You are on page 1of 9

Cellular Signalling 21 (2009) 827–835

Contents lists available at ScienceDirect

Cellular Signalling
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c e l l s i g

Review

Mammalian target of rapamycin complex 1: Signalling inputs, substrates and


feedback mechanisms
E.A. Dunlop, A.R. Tee ⁎
Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, Wales, CF14 4XN, UK

a r t i c l e i n f o a b s t r a c t

Article history: The mammalian target of rapamycin (mTOR) signalling pathway is implicated in the pathogenesis of a
Received 9 December 2008 number of cancers and inherited hamartoma syndromes which have led to mTOR inhibitors, such as
Accepted 2 January 2009 rapamycin, being tested in clinical trials. Knowledge of the mTOR pathway is rapidly expanding. This review
Available online 8 January 2009
provides an update on the most recent additions to the mTOR pathway with particular emphasis on mTORC1
signalling. mTORC1 signalling is classically known for its role in regulating cell growth and proliferation
Keywords:
mTOR
through modulation of protein synthesis. Recent research has identified novel mTORC1 cell signalling
Rheb mechanisms that modulate mitochondrial biogenesis, hypoxia signalling and cell cycle progression and
TSC uncovered novel mTORC1 targets; YY1, HIF and SGK1. It is unsurprising that regulation of mTORC1 is
HIF multifaceted with many positive and negative signalling inputs. We discuss the recent advances that have
YY1 been made to determine the upstream mechanisms that control mTORC1 through hypoxia, energy sensing
SGK1 and nutrient signalling. Also discussed are current findings that have unravelled a series of novel mTORC1-
PRAS40 associated proteins that directly control the activity of mTORC1 and include PRAS40, FKBP38, Rag GTPases
FKBP38
and RalA.
4E-BP1
© 2009 Elsevier Inc. All rights reserved.
S6K1

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
2. mTOR structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
2.1. mTORC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
2.2. mTORC1 associated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
2.3. New mTORC1 substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
2.3.1. YY1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
2.3.2. STAT3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
2.3.3. SGK1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
2.3.4. Hypoxia Inducible Factor (HIF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
3. Upstream control of mTORC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
3.1. Hypoxic input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
3.2. Energy sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
3.3. Mitogenic stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
3.3.1. Phosphatidic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
3.3.2. MAPKAPK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
3.4. Nutrient input into mTORC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
3.4.1. Vps34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
3.4.2. MAP4K3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
3.4.3. Rag GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
3.4.4. RalA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
4. mTORC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833

⁎ Corresponding author. Tel.: +44 29 2074 4055; fax: +44 29 2074 6551.
E-mail address: TeeA@cardiff.ac.uk (A.R. Tee).

0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellsig.2009.01.012
828 E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835

5. Feedback in the mTOR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834


6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834

1. Introduction PI3K/Akt and Ras/MAPK cell signalling pathways regulate mTOR


signalling through TSC2, where Akt (also referred to as PKB) or Rsk,
Mammalian target of rapamycin (mTOR) is a central signalling within the Ras/MAPK pathway, phosphorylates TSC2 on over-lapping
molecule which is upregulated in various cancers and hamartoma and distinct residues that lead to TSC1–TSC2 inactivation. Erk has also
syndromes. Aberrant mTOR signalling in tumours is due to either loss been shown to phosphorylate TSC2, suppressing TSC2 function by
of function of upstream tumour suppressor proteins or activating disrupting the TSC1–TSC2 heterodimer [13]. Within the PI3K/Akt
mutations within oncogenes that feed into the mTOR pathway. The pathway, PTEN functions as a tumour suppressor by directly opposing
outcome in both situations is a propensity for increased cell growth the activity of PI3K through dephosphorylating phosphatidylinositol-
and proliferation. The specific mTOR inhibitor rapamycin, or its 3,4,5-triphosphate (PIP3) [14]. Therefore, loss of function of PTEN,
derivatives, have now been used in a number of clinical trials [1–6], which causes Cowden disease [15,16], leads to an accumulation of PIP3
and while some patients clearly benefited from treatment, efficacy and constitutive activation of downstream PI3K signalling events that
within and between trials has been variable. This observed variability feed onto mTOR through TSC2. Another hamartoma syndrome,
is likely due to the complexity of mTOR signalling involving negative Neurofibromatosis Type 1, is caused by loss of function mutations of
feedback mechanisms and rapamycin-induced inhibition of mTOR- a Ras-GAP called NF1 [17]. Consequently, NF1 inactivation heightens
regulated cellular processes that we do not yet fully understand. signal transduction through the Ras/MAPK pathway and the PI3K/
Studying inherited hamartoma syndromes, where upregulation of Akt/mTOR pathway via cross-talk from Ras. This explains the high
mTOR signalling plays a pivotal role in the pathology of the disease, levels of TSC2 phosphorylation and constitutive mTOR activation
has allowed the complex interplay of many signal transduction identified in NF1-deficient primary cells and human tumours [18].
components that regulate mTOR to be better characterised. Further Both these mitogenically regulated PI3K and MAPK signalling
studies will no doubt lead to the identification of additional drug pathways activate mTOR via impairment of TSC2 function. However,
targets or potential combinatorial drug therapies which could be the energy sensing AMP-dependent protein kinase (AMPK) inhibits
tested in new clinical trials. mTOR signal transduction by phosphorylating and activating TSC2
There are a number of hamartoma syndromes which result from a [19]. AMPK is itself activated by the serine/threonine kinase LKB1/
mutation in a tumour suppressor functioning upstream of mTOR. Tsc1 STK11 which phosphorylates the AMPK α-subunit on Thr172 [20].
and Tsc2 are both classed as tumour suppressors and were originally AMPK-dependent phosphorylation of TSC2 on Thr1227 and Ser1345 [21]
identified as the genes responsible for the hamartoma condition is lost when inactivating mutations within LKB1 occur, giving rise to
Tuberous Sclerosis Complex (TSC) [7,8]. The TSC1 and TSC2 hetero- Peutz–Jeghers Syndrome [22].
dimer inhibits mTOR signalling by acting as a GTPase activating More recently the hamartoma disorder, Birt–Hogg–Dubé (BHD)
protein (GAP) towards the small GTPase Ras homolog enriched in syndrome was linked to the mTOR and AMPK pathways via the BHD
brain (Rheb) [9–12]. Rheb potently activates mTOR when in a GTP- gene product Folliculin (FLCN). Both FLCN and two recently identified
bound state and TSC1–TSC2 inhibits mTOR indirectly by reverting FLCN interacting proteins (FNIP1 and FNIP2) were suggested to be
Rheb to an inactive GDP-bound form [9]. Numerous signalling involved in nutrient and energy sensing through AMPK and mTOR
pathways converge on TSC2, which places the TSC1–TSC2 hetero- [23–25], where FLCN and FNIP1 are phosphorylated in an AMPK- and
dimer as a central coordinator of mTOR signal transduction. Both the mTOR-dependent manner. Linking these syndromes to mTOR, which

Fig. 1. Tumour suppressors feeding into mTORC1. mTORC1 coordinates a number of mitogenic and energy sensing pathways. The tumour suppressors mutated in various hamartoma
syndromes are highlighted.
E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835 829

