Cell Death in Therapy 2015; 1: 11–22
Review
Open Access
Israel C. Nnah#, Khoosheh Khayati#, Radek Dobrowolski*
Cellular metabolism and lysosomal mTOR
signaling
Abstract: Over the last few years extensive studies have
linked the activity of mTORC1 to lysosomal function. These
observations propose an intriguing integration of cellular
catabolism, sustained by lysosomes, with anabolic
processes, largely controlled by mTORC1. Interestingly,
lysosomal function directly affects mTORC1 activity and
is regulated by ZKSCAN3 and TFEB, two transcription
factors and substrates of mTORC1. Thus, the lysosomal
mTOR signaling complex represents a hub of cellular
energy metabolism, and its dysregulation may lead to a
number of human diseases. Here, we discuss the recent
developments and highlight the open questions in this
growing field.
Keywords: Lysosomes, mTORC1, autophagy, endocytosis,
TFEB, ZKSCAN3
DOI 10.1515/cdth-2015-0001
Received November 24, 2014; accepted December 29, 2014
Glossary
mTORC: mechanistic target of Rapamycin complex
Deptor: DEP domain containing mTOR-interacting protein
mLST8/GbL: mammalian lethal with sec-13 protein 8/ G
protein beta subunit-like
mSin1: mammalian stress-activated map kinaseinteracting protein 1
Pras40: proline-rich Akt substrate 40 kDa
Protor1/2: protein observed with rictor 1 and 2
Raptor: regulatory-associated protein of mTOR
Rictor: rapamycin-insensitive companion of mTOR
*Corresponding author: Radek Dobrowolski: Federated Department
of Biological Sciences, Rutgers University/New Jersey Institute of
Technology, Newark, NJ 07102, E-mail: [email protected]
Israel C. Nnah, Khoosheh Khayati: Federated Department of Biological Sciences, Rutgers University/New Jersey Institute of Technology,
Newark, NJ 07102
#
Equal author contribution
1 Introduction
Lysosomes are membrane-bound organelles responsible
for breaking down nutrients, which are taken up from
the extracellular space by endocytosis or from the
cytoplasm by autophagy. As such, lysosomes maintain
cellular homeostasis and mediate many physiological
processes including the clearance of debris and damaged
organelles, energy metabolism, lipid homeostasis, and
plasma membrane repair [1–3]. In addition to housing
integral membrane proteins that contribute to lysosomal
function and membrane integrity, lysosomes contain
more than 50 soluble hydrolases needed for the digestion
of proteins, lipids, nucleic acids and carbohydrates [2].
Collectively, these proteins allow lysosomes to maintain
a pH of 4.5, a calcium concentration of 0.4 - 0.6 mM,
a sodium concentration of 145 mM, and a membrane
potential of 19 mV (Figure 1) [2, 4–6]. Since the discovery
of lysosomes by Christian de Duve in the early 1950s,
the sorting, trafficking, and biogenesis of lysosomal
proteins have been studied extensively using a variety
of methodological approaches [5, 7–9]. Recent studies
broaden the functional characterization of lysosomes;
rather than simple endpoints of the cellular digestive
system, lysosomes are dynamic organelles with their own
signaling complex: the mechanistic target of Rapamycin
complex 1 (mTORC1). This review summarizes recent
findings on lysosomal mTOR function, its contributions to
cellular metabolism, homeostasis, and its dysregulation
in human disease.
