Saccharomyces cerevisiae. Genetics 170: 1515-1524, 2005.
117. Urano J, Comiso MJ, Guo L, Aspuria PJ, Deniskin R, Tabancay AP, Jr.,
Kato-Stankiewicz J, and Tamanoi F. Identification of novel single amino acid changes
that result in hyperactivation of the unique GTPase, Rheb, in fission yeast. Mol Microbiol
58: 1074-1086, 2005.
118. Wang H, Kubica N, Ellisen LW, Jefferson LS, and Kimball SR.
Dexamethasone represses signaling through the mammalian target of rapamycin in
muscle cells by enhancing expression of REDD1. J Biol Chem 281: 39128-39134, 2006.
119. Wang Y, Nakashima N, Sekiguchi T, and Nishimoto T. Saccharomyces
cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through
Saccharomyces cerevisiae Ran-binding protein, Yrb2p. Biochem Biophys Res Commun
336: 639-645, 2005.
120. Watanabe R, Tambe Y, Inoue H, Isono T, Haneda M, Isobe K, Kobayashi T,
Hino O, Okabe H, and Chano T. GADD34 inhibits mammalian target of rapamycin
signaling via tuberous sclerosis complex and controls cell survival under bioenergetic
stress. Int J Mol Med 19: 475-483, 2007.
121. Weng QP, Andrabi K, Kozlowski MT, Grove JR, and Avruch J. Multiple
independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol 15:
122. Weng QP, Kozlowski M, Belham C, Zhang A, Comb MJ, and Avruch J.
Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific
anti-phosphopeptide antibodies. J Biol Chem 273: 16621-16629, 1998.
123. Wu B, Ottow K, Poulsen P, Gaber RF, Albers E, and Kielland-Brandt MC.
Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p.
J Cell Biol 173: 327-331, 2006.
124. Yamagata K, Sanders LK, Kaufmann WE, Yee W, Barnes CA, Nathans D,
and Worley PF. rheb, a growth factor- and synaptic activity-regulated gene, encodes a
novel Ras-related protein. J Biol Chem 269: 16333-16339, 1994.
125. Zhang H, Stallock JP, Ng JC, Reinhard C, and Neufeld TP. Regulation of
cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev 14: 2712-2724,
126. Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, and Pan D. Rheb is a direct
target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 5: 578-581,
Stocker H, Radimerski T, Schindelholz B, Wittwer F, Belawat P, Daram P,
Tor signaling requires a functional class C Vps protein complex in Saccharomyces
cerevisiae. Genetics 176: 2139-2150, 2007.
Zurita-Martinez SA, Puria R, Pan X, Boeke JD, and Cardenas ME. Efficient
Figure 1. Candidate mechanisms for Rheb-GTP activation of mTORC1.
The segment of mTOR from ~AA1967-2500 is pictured; the cleft in the rectangle divides
the catalytic domain into upper (2147-2300) and lower (2301-2430) lobes. The loop
represents the FKBP12/rapamycin binding (FRB) domain (AA 2014-2115). Insulin and
growth factors promote Rheb GTP charging by inhibition of the Tuberous Sclerosis
Complex, a Rheb GTPase activator, as illustrated in c. Three models for mTORC1
activation by Rheb-GTP are illustrated. In a Rheb binds to the upper lobe of the catalytic
domain and when GTP charged, enables mTOR activation. Amino acid withdrawal,
through an effect on the lower lobe, interferes with Rheb binding to the upper lobe. In b
the ability of Rheb-GTP to promote activation of phospholipase D1 generates
phosphatidic acid (PA), which binds to the FRB domain, promoting activation. In c the
Rheb-GTP binds to the mTOR inhibitor, FKBP38, competing with and displacing it from
mTOR, disinhibiting the mTOR kinase activity. The site of action of amino acids in b
and c is unknown.
Figure 2. A possible mechanism of action of the rag GTPases in amino acid
regulation of mTORC1. The rag GTPases exist as an obligatory heterodimer. GTP
charging of the ragA/B partner, possibly dependent on amino acid sufficiency, promotes
the association of the rag heterodimer with mTORC1 and its translocation to a membrane
compartment enriched in Rheb, thereby enabling mTORC1 activation when Rheb is GTP
charged. Based on data in refs. 61 and 93; see text for details.
RAG A/B Download full-text