is a critical signalling pathway responsible for controlling cell growth, a phenylalanine followed by two hydrophobic and two acidic residues.
may help explain why mutations within these tumour suppressors It was discovered that this motif was essential for interaction of Raptor
cause the hamartoma phenotype. Fig. 1 illustrates how these tumour with mTORC1 substrates [47,48]. It is possible that other putative
suppressors regulate the mTOR pathway. mTORC1 substrates may possess a TOS motif to facilitate Raptor
binding. Raptor may make multiple interactions with mTORC1
2. mTOR structure and function substrates. Indeed, in addition to its TOS motif, 4E-BP1 also contains
a RAIP motif which is necessary for efficient phosphorylation of this
mTOR is a member of the phosphoinositide 3-kinase related kinase substrate by mTORC1 in vivo [49] and was later shown to regulate
(PIKK) family, whose catalytic domain has significant amino acid Raptor interaction [50].
homology to that of the phosphoinositide 3-kinases (PI3Ks) [26].
Unlike PI3K family members which phosphorylate lipids, the PIKK 2.2. mTORC1 associated proteins
family, of which there are six mammalian members (ATM, ATR, DNA-
PKcs, mTOR, SMG1 and TRRAP) function as serine/threonine kinases, The small G-protein Rheb activates mTORC1 when it is GTP-bound
with the exception of the catalytically inactive TRRAP [27]. Like other [11] and farnesylated [12]. While activation of mTORC1 requires GTP-
PIKK family members, mTOR contains a number of HEAT repeats bound Rheb, the interaction of Rheb with mTOR is independent of the
within its N-terminus (named after Huntingtin, Elongation factor 3, A nucleotide binding state of Rheb [51,52]. Rheb has also been reported
subunit of protein phosphatase 2A, and TOR1, the proteins in which to bind to other mTORC1 components in vitro, namely directly to
the repeat was first identified) [28]. mTOR also possesses a FRAP, ATM mLST8 and to the carboxylterminal WD propeller segment of Raptor
and TRRAP (FAT) domain and a FAT C-terminal (FATC) domain [29], [53]. As these Rheb interaction studies were carried out with over-
named after the family members who contain these motifs. Both HEAT expressed proteins, it remains to be determined whether this holds
and FAT domains are distinct functional motifs thought to mediate true in vivo.
protein interactions. Unlike other PIKK family members, mTOR More recently, FKBP38 and proline-rich Akt substrate of 40 kDa
possesses a FRB (FKBP12-rapamycin binding) domain that is the (PRAS40) were identified as additional components of mTORC1.
minimal region required to bind FKBP12/rapamycin (a specific mTOR FKBP38 belongs to the same protein family as FKBP12, which interacts
inhibitor) and lies immediately N-terminal to the kinase domain [30]. with the FRB domain of mTOR as a FKBP12/rapamycin complex
Recent evidence has also indicated the presence of a nuclear shuttling [54,55]. Similarly, FKBP38 binds to the FRB domain of mTOR and
signal within mTOR involving Leu545 and Leu547 [31], although the inhibits the ability of mTORC1 to signal to downstream targets. Unlike
mechanism behind mTOR nuclear transport is yet to be elucidated. FKBP12, the FKBP38 inhibition does not require the presence of
mTOR is found in two cellular complexes, termed mTOR complex 1 rapamycin. The mTORC1-FKBP38 interaction is strengthened in cells
(mTORC1) and mTORC2. deprived of amino acids or serum [56]. It was shown that active Rheb-
GTP binds FKBP38 with a higher affinity than Rheb-GDP [56] and the
2.1. mTORC1 GTP-bound form prevents the FKBP38-mTORC1 interaction, thus
relieving the inhibition of mTORC1 [56]. Further analysis revealed
The core component of mTORC1 is the regulatory associated that the switch I region of Rheb was critical for FKBP38 interaction
protein of mTOR (Raptor) that is thought to bind to the HEAT repeats [57]. The FKBP38-mTOR and FKBP38-Rheb interactions were con-
within mTOR [32]. mTORC1 also contains lethal with sec13 protein 8 firmed by another group, but with some contrasting observations [58].
(LST8, also known as GβL), which was identified as a Tor interactor in They reported that the FKBP38 interaction with Rheb did not differ
both yeast [33,34] and mammalian cells [35] and plays a role in depending on whether Rheb was GDP or GTP-bound, nor was the
stabilising mTOR–Raptor interactions [35]. FKBP38 interaction with mTOR altered upon nutrient withdrawal [58].
The first described and best characterised substrates of mTORC1 They, and others, have not found any evidence of FKBP38 inhibiting
are eukaryotic initiation factor 4E-binding proteins 1, 2 and 3 (4E-BP1, the phosphorylation of mTORC1 substrates when FKBP38 is over-
2 and 3) [36,37] and the p70 S6 kinases (S6K1 and S6K2) [38]. 4E-BPs expressed in mammalian cells [58,59]. Such conflicting data will
function as translation repressors when unphosphorylated. mTORC1 hopefully be resolved as other groups study the interaction.
phosphorylation of 4E-BP1 on four Ser/Thr residues causes 4E-BP1 to The precise role of PRAS40 within the mTORC1 signalling pathway is
dissociate from eIF4E which is bound to the m7GpppN cap moiety also subject to debate as it has been described as either an mTORC1
located at the 5′-end of mRNAs. Through repression of 4E-BP1, mTOR inhibitor [60,61] or mTORC1 substrate [62,63] or both [64]. It is clear that
drives cell growth and proliferation by enhancing eIF4E-mediated PRAS40 contains a TOS motif which is important for its interaction with
translation of these mRNAs. S6K1 also regulates cell growth and Raptor, and mutations to key residues within this motif adversely affect
proliferation, which is dependent on mTORC1 signalling [39,40]. Full binding [61–63]. In vivo, the PRAS40–Raptor interaction was shown to
activation of S6K1 requires Thr389 phosphorylation within the linker require mTOR [61], while in vitro interactions revealed PRAS40 binding
region of S6K1 by mTORC1. Although S6K1 was first identified as a to the mTOR kinase domain [65]. Such evidence suggests multiple
kinase of ribosomal protein S6 (rpS6), the functional purpose of rpS6 protein–protein interactions between PRAS40 and both mTOR and
phosphorylation by S6K1 is still unresolved. Further targets of S6K1 Raptor. It is known that amino acid or glucose withdrawal strengthens
include SKAR [41] which recruits activated S6K1 to the exon-junction the mTOR–Raptor association [32]. This trend is also observed with
complex of mRNAs thereby enhancing translation efficiency gained by PRAS40–Raptor binding, as the association is enhanced in conditions of
splicing [42]. Another target is eIF4B which becomes phosphorylated amino acid withdrawal [63] or glucose withdrawal [65]. PRAS40 is
on Ser422 by S6K1, increasing its association with eIF3 [43,44]. S6K1 phosphorylated on Thr246 by Akt and this modification is known to be
also phosphorylates eukaryotic elongation factor 2 kinase (eEF2K). important for mediating mTOR activation by insulin [65]. Akt phosphor-
Phosphorylation of eEF2K by S6K1 renders eEF2K less active, resulting ylation of PRAS40 allows it to bind 14-3-3 protein [66], although the
in the dephosphorylation and activation of its substrate eEF2 and physiological significance of 14-3-3 binding to phosphorylated PRAS40
promotion of the elongation phase of protein synthesis [45]. Thus by is not yet known. It was postulated that 14-3-3 binding to the Akt-
acting through a number of downstream targets, 4E-BPs and S6Ks phosphorylated Thr246 site sequestered PRAS40 away from mTORC1,
influence the level of protein synthesis in cells. thus relieving any inhibitory action that PRAS40 has on mTORC1.
Analysis of the amino acid composition of S6K and 4E-BP proteins However, an in vitro mTORC1 kinase assay in the presence of 14-3-3
revealed a common motif within their structure, known as the TOR revealed that the binding of 14-3-3 to PRAS40 was not necessary to
signalling (TOS) motif [46]. This is a five amino acid motif consisting of relieve the inhibitory action of PRAS40 on mTORC1 [60].
830 E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835