2 mTOR and Its Complexes
mTOR, a member of the phosphoinositide 3-kinase (PI3K)related kinase family, is a serine/threonine kinase that
interacts with several proteins to form two structurally
and functionally distinct complexes named mTORC1 and
mTORC2 [1, 10–13]. Both complexes are responsive to
various upstream signals with differential integrations and
downstream outputs. Over the years, mTORC1 has been
shown to control cellular metabolic processes including
© 2015 Israel C. Nnah et al. licensee De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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Figure 1: Lysosomal Homeostasis. Inactivation of mTORC1 is largely regulated by its dissociation from the lysosomal membrane and subsequent replacement by the TSC complex. This process depends on lysosomal homeostasis controlled by v-ATPase, the proton pump which
maintains a pH of 4.5, two-pore channels (TPC), responsible for mTORC1-dependent Na+ efflux, the transient receptor potential cation
channel (TRPML 1-3), calcium efflux channels from the mucolipin protein family, and calcium-proton exchangers. The interplay of all these
channels maintain the lysosomal membrane potential at 19 mV, the calcium concentrations at 0.4 – 0.6 mM and the sodium at 145 mM.
protein and lipid synthesis, lysosomal biogenesis, and
autophagy. Compared to mTORC1, less is known about
mTORC2 function. The sensitivity of mTORC2 to amino
acids is discussed in the field and a number of conflicting
studies exist [14–17]. However, it is widely accepted that
mTORC2 reacts to growth factors, such as insulin, through
a poorly understood mechanism that requires the PI3K
pathway [18]. Although the mechanisms underlying
mTORC2 function are unclear, conclusive studies have
demonstrated that the complex is involved in cell survival,
apoptosis and proliferation [14, 19–22].
The composition of both mTOR complexes is well
characterized. In addition to the mTOR catalytic subunit,
both complexes contain: Deptor, an inhibitor of mTOR
[23], mLST8 (also known as GbL) [24, 25], and the Tti1/
Tel2 complex [26], two structural proteins. Pras40, also
an mTOR inhibitor, and Raptor are exclusive to mTORC1
[27–32], whereas Rictor, mSin1 and Protor1/2 are mTORC2specific components [28, 33, 34]. Importantly, both
complexes are susceptible to such mTOR inhibitors as
Rapamycin or Torin [35, 36]. Given the lack of allosteric
inhibitors for the two complexes, specific inhibition
of either mTORC1 or mTORC2 requires the use of nonpharmacological strategies such as RNAi mediated knock
down of either Raptor or Rictor, respectively.
3 Lysosomes and mTORC1
Signaling
mTORC1 has been shown to integrate major intracellular
and extracellular signaling pathways including growth
factors signaling, amino acids sensing, energy status,
and cellular stress (Figure 3). Such mTORC1-mediated
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signal integration allows for well-controlled regulation of
anabolic and catabolic processes, such as protein and lipid
synthesis, autophagy, lysosomal biogenesis, and energy
metabolism [1]. The complex localizes to lysosomal surfaces
through an amino acid-dependent process involving the
heterodimeric RagA/B-RagC/D GTPases [37, 38] (discussed
later). When localized on lysosomal membranes, mTORC1 is
activated by the membrane-tethered Ras homolog enriched
in brain (Rheb) protein [39, 40]. Thus, two successive
events are required for mTORC1 activation: translocation of
the complex to the lysosomal surface (regulated by amino
acids) and activation by the GTP-bound Rheb (differentially
regulated by cellular signaling).
mTORC1 signaling is dependent on a number of
lysosomal membrane proteins which form the lysosome
nutrient sensing (LYNUS) machinery. Components of this
machinery include the following proteins and affect the
indicated functions: vacuolar ATPase (v-ATPase), the
proton pump that acidifies the lysosomal lumen [41],
Ragulator protein complex, which acts as a guanine
nucleotide exchange factor (GEF) for Rag A and B GTPases
[42], a heterodimeric RagA/B-RagC/D GTPases, which
relay amino acid-induced signals to mTORC1 [42], and
endolysosomal ATP-sensitive sodium (Na+) permeable
channel, which regulates lysosomal membrane potential,
pH stability and amino acid homeostasis by responding
to ATP levels [43]. Comprehensive characterization of the
LYNUS tethering components suggests dependency of
mTORC1 on lysosomal activity. Therefore, it is evident that
lysosomal homeostasis affects many cellular activities
downstream of mTORC1.