Studies implicated PRAS40 as a negative regulator of mTORC1 as mTORC1 substrate. More recent work revealed that the expression
they revealed that PRAS40 inhibited mTORC1-mediated phosphor- of mitochondrial genes is controlled by mTORC1 [69]. These
ylation of 4E-BP1 [64] and S6K1 [60] in vitro, and inhibited S6K1 investigations into mitochondrial biogenesis led to the discovery
phosphorylation when PRAS40 was over-expressed in cells [60]. that the activity of YY1 (Yin Yang 1), which is a zinc-finger GLI-
Furthermore, the interaction of PRAS40 with mTORC1 was reduced Kruppel class of transcription factor [70], was regulated by mTORC1
upon activation of the PI3K/Akt pathway via insulin stimulation [69]. Both mTOR and Raptor were shown to associate with YY1, with
[61,65]. This finding strengthens the idea that PRAS40 inhibits the interactions requiring both the amino and carboxy termini of YY1
mTORC1 as disruption of mTORC1–PRAS40 binding via PI3K/Akt [69] revealing that YY1 could be a direct substrate of mTORC1.
signalling would lead to higher levels of mTORC1 activity. However, Peroxisome-proliferator-activated receptor coactivator (PGC)-1α is
to contradict this viable mechanism, the mTORC1 inhibitor rapa- known to regulate the expression of many mitochondrial genes and
mycin also decreases the association of PRAS40 with mTORC1 com- was found to function as a transcriptional coactivator for YY1 in an
ponents [61,63]. Rapamycin is known to weaken the interaction of mTOR-dependent manner, with mTOR appearing to regulate the
mTOR–Raptor complexes [32] so it was suggested that the decrease function of YY1 and PGC-1α by directly altering their physical
in PRAS40–mTOR affinity following rapamycin treatment was interaction [69]. The model of action proposed by the authors is that
caused through destabilisation of the mTOR–Raptor complex [65]. a decrease in mTOR activity inhibits YY1-PGC-1α function, leading to
This would fit with the model that Raptor recruits substrates to decreased expression of mitochondrial genes [69].
mTORC1, and so loss of Raptor binding disrupts the recruitment of
PRAS40 to mTORC1. Additionally, there are conflicting reports over 2.3.2. STAT3
whether the TOS mutant of PRAS40, which cannot bind Raptor still Signal transducer and activator of transcription 3 (STAT3) is a
inhibits mTORC1-mediated phosphorylation of 4E-BP1 [61,62]. transcription factor which is activated by cytokine stimulation of
Experiments involving PRAS40 small interfering RNA (siRNA) certain receptor tyrosine kinases (RTK), such as epidermal growth
have also yielded contradictory results, with some indicating factor receptor, or non-RTKs via the Janus kinase family [71]. Cytokine
PRAS40 knockdown leads to increased phosphorylation of S6K1 stimulation results in phosphorylation of STAT3 on Tyr705, but
and 4E-BP1 [63], while others report a decrease in phosphorylation additional phosphorylation on Ser727 is required for full transcrip-
of mTORC1 substrates [62]. tional activity of STAT3 [71]. An investigation into the kinase
Other groups have shown that PRAS40 is a substrate for mTORC1, responsible for the phosphorylation of STAT3 on Ser727 revealed that
being phosphorylated at Ser183 both in vitro and in vivo [63]. This this phosphorylation event could be inhibited by rapamycin. A STAT3
mTORC1-mediated phosphorylation is reduced by mutation of the peptide was efficiently phosphorylated on Ser727 by mTOR, but not by
TOS motif [62]. Phosphopeptide mapping revealed two further a kinase-inactive mTOR mutant or S6K1 [72], thus directly implicating
mTORC1 phosphorylation sites on PRAS40 at Ser212 and Ser221 [67]. mTOR in STAT3 activation. mTOR and STAT3 have also been shown to
mTORC1-mediated PRAS40 phosphorylation is positively influenced associate in neural stem/progenitor cells [73]. STAT3 contains two
by Rheb and negatively affected by TSC1–TSC2, just like other mTORC1 putative TOS motifs [50], indicating a possible binding site for Raptor,
substrates [63]. However, there is conflicting data over whether although this has not yet been confirmed experimentally. STAT3 might
PRAS40 and other TOS-containing substrates compete for binding to also be indirectly regulated by mTOR based on evidence from shRNA
Raptor [61–63]. Mutation of Ser221 to Ala increases the inhibitory experiments, where knockdown of mTOR reduced the level of STAT3
activity of PRAS40 towards mTORC1, leading the authors of this study expression in MCF7 breast cancer cells [74].
to propose that after mTORC1 activation by upstream regulators,
PRAS40 is phosphorylated directly by mTOR, thus contributing to the 2.3.3. SGK1
relief of PRAS40-mediated substrate competition [67]. It has been known for a long time that rapamycin treatment
Such differing reports over the exact role of PRAS40 within the upregulates the cyclin-dependent kinase inhibitor, p27 (KIP1), leading
mTORC1 pathway may have arisen because of the varying techniques to G1 arrest. However, the exact role of mTOR within this process was
used by the authors (in vivo and in vitro analysis), as some may be not understood. A recent report identified the serum- and glucocorti-
more physiological than others. Over-expression of PRAS40 may be coid-inducible kinase (SGK1) as a potential mTORC1 substrate [75]
contributing to non-physiological observations, and therefore further that regulates cell cycle progression through its action on p27. The
in vivo studies may help to clarify the role of PRAS40 and solve some of authors showed that SGK1 bound both mTOR and Raptor where this
the discrepancies in the literature. association did not require a putative TOS motif or prior SGK1
phosphorylation [75]. mTOR-induced SGK1 phosphorylation at Ser422,
2.3. New mTORC1 substrates within the hydrophobic motif led to phosphorylation of p27 at Thr157
and was rapamycin sensitive [75]. Phosphorylation and mislocalisa-
It was discovered that mTOR has a wider function in cellular tion of the SGK1 substrate p27 in mTOR-overexpressing cells were
processes than just being restricted to controlling protein synthesis. To attenuated when either Raptor or Rictor expression was knocked
participate in these cellular events, it must signal through substrates down [75], suggesting that both mTORC1 and mTORC2 are involved in
other than S6K and 4E-BP. Current research in this area has uncovered modulating SGK1.
potential mTORC1 substrates which link the complex to mitochondrial In contrast, another study established mTORC2 as the cellular
biogenesis, cell cycle progression and hypoxia. mediator of SGK1 phosphorylation as they found phosphorylation
of the hydrophobic motif and SGK1 activity was ablated in cells
2.3.1. YY1 with intact mTORC1 signalling but compromised mTORC2 signal-
The activity of mTORC1 was shown to directly correlate with ling [76]. Additionally, they report that SGK1 phosphorylation was
mitochondrial metabolism [68]. The rates of oxygen consumption and resistant to rapamycin treatment and that immunoprecipitated
the mitochondrial oxidative capacity were shown to be down mTORC2 phosphorylated SGK1 at Ser422 in vitro. They highlight that
regulated when mTORC1 was inhibited with rapamycin. S6K1 was reduced phosphorylation of SGK1 substrates, such as FOXO3, is
shown not to be responsible as mitochondrial metabolism was observed in mTORC2-deficient cells [77] and is an outcome that is
unaltered when S6K1 expression was knocked down in cells by explained by mTORC2-mediated activation of SGK1. Further re-
siRNA [68]. Given that mTOR and Raptor were present in mitochon- search into this area should clarify whether SGK1 is a substrate of
drial fractions [68], it was postulated that mTORC1 might regulate mTORC1 or mTORC2, or indeed whether both complexes play a role
mitochondrial metabolism via a novel mitochondria-localised in its regulation.
E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835 831

2.3.4. Hypoxia Inducible Factor (HIF) 3. Upstream control of mTORC1


HIF-α is stabilised under hypoxic conditions, allowing it to bind
HIF-β (also called aryl hydrocarbon receptor nuclear translocator 3.1. Hypoxic input
(Arnt)). As a dimer, HIF-α/HIF-β translocates to the nucleus and
induces expression of genes involved in angiogenesis, erythropoiesis, Hypoxia downregulates mTORC1 signalling and was thought to be
and glucose metabolism. As patients with mTOR-associated hamar- due to a mechanism that occurred independently of HIF [90]. Indeed,
toma diseases develop highly vascularised tumours it is likely that an it is evident that inhibition of mTOR by serum-withdrawal is more
increased level of HIF-mediated gene expression occurs. A number of rapid under hypoxia and reveals a negative input from hypoxia to
studies have identified the mTOR pathway as a positive regulator of mTOR [91]. Inhibition of mTOR is also indirectly regulated by HIF
HIF-1α, with evidence for regulation at the level of transcription, through Redd1 and Redd2 (also known as RTP801 and RTP801L) as
translation and protein stability. expression of both Redd1 and Redd2 are upregulated by HIF. Redd1 is
There is a question over whether HIF is regulated directly by mTOR, necessary for hypoxia-induced down-regulation of mTOR where this
or indirectly, via the mTORC1 substrate, S6K1. A correlative study downregulation requires functional TSC2 [85]. When over-expressed,
showed that rapamycin selectively suppressed translation of mRNAs Redd1 and Redd2 proteins inhibit the phosphorylation of the mTORC1
containing a 5′ terminal polypyrimidine tract (5′TOP) as well as signalling targets, 4E-BP1 and S6K1, and so it was postulated that
reducing rpS6 phosphorylation [78]. From this work, it was postulated Redd1 and Redd2 activated TSC2 within cells [85,92]. Further work
that rpS6 controlled the translation of 5′TOP mRNAs, which was up- revealed that Redd1 bound to 14-3-3 proteins. 14-3-3 binding to TSC2
regulated when rpS6 was phosphorylated by S6K1. As the 5′ untrans- is known to inactivate the TSC1-TSC2 heterodimer. The 14-3-3 binding
lated region (UTR) of HIF-1α has tracts of 8, 9 and 17 pyrimidines sites at Ser939 and Ser981 within TSC2 are Akt-mediated phosphoryla-
downstream of nucleotide +32 [79] it was proposed that S6K1 might tion sites so 14-3-3/TSC2 complexes occur after mitogenic activation
also regulate HIF-1α translation. More recent reports from a mouse of Akt [93]. It is possible that Redd1 activates TSC2 by competing for
S6K1−/− ES cell line, lymphoblastoid cell lines [80], amino acid- the binding of 14-3-3, as 14-3-3/Redd1 complexes would be favoured
starved HEK293 cells [81], cells derived from S6K1−/−/S6K2−/− mice over inactive 14-3-3/TSC2 complexes. In line with this mechanism,
[82] and MEFs with all five rpS6 phosphorylation sites mutated [83] Redd1-mediated inhibition of mTORC1 occurs even under constitutive
indicate that S6K1 activation and subsequent phosphorylation of rpS6 Akt activation and thus hypoxic stress is able to override and repress
are dispensable for the translation of 5′TOP mRNAs [80]. Nevertheless, mitogenic activation of mTORC1 [94].
inhibition of mTOR with rapamycin is known to repress mitogenic Bnip3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) has also
stimulation of 5′TOP mRNA translation [78,82]. It is currently unclear been implicated in the mTORC1 hypoxic response. It is a member of the
how mTOR regulates 5′TOP mRNA translation and whether HIF-1α Bcl-2 superfamily, is enhanced by HIF-1α during hypoxia [95] and is
mRNA is regulated as a bona fide 5′TOP message. Additional studies known to upregulate autophagy [96]. Knockdown of Bnip3 prevents
need to be carried out to examine whether HIF-1α translation is truly hypoxia-induced repression of S6K and rpS6 phosphorylation [97],
regulated by the mTOR/S6K1 pathway. suggesting it is involved in hypoxic signalling through mTORC1. Unlike
Studies have also shown a link between mTOR and HIF-1α mRNA Redd1, which appears to act at the level of TSC2, Bnip3 interacts with
levels and protein stability. Analysis of TSC2−/− MEFs, which possess Rheb and decreases the levels of actively loaded Rheb bound to GTP.
heightened mTOR activity, revealed increased HIF-1α protein levels Consequently, Bnip3 inhibits Rheb's ability to activate mTORC1 and
compared to wild-type MEFs, which were at least partly due to subsequently causes S6K dephosphorylation [97].
changes in HIF-1α mRNA expression [84]. In the TSC2−/− cells, HIF-
1α levels remained inappropriately elevated following prolonged 3.2. Energy sensing
hypoxia [84,85]. This could be reversed with rapamycin treatment,
indicating that this phenomenon is due to increased mTOR activity It is important that mTORC1 is not active when ATP is limiting, to
[85]. HIF-1α expression and activity was also found to be upregulated reduce energy demanding cell processes such as protein synthesis. For
following mitogenic stimulation and activation of the PI3K/Akt/mTOR this reason the pathway is sensitive to input from AMPK. AMPK becomes
pathway in prostate cancer cells [86], while rapamycin treatment active in response to energy stress, when cellular ATP levels decline.
repressed the basal expression of HIF-1α in both normoxic and AMPK is activated by phosphorylation of Thr172 in its activation loop by
hypoxic prostate cancer cells [87]. Evidence for mTOR playing a role in LKB1 [98]. AMPK then phosphorylates TSC2 on Ser1345 and Thr1227,
HIF-1α protein synthesis comes from experiments which showed that which promotes TSC2's ability to inhibit mTORC1 [21]. However, this
mitogenic stimulation of MCF7 human breast cancer cells by heregulin does not fully explain the actions of AMPK, as TSC2 null cells display
(an epidermal growth factor-like growth factor) activates mTOR and some inhibition of mTORC1 in response to ATP depletion, glycolytic
increases the rate of HIF-1α protein synthesis, while not affecting HIF- inhibitors or AMPK activators, indicating the presence of a TSC2-
1α protein stability [88]. independent mechanism [99,100]. Bioinformatic analysis revealed
Evidence for direct targeting of HIF by mTOR comes from analysis Raptor as a potential AMPK substrate and experimental work confirmed
revealing that HIF contains a TOS motif (FVMVL) which is required for that Raptor was phosphorylated both in vivo and in vitro by AMPK [100].
its interaction with Raptor [89]. Under hypoxic conditions, Rheb- The phosphorylation sites were identified as Ser792 and Ser722 and
induced activation of mTOR potently enhances the transcriptional mutation of both these sites prevented AMPK agonists from fully
activity of HIF-1α. In this study, mTOR did not appear to influence the suppressing mTORC1 activity in both TSC2 wild-type and TSC2 null cells
stability of HIF-1α [89] although others have shown that the [100]. This phosphorylation of Raptor by AMPK induces 14-3-3 binding
suppressive effect of rapamycin on HIF-1α protein levels in hypoxic to Raptor and is necessary for mTORC1 inhibition [100]. Thus it appears
conditions is primarily due to impairment of the mechanism that AMPK inhibits mTORC1 function through two distinct mechanisms.
responsible for the stabilisation of the protein [87]. Over-expressed, The repression of mTORC1 by AMPK-mediated phosphorylation events
HIF-1α lacking a functional TOS motif still translocates to the nucleus are summarised in Fig. 2, and show the role that 14-3-3 proteins play to
but is unable to form a functional transcriptional complex with its regulate mTORC1.
coactivator CBP/p300 suggesting that mTOR enhances HIF-1α While short-term hypoxia, discussed in Section 3.1, does not affect
mediated-transcription through assembly of the HIF-1α transcrip- cellular energetics [85,90], severe oxygen depletion dramatically
tional machinery [89]. Further work will help better define how mTOR reduces the energy levels of the cell [86]. Hypoxic cells rely on
signalling modulates HIF, which appears to operate at the levels of anaerobic respiration to generate ATP, which is less efficient than the
gene expression, translation, protein stability and activity. mitochondrial respiratory chain and as a result the ratio of AMP:ATP
832 E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835