The discovery of the LYNUS machinery has made
a major impact on the mTOR field and has contributed
immensely to our understanding of cellular metabolism
in normal and pathological conditions. Indeed, studying
LYNUS changes our view of lysosomes; rather than simple
endpoints of cellular clearance, lysosomes are being
characterized as sensors and regulators of major cellular
functions including growth, energy metabolism, protein/
lipid biosynthesis, autophagy, and lysosomal exocytosismediated plasma membrane repair [3].
4 Regulation of Auto-Lysosomal
Biogenesis
The transcription factor EB (TFEB) protein, a member
of the basic helix-loop-helix leucine zipper family of
transcription factors shown to control expression of many
lysosomal genes, is considered a “master regulator” of
lysosomal biogenesis [44]. Upon activation, TFEB directly
Lysosomal Signaling in Cellular Homeostasis 13
binds to a 10-base pair DNA motif (GTCACGTGAC), known
as the Coordinated Lysosomal Expression and Regulation
(CLEAR) element, in the promoter regions of many
lysosomal and autophagosomal genes, thereby promoting
their transcription [44]. For many years, the expression of
lysosomal genes was thought to be a constitutive process.
Recent studies have revealed that cells constantly regulate
lysosomal function and number in response to their
energetic or degradative needs [45]. As mTORC1 is a wellestablished sensor and regulator of cellular energy status
[1, 46], it is unsurprising that the complex negatively
regulates lysosomal biogenesis through inhibition of TFEB.
Specifically, in fed cells active mTORC1 phosphorylates
TFEB at Serine 211 (S211), the amino acid residue that
mediates TFEB interaction with cytosolic chaperon 14-3-3,
thereby preventing nuclear entry of the transcription
factor [47]. Conversely, in nutrient-deprived cells, the S211
of TFEB remains unphosphorylated allowing TFEB to
translocate to the nucleus to induce transcription of the
CLEAR network genes (Figure 2). Coordinated expression
of the CLEAR gene network promotes autophagosomal
and lysosomal biogenesis [44].
An additional regulatory layer of auto-lysosomal
biogenesis is maintained by ZKSCAN3, a member of the zinc
finger transcription factors harboring Kruppel-associated
box (KRAB) and SCAN domains [48]. Members of this
protein family bind to DNA in a zinc-dependent manner
to repress gene transcription. ZKSCAN transcription
factors play a role in several cellular functions including
maintenance of the nucleolus, cell proliferation, and
apoptosis [49]. Recently, ZKSCAN3 was described to
be the key transcriptional repressor of autophagy [48,
50]. Further, this factor transcriptionally attenuates the
expression of more than sixty genes encoding proteins
involved in various steps of autophagy and lysosomal
biogenesis and function [48]. Interestingly, mTORC1
phosphorylates ZKSCAN3 to regulate its subcellular
localization. In fed conditions, when mTORC1 is active,
phosphorylated ZKSCAN3 shuttles into the nucleus
to repress autophagy genes; under nutrient-deprived
conditions, ZKSCAN3 accumulates in the cytoplasm
enabling persistence of autophagy [48].
Thus, TFEB and ZKSCAN3 are differentially regulated
by mTORC1 to fine-tune the auto-lysosomal system.
However, several key aspects of TFEB and ZKSCAN3
regulation remain unclear. The extent to which ZKSCAN3
interacts with the CLEAR gene network to repress TFEBresponsive genes remains unknown. The molecular cues
guiding ZKSCAN3 and mTORC1 interaction in normal and
disease conditions are currently under investigation.
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Figure 2: Lysosomes and mTORC1 Signaling. The lysosomal membrane harbors vATPase, Ragulator, and the Rags (A/B and C/D) to tether
mTORC1 to the proximity of Rheb, a small GTPase inhibited by TSC. Only membrane-tethered mTORC1 can be activated by Rheb to phosphorylate the two transcription factors: TFEB and ZKSCAN3. Since phosphorylated TFEB binds to 14-3-3 proteins and remains in the cytoplasm,
phosphorylated ZKSCAN3 translocates into the nucleus to inhibit autophagy genes. During starvation, mTORC1 dissociates from the
membranes and is rendered inactive. Under these conditions, TFEB remains unphosphorylated and localized to the nucleus where it binds
to the CLEAR gene network to promote lysosomal and autophagosomal gene expression. Thus, TFEB restores lysosomal activity and cellular
homeostasis by inducing autophagy and cellular clearance.
5 Lysosomal Sensing of Cellular
Signaling
The recruitment and activation of mTORC1 on the
lysosomal surface is driven by two signaling axes: the
amino acid-Rag-GTPase axis and the tuberous sclerosis 1
and 2 (TSC1/2)-Rheb-GTPase axis. The TSC1/2 heterodimer,
also known as hamartin/tuberin, is the key upstream
regulator of mTORC1 activity [1, 51–54]. A myriad of studies
have demonstrated that active (GTP-bound) Rheb directly
interacts with mTORC1 and stimulates its kinase activity
[39, 40, 55]. Conversely, TSC1/2 negatively regulates
mTORC1 by converting Rheb from its GTP-bound active
form to its GDP-bound inactive state. Thus, TSC1/2 acts as
a GTPase-activating protein (GAP) for Rheb [40]. To date,
other direct regulators of Rheb activity have not been
identified, and a GEF for Rheb has yet to be discovered.
The TSC complex acts as a central hub and conduit
(Figure 3), integrating and conveying several upstream
signals that impact mTORC1 activity. Insulin-like growth
factor 1 (IGF1) is a well-characterized signal, which
stimulates the PI3K and Ras pathways upstream of TSC1.
Protein kinase B (Akt/PKB), extracellular-signal-regulated
kinase 1/2 (ERK1/2), and the ribosomal S6 kinase (RSK1)
are the effector kinases of IGF1 signaling and directly
phosphorylate and inactivate TSC1/2 [56, 57]. Akt also
activates mTORC1 in a TSC-independent manner by
phosphorylating PRAS40, an inhibitor of mTORC1, leading
to the dissociation of the protein from the complex [27,
30, 58]. PRAS40 inhibits cell growth, S6 kinase 1 (S6K1)
phosphorylation, and Rheb-induced activation of the
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Lysosomal Signaling in Cellular Homeostasis 15
Figure 3: Sensing of Cellular Signaling by TSC Complex and mTORC1. The TSC complex represents a hub for incoming cellular signals ranging
from hypoxia, DNA damage, and energy levels to growth factors, Wnt, and TNF signals. Signaling mediators and/or kinases modulate the
GAP activity of the TSC complex to inhibit or activate Rheb. An inactive TSC complex results in active mTORC1 thereby promoting protein and
lipid synthesis, energy metabolism, and inhibition of lysosomal biogenesis and autophagy.
mTORC1 pathway [27]. In vitro, PRAS40 prevents Rheb-induced increase in mTORC1 kinase activity [27]. Thus,
IGF1 signals to mTORC1 through Rheb- and PRAS40mediated inputs. Mechanistically, however, it is unclear
how insulin is able to activate mTORC1 through the
coordinated regulation of both inputs.
Despite the importance of the TSC1/2 complex in
mTORC1 activity modulation, recent studies indicate
that mTORC1 may be regulated independently of TSC1/2
via G-protein coupled receptor (GPCR)-mediated calcium
(Ca2+) signaling. Specifically, the neuropeptide orexin, lack
of which induces the chronic sleep disorder narcolepsy,
signals through the orexin/GPCR to affect mTORC1
activity independently of TSC1/2. Further, the receptor
was described as an Erk/Akt-independent, calciumstimulated, v-ATPase-mediated mTORC1 activator [59].