increases and AMPK becomes active. A dominant negative form of [102]. Two additional mitogenic signalling pathways have recently
AMPK is sufficient to block hypoxia-induced hypophosphorylation of been shown to integrate with mTOR signalling, as described below.
S6K, indicating that AMPK activation by hypoxia promotes mTOR
inhibition [91]. It appears that inhibition of mTOR through hypoxic 3.3.1. Phosphatidic acid
signals occurs at multiple levels in order to quickly and efficiently Phosphatidic acid (PA) was found to stimulate S6K1 activation and
reduce mTORC1-mediated signalling. 4E-BP1 phosphorylation in serum-starved cells in a rapamycin-sensitive
An additional parallel pathway involving Redd1 also operates to manner, implicating it as an upstream effector of mTOR [103]. Mutation
repress mTORC1 signalling in response to energy stress. Redd1 is of Arg2109 to Ala within the FRB domain of mTOR diminishes the affinity
induced in response to hypoxia (described in Section 3.1) and for PA, as does high ionic strength (500 mM NaCl), suggesting an
similarly, Redd1 mRNA is upregulated following glucose withdrawal electrostatic interaction between FRB and PA [103]. Analysis of the
or cellular ATP depletion. Redd1−/− mouse embryonic fibroblasts do amino acid residues of the FRB domain involved in rapamycin and PA
not dephosphorylate S6K1 following energy stress, indicating that binding indicates a substantial overlap of the binding sites [104,105],
Redd1 is required for proper response to energy stress [101]. However, suggesting that competition for binding may influence mTOR activation
Redd1 is not required for AMPK activation and is not involved in by PA. Phospholipase D1 (PLD1) is responsible for cellular production of
AMPK-mediated phosphorylation of TSC2, suggesting it functions in PA and a recent study [106] provides a mechanism for how PLD1 and PA
parallel to AMPK [101]. Stress-induced Redd1 function requires TSC2 integrate into the mTOR pathway. Overexpression of Rheb activates
as Redd1 overexpression in TSC2−/− cells has no effect on S6K1 PLD1 and conversely Rheb knockdown diminishes PLD activation and
phosphorylation levels [101]. Presumably, by activating several reduces S6K1 phosphorylation [106]. A farnesylation-deficient mutant
distinct negative pathways in response to cellular energy depletion, of Rheb (C181S) had a diminished ability to activate PLD1 [106],
cells are able to more effectively shut down mTORC1 signalling and indicating the importance of this C-terminal post-translational mod-
conserve energy supplies. ification for Rheb to induce PLD1 activation. Interestingly, PLD1
knockdown experiments showed that PLD1 was required for Rheb
3.3. Mitogenic stimuli activation of mTOR, and that the effects of PLD1 knockdown were
rescued by the addition of exogenous PA [106]. Rheb was also shown to
It is well documented that growth factors activate mTOR through physically interact with PLD1 [106]. These observations point to Rheb
the PI3K–Akt pathway and via ERK-RSK signalling. Both Akt and RSK acting upstream of PLD1 on a linear pathway [106]. As PLD1 is mostly
function by phosphorylating specific sites on TSC2 (Ser939 and Thr1462 localised to intracellular membrane structures, a model was proposed
in the case of Akt, Ser1798 and to a lesser extent the two Akt sites in the whereby the membrane localisation of mTOR confers its proximity to
case of RSK), thus inactivating it and allowing mTOR to become active PLD1 [107]. A possible mechanism is that Rheb activation stimulates
PLD1 to produce PA which, due to its appropriate cellular localisation,
can then bind to and activate mTOR.
In addition to its action on PLD1 and mTORC1, Rheb also associates
with B-Raf kinase in vivo. This inhibits B-Raf kinase activity thereby
preventing B-Raf-dependent activation of the transcription factor Elk-1
[108]. The action of Rheb on B-Raf is independent of its farnesylation
status [109]. Rheb also inhibits Ser338 phosphorylation of C-Raf, thought
to be a result of Rheb-induced disruption of B-Raf/C-Raf heterodimer-
isation. This role of Rheb is independent of its action on mTORC1 [110].
These findings place PLD, FKBP38, B-Raf and C-Raf, as downstream
effectors of Rheb, so inactivation of the upstream TSC1–TSC2 complex
can lead to a number of Rheb-activated mTORC1-dependent and
independent signalling events. These are summarised in Fig. 3.

3.3.2. MAPKAPK2
Serum, as well as stress responses, activate the p38 MAP kinase
pathway, which has been linked to the mTOR pathway through
MAPKAPK2 (MK2). MK2 is phosphorylated and activated by p38, and
once active, can phosphorylate Ser1210 on TSC2 [111]. Phosphorylation
of this TSC2 site has been shown to increase the binding of TSC2 to 14-
3-3 [111–113]. Binding of 14-3-3 to TSC2 is also enhanced by pathways
that include P13K/Akt. While 14-3-3 binding to the Akt phosphoryla-
tion sites (Ser939 and Ser981) has been shown to inactivate TSC2 by
cytosolic translocation of TSC2 away from cellular membrane tethered
TSC1 [93], the effects of 14-3-3 binding to the MK2 phosphorylation
site are less well-characterised. 14-3-3-mediated modulation of TSC1–
TSC2 activity in response to serum-activated pathways may explain
Fig. 2. Activation and repression of mTORC1 by phosphorylation and 14-3-3 association. how mTORC1 is sensitive to serum input.
(a) During unstimulated (serum-starved) conditions TSC1–TSC2 heterodimers are
located to intracellular membrane compartments and function as a RhebGAP, reverting
3.4. Nutrient input into mTORC1
Rheb to an inactive GDP-bound form. Negative regulators, PRAS40 and FKBP38 interact
and repress mTORC1. During states of low energy supply, AMPK dominantly inhibits
mTORC1 via a duel mechanism: phosphorylation of TSC2 and Raptor, that activates TSC2 One of the many signals which mTORC1 responds to is the nutrient
and represses mTORC1 via 14-3-3 binding to Raptor, respectively. (b) Upon mitogenic status of the cell. It is essential that mTORC1 coordinates its activity in
stimulation, Akt phoshorylates TSC2 and PRAS40. Binding of 14-3-3 to TSC2 causes its response to nutrient availability. A cell must conserve resources when
cytoplasmic translocation away from TSC1 and consequently active Rheb-GTP is
favoured. Rheb-GTP is speculated to interact with the mTORC1 repressor protein
nutrients are scarce and so it is not desirable for mTORC1 to stimulate
FKBP38 leading to mTORC1 activation. 14-3-3 binding to PRAS40 is also thought to further protein synthesis under such conditions. Of the amino acids,
relieve mTORC1 inhibition by PRAS40. one branched chain amino acid leucine has been identified as having
E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835 833

signalling to mTORC1. This family of four proteins are Ras-related


small GTPases which function as heterodimers in mammals. They
were found to be capable of interacting with Raptor, but not Rictor,
thus identifying them as mTORC1 associated proteins [120]. Amino
acid stimulation increases the levels of GTP bound to RagB, while
stable expression of an active RagBGTP mutant eliminates the
sensitivity of the mTORC1 pathway to both leucine and total amino
acid withdrawal [120]. In mammalian cells, rapamycin blocks Rag-
induced S6K phosphorylation, indicating that these proteins function
upstream of mTORC1 [121]. Work with Drosophila revealed that under
nutrient starved conditions, expression of wild-type RagA resulted in
a modest increase in larval fat body cell size, while expression of
RagAGTP increased cell size 3-fold. Little effect was observed under
normal-fed conditions, indicating a specific role for RagA in nutrient
response [121]. The Rag proteins appear to mediate their effects by
regulating the cellular localisation of mTOR in response to amino
Fig. 3. Upstream repressors and downstream effectors of Rheb. When farnesylated and
acids. It has been proposed that in nutrient-rich conditions, mTOR is
GTP-bound, Rheb inhibits FKBP38 and activates PLD1, thus activating mTORC1 through two
mechanisms; relieving the inhibition from FKBP38 and activating mTORC1 through the localised by the Rag proteins to the same cellular compartment as its
production of phosphatidic acid (PA). Rheb also inhibits the actions of B-Raf and C-Raf and activator, Rheb [120]. A potential role for the Rag proteins in
signals via RalA to mTORC1, through an undefined mechanism. autophagy has also been proposed, with RagAGTP and RagCGDP
inhibiting conversion of the autophagy marker LC3 in response to
the most influential role in mTOR stimulation, with leucine with- amino acid starvation [121]. The model of action proposed is that the
drawal being almost as effective as complete amino acid starvation on Rag proteins function between amino acids and mTORC1 and in
mTOR signalling [114]. While it is not yet fully understood how parallel to the TSC1–TSC2/Rheb input to mTORC1 [120,121].
mTORC1 responds to nutrient input, some important mediators of the
effect have been uncovered. 3.4.4. RalA
The most recent GTPase to be linked to the mTORC1 nutrient input
3.4.1. Vps34 is RalA. RNAi knockdown of RalA robustly inhibited phosphorylation
The first protein to be linked to nutrient-sensing within the mTORC1 of both S6K and 4E-BP1 following amino acid or glucose stimulation of
pathway was vacuolar protein sorting 34 (Vps34). It was shown that HeLa cells [59]. The same results were obtained when the expression
overexpression of Vps34 activates the mTORC1 downstream effector, of RalGDS, the upstream activator and guanine exchange factor of RalA
S6K1, but this effect was independent of insulin stimulation [115,116]. was knocked down, and indicates a role for the RalGDS/RalA pathway
The activity of Vps34 was markedly depressed by amino acid or glucose in nutrient activation of mTORC1. Activation of RalA was nutrient-
starvation, but rapamycin treatment did not affect its activity [115], thus dependent where RalA became predominantly GTP-bound following
indicating a role for it in integrating nutrient signals upstream of incubation of cells with amino acids. Further analysis revealed that
mTORC1. Recent work has implicated intracellular calcium ([Ca2+]i) in RalA functioned downstream of Rheb (shown in Fig. 3) as the mTORC1
this process with evidence that amino acid stimulation leads to a rise in inhibition observed following Rheb knockdown or expression of a
[Ca2+]i, while calcium chelators inhibit phosphorylation of S6K1 in dominant negative form of Rheb could be partially reversed by
amino acid-stimulated cells [117]. The mechanism uncovered is that expression of a constitutively active RalA mutant [59]. The exact
rising [Ca2+]i stabilises the binding of Ca2+/calmodulin to a conserved mechanism for how RalA feeds into mTORC1 and integrates with the
motif in Vps34, thereby activating Vps34. This alters the conformation of Rheb input is yet to be determined.
the Vps34-associated mTORC1 signalosome, allowing other factors to It is not known whether these nutrient sensitive regulators function
bind and fully activate mTORC1. The result is that amino acid stimulation in a mutually exclusive fashion or whether they co-ordinate with one
increases [Ca2+]i which induces mTORC1 signalling in a Vps34- another to finely adjust the permissive nutrient input to mTORC1.
dependent manner [117]. Whether such a mechanism is conserved Further work into their functions could help elucidate exactly where in
between species remains to be determined as a report looking at Vps34 the pathway between amino acids and mTORC1 they operate and the
function in Drosophila concluded that kinase dead mutants of Vps34 did mechanism by which they exert their effects on mTORC1.
not disrupt TOR-dependent signalling [118].
4. mTORC2
3.4.2. MAP4K3
A dsRNA screen to co-suppress Drosophila protein kinases alongside mTOR is also found complexed with mLST8 along with the
dTsc1 identified knock-down of CG7097 as contributing to loss of dS6K rapamycin insensitive companion of TOR (Rictor), mSin1 and PRR5,
phosphorylation [119]. It was found that depletion of the mammalian in a complex known as mTORC2 [122–126]. This complex was found to
ortholog, MAP4K3 (also known as germinal centre-like kinase (GLK)) be the kinase responsible for phosphorylating Ser473 in Akt [127,128].
had a similar effect on mammalian S6K1 phosphorylation. Over- Knockdown of mTORC2 components prevents actin polymerisation
expression of MAP4K3 activates phosphorylation of both S6K1 and 4E- and cell spreading, indicating that like yeast Tor2, mTORC2 has a role
BP1 in a rapamycin-sensitive manner. Conversely, knock-down of in actin cytoskeleton organisation [123]. It was speculated that this
MAP4K3 suppresses the S6K1 phsophorylation normally induced by process could be mediated by mTORC2 signalling through Rho and Rac
amino acid restimulation, implicating it as a nutrient regulated kinase [123]. Further substrates of mTORC2 are being identified, with PKC
within the mTORC1 pathway [119]. A marked decrease in cell size was recently shown to be regulated by mTORC2 [129]. In Rictor or mSin1
also apparent following MAP4K3 knock-down, which was at a similar null cells, PKCα protein levels and phosphorylation were markedly
level to that seen when the mTORC1 activator Rheb was depleted [119]. reduced. The mTORC2 components were found to be important in
both the phosphorylation of the turn and hydrophobic motifs of
3.4.3. Rag GTPases conventional PKCs and the novel PKCɛ and this phosphorylation is
In two recent papers, Sancak et al. [120] and Kim et al. [121] critical for PKC kinase stability and function [129]. mTORC2 is
identified the Rag GTPases as important mediators of amino acid generally described as being rapamycin-insensitive, but it is now
834 E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835