This study demonstrated that the orexin/GPCR activates
mTORC1 pathways in mouse brains and multiple
recombinant cell lines that express orexin 1 and orexin 2
receptors. The orexin/GPCR coordinates extracellular Ca2+
influx and the v-ATPase/Rag GTPases to activate mTORC1
excluding the involvement of Akt and Erk [59]. However, it
is unclear whether orexin/GPCR-mediated Ca2+ signaling
is relayed through v-ATPase or if its function is simply
required to sense amino acid levels in cells. Thus, further
research on this important GPCR will help clarify and
delineate how upstream mTORC1 regulators route signals
affecting the complex’s function.
The canonical Wnt signaling pathway regulates cell
proliferation, differentiation, growth, and development
[60], and is highly dependent on the endo-lysosomal
system [61, 62]. Wnt signaling also activates mTORC1
through TSC1/2 by inhibiting glycogen synthase kinase
3β (GSK3β), which phosphorylates and promotes TSC1/2
activity [63]. Interestingly, GSK3β phosphorylates the
microtubule-associating protein tau, which is implicated
in Alzheimer’s disease [64, 65]. Relatedly, upregulation of
mTORC1 has been associated with increased translation
of tau [66].
Pro-inflammatory cytokines also modulate mTORC1
signaling [67]. Tumor necrosis factor-α (TNFα) is one
of the best-characterized cytokines as it is secreted by
many pro-inflammatory cell types, such as macrophages,
lymphocytes, and fibroblasts [68]. TNFα induces cell
proliferation, differentiation, or apoptosis in a cell
type-specific manner via its receptors, the ubiquitously
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expressed TNFR1 (p55) or the immune system-specific
TNFR2 (p75), and various downstream effectors [69].
Despite thorough studies of the functional role of TNFα,
the mechanism by which the cytokine affects mTORC1
activity remains unclear. A recent study of the mechanism
in MCF-7 cells indicated that TNFα signals independently
of PI3K/Akt to induce IκB kinase (IKK) β-mediated
inactivation of TSC1/TSC2 to promote mTORC1 activity
(Figure 3; [67]). Indeed, suppression of TSC1 by TNFα
may help clarify how inflammation may support tumor
angiogenesis. However, TNFα-mediated modulation of
mTORC1 may not be PI3K-Akt independent and may be
mediated by other IKK isoforms in different cell types [70,
71].
Cellular stressors such as low energy, hypoxia,
endoplasmic reticulum (ER) stress, and DNA damage act
in part through TSC1/2 to negatively modulate mTORC1
activity. Adenosine monophosphate-activated protein
kinase (AMPK) is a sensor of cellular stress [1]. In response
to hypoxia or low energy state, AMPK phosphorylates TSC2
and increases its GAP activity toward Rheb [39]. Similar
to Akt signal transduction, AMPK also signals directly to
mTORC1 by phosphorylating Raptor and subsequently
inducing allosteric inhibition of mTORC1 [72]. P53mediated DNA damage response signals induce the
expression of TSC2 and phosphatase and tensin homolog
deleted on chromosome 10 (PTEN). These proteins
function to down-regulate PI3K-mTORC1 pathways
[73, 74], and activate AMPK through a mechanism that
depends on the induction of stress inducible proteins
known as sestrins [1, 46]. These signals regulate mTORC1
through the TSC complex, but how TSC integrates these
signals to control Rheb is unknown. It will be of relevance
to understand how TSC1/2 is able to assign precedence to
multiple incoming upstream-signals, how the complex
differentiates which signals are more dominant, and
whether a cell-type specific regulatory mechanism exists.