becoming apparent that while short-term rapamycin treatment does References


not inhibit this complex, longer-term treatment leads to dissociation
of mTORC2 [130], which is mediated by dephosphorylation of Rictor
[1] R.J. Motzer, B. Escudier, S. Oudard, T.E. Hutson, C. Porta, S. Bracarda, V. Grünwald, J.A.
and mSin1 [131]. In certain cell types the dissociation of the complex Thompson, R.A. Figlin, N. Hollaender, G. Urbanowitz, W.J. Berg, A. Kay, D. Lebwohl, A.
upon long-term rapamycin exposure is sufficient to decrease levels of Ravaud, RECORD-1 Study Group, Lancet 372 (2008) 449.
intact mTORC2 below those needed to maintain Akt phosphorylation [2] M. Rizell, M. Andersson, C. Cahlin, L. Hafström, M. Olausson, P. Lindnér, Int. J. Clin.
Oncol. 13 (2008) 66.
[130]. [3] T.F. Cloughesy, K. Yoshimoto, P. Nghiemphu, K. Brown, J. Dang, S. Zhu, T. Hsueh, Y.
Although the TSC1–TSC2 heterodimer plays a vital role in Chen, W. Wang, D. Youngkin, L. Liau, N. Martin, D. Becker, M. Bergsneider, A. Lai, R.
modulating mTORC1 activity, it was unknown what role, if any, it Green, T. Oglesby, M. Koleto, J. Trent, S. Horvath, P.S. Mischel, I.K. Mellinghoff, C.L.
Sawyers, PLoS Med. 5 (2008) e8.
played in the regulation of mTORC2. Recent work revealed that cells [4] M.M. Mita, A.C. Mita, Q.S. Chu, E.K. Rowinsky, G.J. Fetterly, M. Goldston, A. Patnaik, L.
lacking a functional TSC1–TSC2 complex had impaired activity Mathews, A.D. Ricart, T. Mays, H. Knowles, V.M. Rivera, J. Kreisberg, C.L. Bedrosian, A.W.
towards the mTORC2 substrate Akt, with this being independent of Tolcher, J. Clin. Oncol. 26 (2008) 361.
[5] D.M. Davies, S.R. Johnson, A.E. Tattersfield, J.C. Kingswood, J.A. Cox, D.L.
known feedback mechanisms [132]. TSC1–TSC2 was found to McCartney, T. Doyle, F. Elmslie, A. Saggar, P.J. de Vries, J.R. Sampson, N. Engl. J.
physically associate with mTORC2, but its affect on this complex did Med. 358 (2008) 200.
not involve Rheb, illustrating differences in control of mTORC1 and [6] J.J. Bissler, F.X. McCormack, L.R. Young, J.M. Elwing, G. Chuck, J.M. Leonard, V.J.
Schmithorst, T. Laor, A.S. Brody, J. Bean, S. Salisbury, D.N. Franz, N. Engl. J. Med. 358
mTORC2 [132]. Little else is currently known about upstream mTORC2
(2008) 140.
regulation, but further studies should unravel inputs to this complex. [7] M. van Slegtenhorst, R. de Hoogt, C. Hermans, M. Nellist, B. Janssen, S. Verhoef, D.
Lindhout, A. van den Ouweland, D. Halley, J. Young, M. Burley, S. Jeremiah, K.
5. Feedback in the mTOR pathway Woodward, J. Nahmias, M. Fox, R. Ekong, J. Osborne, J. Wolfe, S. Povey, R.G. Snell, J.P.
Cheadle, A.C. Jones, M. Tachataki, D. Ravine, J.R. Sampson, M.P. Reeve, P. Richardson, F.
Wilmer, C. Munro, T.L. Hawkins, T. Sepp, J.B. Ali, S. Ward, A.J. Green, J.R. Yates, J.
From the signalling components summarised above, it is clear that Kwiatkowska, E.P. Henske, M.P. Short, J.H. Haines, S. Jozwiak, D.J. Kwiatkowski,
the mTOR pathway is complex and responds to a variety of stimuli. It is Science 277 (1997) 805.
[8] European Tuberous Sclerosis Consortium, Cell 75 (1993) 1305.
not surprising that there are multiple feedback mechanisms that [9] Y. Zhang, X. Gao, L.J. Saucedo, B. Ru, B.A. Edgar, D. Pan, Nat. Cell Biol. 5 (2003) 578.
operate in order to refine signal transduction through mTORC1. One of [10] A. Garami, F.J. Zwartkruis, T. Nobukuni, M. Joaquin, M. Roccio, H. Stocker, S.C.
the best characterised negative feedback loops is the action of S6K on Kozma, E. Hafen, J.L. Bos, G. Thomas, Mol. Cell 11 (2003) 1457.
[11] K. Inoki, Y. Li, T. Xu, K.L. Guan, Genes Dev. 17 (2003) 1829.
the PI3K/Akt pathway. Observations of TSC2 null MEFs uncovered the [12] A.R. Tee, B.D. Manning, P.P. Roux, L.C. Cantley, J. Blenis, Cur. Biol. 13 (2003) 1259.
ability of the S6Ks to both suppress IRS-1 transcription and increase [13] L. Ma, Z. Chen, H. Erdjument-Bromage, P. Tempst, P.P. Pandolfi, Cell 121 (2005) 179.
IRS-1 protein phosphorylation at Ser302. The result is that following [14] T. Maehama, J.E. Dixon, J. Biol. Chem. 273 (1998) 13375.
[15] D. Liaw, D.J. Marsh, J. Li, P.L. Dahia, S.I. Wang, Z. Zheng, S. Bose, K.M. Call, H.C. Tsou,
mTORC1 activation, S6K-mediated phosphorylation of IRS-1 impairs M. Peacocke, C. Eng, R. Parsons, Nat. Genet. 16 (1997) 64.
its association with the insulin receptor, thus preventing the activation [16] M.R. Nelen, W.C. van Staveren, E.A. Peeters, M.B. Hassel, R.J. Gorlin, H. Hamm, C.F.
of PI3K following insulin stimulation [133]. Chronic mTORC1 activa- Lindboe, J.P. Fryns, R.H. Sijmons, D.G. Woods, E.C. Mariman, G.W. Padberg, H.
Kremer, Hum. Mol. Genet. 6 (1997) 1383.
tion, such as that observed in TSC2 null cells, also results in lower
[17] G.F. Xu, P. O'Connell, D. Viskochil, R. Cawthon, M. Robertson, M. Culver, D. Dunn, J. Stevens,
levels of IRS-1 transcription, thus causing longer-term suppression of R. Gesteland, R. White, R. Weiss, Cell. 62 (1990) 599.
insulin activation of PI3K [133,134]. [18] C.M. Johannessen, E.E. Reczek, M.F. James, H. Brems, E. Legius, K. Cichowski, Proc.
Another feedback mechanism operates in response to hypoxia. As Natl. Acad. Sci. 102 (2005) 8573.
[19] M.N. Corradetti, K. Inoki, N. Bardeesy, R.A. DePinho, K.L. Guan, Genes Dev. 18
discussed in Section 3.1, hypoxia increases Redd1 mRNA levels and (2004) 1533.
Redd1 protein binds 14-3-3, thus releasing TSC2 and allowing it to [20] S.P. Hong, F.C. Leiper, A. Woods, D. Carling, M. Carlson, Proc. Natl. Acad. Sci. 100
downregulate mTORC1 [94]. However, in TSC2 null cells and TSC (2003) 8839.
[21] K. Inoki, T. Zhu, K.L. Guan, Cell 115 (2003) 577.
patients, mTOR is inappropriately active and the Redd1 feedback input [22] A. Hemminki, D. Markie, I. Tomlinson, E. Avizienyte, S. Roth, A. Loukola, G. Bignell, W.
which would normally operate in response to prolonged hypoxia is Warren, M. Aminoff, P. Höglund, H. Järvinen, P. Kristo, K. Pelin, M. Ridanpää, R.
unable to function due to the lack of functional TSC2. Therefore, mTORC1 Salovaara, T. Toro, W. Bodmer, S. Olschwang, A.S. Olsen, M.R. Stratton, A. de la
Chapelle, L.A. Aaltonen, Nature 391 (1998) 184.
remains inappropriately active and HIF levels remain elevated. [23] M. Baba, S.B. Hong, N. Sharma, M.B. Warren, M.L. Nickerson, A. Iwamatsu, D.
Esposito, W.K. Gillette, R.F. Hopkins, J.L. Hartley, M. Furihata, S. Oishi, W. Zhen, T.R.
6. Summary Burke Jr., W.M. Linehan, L.S. Schmidt, B. Zbar, Proc. Natl. Acad. Sci. 103 (2006)
15552.
[24] H. Hasumi, M. Baba, S.B. Hong, Y. Hasumi, Y. Huang, M. Yao, V.A. Valera, W.M.
Our knowledge of the mTOR signalling pathway is constantly Linehan, L.S. Schmidt, Gene 415 (2008) 60.
evolving and our understanding of the cellular events surrounding [25] Y. Takagi, T. Kobayashi, M. Shiono, L. Wang, X. Piao, G. Sun, D. Zhang, M. Abe, Y.
Hagiwara, K. Takahashi, O. Hino, Oncogene 27 (2008) 5339.
mTOR signalling has become more complex since mTOR was first
[26] C.T. Keith, S.L. Schreiber, Science 270 (1995) 50.
identified as the target of the immunosuppressant, rapamycin. The [27] R.T. Abraham, DNA Repair 3 (2004) 883.
relatively recent discovery that mTOR exists in two separate [28] M.A. Andrade, P. Bork, Nat. Genet. 11 (1995) 115.
complexes with independent inputs and substrates has helped clarify [29] R. Bosotti, A. Isacchi, E.L. Sonnhammer, Trends Biochem. Sci. 25 (2000) 225.
[30] J. Chen, X.F. Zheng, E.J. Brown, S.L. Schreiber, Proc. Natl. Acad. Sci. 92 (1995) 4947.
previous data and will accelerate identification of upstream regulators [31] R.A. Bachmann, J. Kim, A. Wu, I. Park, J. Chen, J. Biol. Chem. 281 (2006) 7357.
and downstream effectors of the two complexes. FKBP38, PRAS40, [32] D.H. Kim, D.D. Sarbassov, S.M. Ali, J.E. King, R.R. Latek, H. Erdjument-Bromage, P.
YY1, PLD1, MAP4K3 and the Rag GTPases are the most recent additions Tempst, D.M. Sabatini, Cell 110 (2002) 163.
[33] R. Loewith, E. Jacinto, S. Wullschleger, A. Lorberg, J.L. Crespo, D. Bonenfant, W.
to the mTORC1 network. However, with the identification of mTORC1- Oppliger, P. Jenoe, M.N. Hall, Mol. Cell 10 (2002) 457.
mediated cell processes that are not solely restricted to protein [34] K.P. Wedaman, A. Reinke, S. Anderson, J. Yates, J.M. McCaffery, T. Powers, Mol.
synthesis, it is clear that further aspects of this signalling cascade Biol. Cell. 14 (2003) 1204.
[35] D.H. Kim, D.D. Sarbassov, S.M. Ali, R.R. Latek, K.V.P. Guntur, H. Erdjument-
remain to be uncovered. Ultimately, dissecting the mTOR signalling Bromage, P. Tempst, D.M. Sabatini, Mol. Cell 11 (2003) 895.
network should highlight therapeutic targets of benefit to patients [36] G.J. Brunn, C.C. Hudson, A. Sekulić, J.M. Williams, H. Hosoi, P.J. Houghton, J.C.
with hamartoma syndromes and cancer. Lawrence Jr., R.T. Abraham, Science 277 (1997) 99.
[37] K. Hara, K. Yonezawa, M.T. Kozlowski, T. Sugimoto, K. Andrabi, Q.P. Weng, M.
Kasuga, I. Nishimoto, J. Avruch, J. Biol. Chem. 272 (1997) 26457.
Acknowledgements [38] E.J. Brown, P.A. Beal, C.T. Keith, J. Chen, T.B. Shin, S.L. Schreiber, Nature 377 (1995) 441.
[39] D.C. Fingar, S. Salama, C. Tsou, E. Harlow, J. Blenis, Genes Dev. 16 (2002) 1472.
[40] D.C. Fingar, C.J. Richardson, A.R. Tee, L. Cheatham, C. Tsou, J. Blenis, Mol. Cell. Biol.
A. Tee and E. Dunlop are supported by the Association for
24 (2004) 200.
International Cancer Research Career Development Fellowship [No. [41] C.J. Richardson, M. Bröenstrup, D.C. Fingar, K. Jülich, B.A. Ballif, S. Gygi, J. Blenis,
06-914/915] and by the Tuberous Sclerosis Association. Curr. Biol. 14 (2004) 1540.
E.A. Dunlop, A.R. Tee / Cellular Signalling 21 (2009) 827–835 835