Collectively, the aforementioned signals modulate
mTORC1 activity to control many cellular processes
downstream of mTORC1 (Figure 3). Protein and lipid
synthesis is the most studied of these mTORC1-mediated
cellular functions. mTORC1 promotes protein synthesis
in eukaryotes by directly phosphorylating eukaryotic
translation initiation factor 4E (elF4E)-binding protein 1
(4E-BP1) and S6K1, which regulate protein translation [75,
76]. The complex regulates the synthesis of lipids required
for cell proliferation through the sterol regulatory elementbinding protein 1/2 transcription factors, which control
gene expression involved in fatty acid and cholesterol
synthesis [77–80]. All these mTOR-mediated effects are
only possible if mTORC1 is tethered to the lysosomal
membrane [38, 42], a condition largely controlled by
amino acids.
6 Lysosomal Amino Acid Sensing
Amino acids serve as building blocks of proteins and
play a critical role in energy homeostasis of the cell. This
latter function is evident when amino acids are converted
to acetyl-CoA for ATP production via the Krebs cycle. As
mTORC1 acts as sensor of amino acid levels to regulate and
maintain cellular homeostasis [1, 46], it is unsurprising
that amino acids regulate mTORC1 activity and its
localization to the lysosome. Indeed, it has been shown
that amino acid withdrawal and replenishment change
cellular localization of mTORC1 from the cytoplasm to
the lysosomal membrane, respectively [38, 42]. Studies
on the role of specific amino acids indicate that arginine
and leucine are necessary yet insufficient for mTORC1
activation. Interestingly, leucine may have the strongest
impact on mTORC1 activity [81]. Mechanisms by which
amino acid signals are conveyed to mTORC1 are currently
under study. Zoncu and colleagues proposed that amino
acids are mainly sensed by an “inside-out” mechanism
of vATPase [82]. The presence of amino acids is relayed
through the Ragulator complex and the Rag GTPases
(RagA/B and RagC/D) to mTORC1 [42]. The Ragulator is
a lysosomal membrane-tethered protein complex and a
direct binding partner of the Rag GTPases. It is thought
that the Ragulator recruits Rag proteins to the lysosome by
changing their GTP/GDP-bound status [38]. Interestingly,
the uptake of L-leucine or other essential amino acids is
mediated by a bidirectional transporter that exchanges
cellular L-glutamine for L-leucine from the extracellular
space [83]. Low L-glutamine levels mediate autophagy
and inhibit growth. A number of tumors with high levels
of L-glutamine can bypass this so-called glutamine
sensitivity, resulting in high mTORC1 activity and growth.
Thus, during well-fed conditions or during inhibition of
protein synthesis (for example caused by cyclohexamide),
amino acids alter binding of GTP/GDP to Rags (RagA/B
GTP, RagC/D GDP-bound) to optimize tethering of mTORC1
to the lysosomal membrane.
Amino acid sensing is independent of the insulin/
PI3K pathway and the Rheb axis. First mechanistic
insight into this insulin/PI3K/Rheb-independence was
demonstrated by investigations using TSC2-knockout
mouse embryonic fibroblasts [84] and lysosomal
membrane-untethered Rheb overexpression systems
[38]. In these studies, overexpressed and free Rheb
proteins interacted with cytosolic mTORC1 to activate its
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function. At endogenous levels, binding of the membrane
tethered Rheb to mTORC1 is reversibly inhibited by
withdrawal of all amino acids or just leucine. Thus,
lysosomal tethering of mTORC1 through the RagulatorRag axis is fundamental in the modulation of Rheb/
TSC-mediated mTORC1 activation. Tethering of mTORC1
to the lysosomal membrane is dependent on cellular
nutrient status, and is a direct consequence of the
complex integration of amino acid transporter function,
endocytosis, and autophagy.