[42] X.M. Ma, S.O. Yoon, C.J. Richardson, K. Jülich, J. Blenis, Cell 133 (2008) 303. [90] A.M. Arsham, J.J. Howell, M.C. Simon, J. Biol. Chem. 278 (2003) 29655.
[43] B. Raught, F. Peiretti, A.C. Gingras, M. Livingstone, D. Shahbazian, G.L. Mayeur, R.D. [91] L. Liu, T.P. Cash, R.G. Jones, B. Keith, C.B. Thompson, M.C. Simon, Mol. Cell 21
Polakiewicz, N. Sonenberg, J.W. Hershey, EMBO J. 23 (2004) 1761. (2006) 521.
[44] D. Shahbazian, P.P. Roux, V. Mieulet, M.S. Cohen, B. Raught, J. Taunton, J.W. [92] M.N. Corradetti, K. Inoki, K.L. Guan, J. Biol. Chem. 280 (2005) 9769.
Hershey, J. Blenis, M. Pende, N. Sonenberg, EMBO J. 25 (2006) 2781. [93] S.L. Cai, A.R. Tee, J.D. Short, J.M. Bergeron, J. Kim, J. Shen, R. Guo, C.L. Johnson, K.
[45] X. Wang, W. Li, M. Williams, N.R. Terada, D. Alessi, C.G. Proud, EMBO J. 20 (2001) 4370. Kiguchi, C.L. Walker, J. Cell Biol. 173 (2006) 279.
[46] S.S. Schalm, J. Blenis, Curr. Biol. 12 (2002) 632. [94] M.P. DeYoung, P. Horak, A. Sofer, D. Sgroi, L.W. Ellisen, Genes Dev. 22 (2008) 239.
[47] H. Nojima, C. Tokunaga, S. Eguchi, N. Oshiro, S. Hidayat, K. Yoshino, K. Hara, N. [95] H.M. Sowter, P.J. Ratcliffe, P. Watson, A.H. Greenberg, A.L. Harris, Cancer Res. 61
Tanaka, J. Avruch, K. Yonezawa, J. Biol. Chem. 278 (2003) 15461. (2001) 6669.
[48] S.S. Schalm, D.C. Fingar, D.M. Sabatini, J. Blenis, Curr. Biol. 13 (2003) 797. [96] M.C. Maiuri, A. Criollo, E. Tasdemir, J.M. Vicencio, N. Tajeddine, J.A. Hickman, O.
[49] A.R. Tee, C.G. Proud, Mol. Cell. Biol. 22 (2002) 1674. Geneste, G. Kroemer, Autophagy 3 (2007) 374.
[50] V.H. Lee, T. Healy, B.D. Fonseca, A. Hayashi, C.G. Proud, FEBS J. 275 (2008) 2185. [97] Y. Li, Y. Wang, E. Kim, P. Beemiller, C.Y. Wang, J. Swanson, M. You, K.L. Guan, J. Biol.
[51] X. Long, Y. Lin, S. Ortiz-Vega, K. Yonezawa, J. Avruch, Curr. Biol. 15 (2005) 702. Chem. 282 (2007) 35803.
[52] E.M. Smith, S.G. Finn, A.R. Tee, G.J. Browne, C.G. Proud, J. Biol. Chem. 280 (2005) [98] R.J. Shaw, M. Kosmatka, N. Bardeesy, R.L. Hurley, L.A. Witters, R.A. DePinho, L.C.
18717. Cantley, Proc. Natl. Acad. Sci. 101 (2004) 3329.
[53] X. Long, Y. Lin, S. Ortiz-Vega, S. Busch, J. Avruch, J. Biol. Chem. 282 (2007) 18542. [99] A. Hahn-Windgassen, V. Nogueira, C.C. Chen, J.E. Skeen, N. Sonenberg, N. Hay,
[54] M.I. Chiu, H. Katz, V. Berlin, Proc. Natl. Acad. Sci. 91 (1994) 12574. J. Biol. Chem. 280 (2005) 32081.
[55] J. Chen, X.F. Zheng, E.J. Brown, S.L. Schreiber, Proc. Natl. Acad. Sci. 92 (1995) 4947. [100] D.M. Gwinn, D.B. Shackelford, D.F. Egan, M.M. Mihaylova, A. Mery, D.S. Vasquez, B.E.
[56] X. Bai, D. Ma, A. Liu, X. Shen, Q.J. Wang, Y. Liu, Y. Jiang, Science 318 (2007) 977. Turk, R.J. Shaw, Mol. Cell 30 (2008) 214.
[57] D. Ma, X. Bai, S. Guo, Y. Jiang, J. Biol. Chem. 283 (2008) 25963. [101] A. Sofer, K. Lei, C.M. Johannessen, L.W. Ellisen, Mol. Cell. Biol. 25 (2005) 5834.
[58] X. Wang, B.D. Fonseca, H. Tang, R. Liu, A. Elia, M.J. Clemens, U.A. Bommer, C.G. [102] P.P. Roux, B.A. Ballif, R. Anjum, S.P. Gygi, J. Blenis, Proc. Natl. Acad. Sci. 101 (2004)
Proud, J. Biol. Chem. 283 (2008) 30482. 13489.
[59] T. Maehama, M. Tanaka, H. Nishina, M. Murakami, Y. Kanaho, K. Hanada, J. Biol. [103] Y. Fang, M. Vilella-Bach, R. Bachmann, A. Flanigan, J. Chen, Science 294 (2001)
Chem. 283 (2008) 35053. 1942.
[60] Y. Sancak, C.C. Thoreen, T.R. Peterson, R.A. Lindquist, S.A. Kang, E. Spooner, S.A. [104] J. Choi, J. Chen, S.L. Schreiber, J. Clardy, Science 273 (1996) 239.
Carr, D.M. Sabatini, Mol. Cell 25 (2007) 903. [105] V. Veverka, T. Crabbe, I. Bird, G. Lennie, F.W. Muskett, R.J. Taylor, M.D. Carr,
[61] L. Wang, T.E. Harris, R.A. Roth, J.C. Lawrence Jr., J. Biol. Chem. 282 (2007) 20036. Oncogene 27 (2008) 585.
[62] B.D. Fonseca, E.M. Smith, V.H.Y. Lee, C. MacKintosh, C.G. Proud, J. Biol. Chem. 282 [106] Y. Sun, Y. Fang, M.S. Yoon, C. Zhang, M. Roccio, F.J. Zwartkruis, M. Armstrong, H.A.
(2007) 24514. Brown, J. Chen, Proc. Natl. Acad. Sci. 105 (2008) 8286.
[63] N. Oshiro, R. Takahashi, K. Yoshino, K. Tanimura, A. Nakashima, S. Eguchi, T. [107] Y. Fang, I.H. Park, A.L. Wu, G. Du, P. Huang, M.A. Frohman, S.J. Walker, H.A. Brown,
Miyamoto, K. Hara, K. Takehana, J. Avruch, U. Kikkawa, K. Yonezawa, J. Biol. Chem. J. Chen, Curr. Biol. 13 (2003) 2037.
282 (2007) 20329. [108] E. Im, F.C. von Lintig, J. Chen, S. Zhuang, W. Qui, S. Chowdhury, P.F. Worley, G.R.
[64] K. Thedieck, P. Polak, M.L. Kim, K.D. Molle, A. Cohen, P. Jenö, C. Arrieumerlou, M.N. Boss, R.B. Pilz, Oncogene 21 (2002) 6356.
Hall, PLoS ONE 2 (2007) e1217. [109] M. Karbowniczek, T. Cash, M. Cheung, G.P. Robertson, A. Astrinidis, E.P. Henske,
[65] E. Vander Haar, S.I. Lee, S. Bandhakavi, T.J. Griffin, D.H. Kim, Nat. Cell Biol. 9 (2007) 316. J. Biol. Chem. 279 (2004) 29930.
[66] K.S. Kovacina, G.Y. Park, S.S. Bae, A.W. Guzzetta, E. Schaefer, M.J. Birnbaum, R.A. [110] M. Karbowniczek, G.P. Robertson, E.P. Henske, J. Biol. Chem. 281 (2006) 25447.
Roth, J. Biol. Chem. 278 (2003) 10189. [111] Y. Li, K. Inoki, P. Vacratsis, K.L. Guan, J. Biol. Chem. 278 (2003) 13663.
[67] L. Wang, T.E. Harris, J.C. Lawrence Jr., J. Biol. Chem. 283 (2008) 15619. [112] S.D. Shumway, Y. Li, Y. Xiong, J. Biol. Chem. 278 (2003) 2089.
[68] S.M. Schieke, D. Phillips, J.P. McCoy Jr., A.M. Aponte, R.F. Shen, R.S. Balaban, T. [113] Y. Li, K. Inoki, R. Yeung, K.L. Guan, J. Biol. Chem. 277 (2002) 44593.
Finkel, J. Biol. Chem. 281 (2006) 27643. [114] K. Hara, K. Yonezawa, Q.P. Weng, M.T. Kozlowski, C. Belham, J. Avruch, J. Biol.
[69] J.T. Cunningham, J.T. Rodgers, D.H. Arlow, F. Vazquez, V.K. Mootha, P. Puigserver, Chem. 273 (1998) 14484.
Nature 450 (2007) 736. [115] M.P. Byfield, J.T. Murray, J.M. Backer, J. Biol. Chem. 280 (2005) 33076.
[70] Y. Shi, E. Seto, L.S. Chang, T. Shenk, Cell 67 (1991) 377. [116] T. Nobukuni, M. Joaquin, M. Roccio, S.G. Dann, S.Y. Kim, P. Gulati, M.P. Byfield, J.M.
[71] Z. Wen, Z. Zhong, J.E. Darnell Jr., Cell 82 (1995) 241. Backer, F. Natt, J.L. Bos, F.J. Zwartkruis, G. Thomas, Proc. Natl. Acad. Sci. 102 (2005)
[72] K. Yokogami, S. Wakisaka, J. Avruch, S.A. Reeves, Curr. Biol. 10 (2000) 47. 14238.
[73] B. Wang, Z. Xiao, B. Chen, J. Han, Y. Gao, J. Zhang, W. Zhao, X. Wang, J. Dai, PLoS [117] P. Gulati, L.D. Gaspers, S.G. Dann, M. Joaquin, T. Nobukuni, F. Natt, S.C. Kozma, A.P.
ONE 3 (2008) e1856. Thomas, G. Thomas, Cell Metab. 7 (2008) 456.
[74] J. Zhou, J. Wulfkuhle, H. Zhang, P. Gu, Y. Yang, J. Deng, J.B. Margolick, L.A. Liotta, E. [118] G. Juhasz, J.H. Hill, Y. Yan, M. Sass, E.H. Baehrecke, J.M. Backer, T.P. Neufeld, J. Cell
Petricoin, Y. Zhang, Proc. Natl. Acad. Sci. 104 (2007) 16158. Biol. 181 (2008) 655.
[75] F. Hong, M.D. Larrea, C. Doughty, D.J. Kwiatkowski, R. Squillace, J.M. Slingerland, [119] G.M. Findlay, L. Yan, J. Procter, V. Mieulet, R.F. Lamb, Biochem. J. 403 (2007) 13.
Mol. Cell 30 (2008) 701. [120] Y. Sancak, T.R. Peterson, Y.D. Shaul, R.A. Lindquist, C.C. Thoreen, L. Bar-Peled, D.M.
[76] J.M. García-Martínez, D.R. Alessi, Biochem. J. 416 (2008) 375. Sabatini, Science 320 (2008) 1496.
[77] D.A. Guertin, D.M. Stevens, C.C. Thoreen, A.A. Burds, N.Y. Kalaany, J. Moffat, M. [121] E. Kim, P. Goraksha-Hicks, L. Li, T.P. Neufeld, K.L. Guan, Nat. Cell Biol. 10 (2008)
Brown, K.J. Fitzgerald, D.M. Sabatini, Dev. Cell 11 (2006) 859. 935.
[78] H.B. Jefferies, C. Reinhard, S.C. Kozma, G. Thomas, Proc. Natl. Acad. Sci. 91 (1994) 4441. [122] D.D. Sarbassov, S.M. Ali, D.H. Kim, D.A. Guertin, R.R. Latek, H. Erdjument-
[79] N.V. Iyer, S.W. Leung, G.L. Semenza, Genomics 52 (1998) 159. Bromage, P. Tempst, D.M. Sabatini, Curr. Biol. 14 (2004) 1296.
[80] M. Stolovich, H. Tang, E. Hornstein, G. Levy, R. Cohen, S.S. Bae, M.J. Birnbaum, O. [123] E. Jacinto, R. Loewith, A. Schmidt, S. Lin, M.A. Rüegg, A. Hall, M.N. Hall, Nat. Cell
Meyuhas, Mol. Cell. Biol. 22 (2002) 8101. Biol. 6 (2004) 1122.
[81] H. Tang, E. Hornstein, M. Stolovich, G. Levy, M. Livingstone, D. Templeton, J. [124] E. Jacinto, V. Facchinetti, D. Liu, N. Soto, S. Wei, S.Y. Jung, Q. Huang, J. Qin, B. Su, Cell
Avruch, O. Meyuhas, Mol. Cell. Biol. 21 (2001) 8671. 127 (2006) 125.
[82] M. Pende, S.H. Um, V. Mieulet, M. Sticker, V.L. Goss, J. Mestan, M. Mueller, S. [125] M.A. Frias, C.C. Thoreen, J.D. Jaffe, W. Schroder, T. Sculley, S.A. Carr, D.M. Sabatini,
Fumagalli, S.C. Kozma, G. Thomas, Mol. Cell. Biol. 24 (2004) 3112. Curr. Biol. 16 (2006) 1865.
[83] I. Ruvinsky, N. Sharon, T. Lerer, H. Cohen, M. Stolovich-Rain, T. Nir, Y. Dor, P. [126] S.Y. Woo, D.H. Kim, C.B. Jun, Y.M. Kim, E.V. Haar, S.I. Lee, J.W. Hegg, S. Bandhakavi,
Zisman, O. Meyuhas, Genes Dev. 19 (2005) 2199. T.J. Griffin, D.H. Kim, J. Biol. Chem. 282 (2007) 25604.
[84] J.B. Brugarolas, F. Vazquez, A. Reddy, W.R. Sellers, W.G. Kaelin Jr., Cancer Cell 4 [127] D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Science 307 (2005) 1098.
(2003) 147. [128] R.C. Hresko, M. Mueckler, J. Biol. Chem. 280 (2005) 40406.
[85] J. Brugarolas, K. Lei, R.L. Hurley, B.D. Manning, J.H. Reiling, E. Hafen, L.A. Witters, L.W. [129] T. Ikenoue, K. Inoki, Q. Yang, X. Zhou, K.L. Guan, EMBO J. 27 (2008) 1919.
Ellisen, W.G. Kaelin Jr., Genes Dev. 18 (2004) 2893. [130] D.D. Sarbassov, S.M. Ali, S. Sengupta, J.H. Sheen, P.P. Hsu, A.F. Bagley, A.L.
[86] H. Zhong, K. Chiles, D. Feldser, E. Laughner, C. Hanrahan, M. Georgescu, J.W. Markhard, D.M. Sabatini, Mol. Cell 22 (2006) 159.
Simons, G.L. Semenza, Cancer Res. 60 (2000) 1541. [131] M. Rosner, M. Hengstschläger, Hum. Mol. Genet. 17 (2008) 2934.
[87] C.C. Hudson, M. Liu, G.G. Chiang, D.M. Otterness, D.C. Loomis, F. Kaper, A.J. Giaccia, [132] J. Huang, C.C. Dibble, M. Matsuzaki, B.D. Manning, Mol. Cell. Biol. 28 (2008) 4104.
R.T. Abraham, Mol. Cell. Biol. 22 (2002) 7004. [133] L.S. Harrington, G.M. Findlay, A. Gray, T. Tolkacheva, S. Wigfield, H. Rebholz, J.
[88] E. Laughner, P. Taghavi, K. Chiles, P.C. Mahon, G.L. Semenza, Mol. Cell. Biol. 21 Barnett, N.R. Leslie, S. Cheng, P.R. Shepherd, I. Gout, C.P. Downes, R.F. Lamb, J. Cell
(2001) 3995. Biol. 166 (2004) 213.
[89] S.C. Land, A.R. Tee, J. Biol. Chem. 282 (2007) 20534. [134] O.J. Shah, Z. Wang, T. Hunter, Curr. Biol. 14 (2004) 1650.