7 Additional Mechanisms of
Lysosomal Amino Acid Sensing
In addition to the aforementioned vATPase-mediated
“inside-out mechanism”, several other processes are
involved in lysosomal amino acid sensing. A recentlydescribed pathway involves leucyl-tRNA synthetase
(LeuRS), a member of amino acid tRNA synthetases,
enzymes known to promote loading of tRNAs with their
cognate amino acids [85]. LeuRS was proposed to be a GAP
for RagD in the lysosomal LYNUS complex [86]. However,
these findings could not be completely reproduced by
other groups who subsequently proposed alternative
mechanisms. For instance, Tsun and colleagues suggested
that the tumor suppressor protein folliculin (FLCN), in
complex with the two paralogs FNIP1/2, exhibits GAP
activity towards the RagC and RagD GTPases [87]. Upon
amino acid withdrawal, FLCN/FNIP localizes to the
lysosomal membrane and amino acid replenishment
displaces the FLCN/FNIP complex into the cytoplasm.
Interestingly, FNIPs directly bind to AMPK, known as a
cellular energy sensor strictly regulating the AMP/ATP
ratios. This is consistent with the observation that FLCN
affects mTORC1 activity by binding and activating RagC/D
[87]. However, it is curious that both the RNAi mediated
knock-down of LeuRS and FLCN, as presented by Han or
Tsun and colleagues, respectively, prevented the amino
acid-induced translocation of mTOR to lysosomes and
consequently its activity. Thus, LeuRS and FLCN may act
in concert to promote mTORC1 activity.
GATOR is another tumor suppressor protein complex
recently characterized as a GAP for Rags A and B. GATOR
is composed of the subcomplexes GATOR1, the GAPcontaining subcomplex, and GATOR2. GATOR2 negatively
regulates GATOR1 to render mTORC1 unresponsive to
amino acids [88]. Some glioblastoma, lung, ovarian, and
breast cancer cells have been found to carry inactivating
mutations in GATOR1 subunits [88–91]. These mutations
make cells insensitive to amino acid depletion and
Lysosomal Signaling in Cellular Homeostasis 17
hypersensitize them to Rapamycin. Interestingly,
Rapamycin hypersensitivity is a common characteristic of
tumor cells in which viability is minimally maintained due
to high mTORC1 activity [88]. It is of interest to determine
how this high mTORC1 signaling integrates with glycolytic
and other metabolic pathways in tumor cells, as such
mechanistic insight will instruct the development of
toxicity- and adverse effect-free anticancer drugs.
A recent study identified the stress-sensing proteins
called guanine nucleotide dissociation inhibitors (GDIs)
for RagA/B [92] through GATOR2 [93]. Sestrins, regulated
by p53 (sestrin 1 and 2) and the transcription factor Foxo
(sestrin 1 and 3), sense mainly oxidative and nutritional
stress. Overexpression of sestrins inhibits mTORC1
tethering to lysosomes, whereas deletion of all three
sestrin isoforms prevents the mTORC1 inactivation during
starvation resulting in reduced survival during postnatal
fasting.
Several cellular proteins, such as Ragulator (the GEF
for Rags), folliculin/FNIP (GAP for RagC/D), the GATORs
(GAPs for RagA/B), and sestrins (GDIs for RagA/B)
collectively regulate amino-acid-mediated tethering of Rag
GTPases to mTORC1. Although the current understanding
of the mTORC1 amino acid sensing machinery seems
robust. However, how these factors interact with each
other and how leucine concentrations are precisely sensed
remains to be clarified.
8 mTORC1 Association With Other
Organelles
mTORC1 predominantly associates with lysosomal
markers. However, emerging evidence suggests that
mTORC1 may also associate with other organelles [94].
Mitochondria and the nucleus have been proposed as
other sites harboring pools of mTORC1 [95–98]. A series of
convincing studies associate mTORC1 to stress granules,
the membrane-free aggregations of cytoplasmic proteins
and RNA. In response to stress, mTORC1 is sequestered
to these compartments and kept inactive to arrest cell
growth under stress conditions [28, 99, 100]. It is of
interest to compare the functional importance of mTORC1
associations with lysosomes with those of other organelles.
Whether mTORC1 sub-cellular localization impacts its
activity, specificity, or responsiveness to incoming signals
needs to be addressed in future studies.
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9 Conclusions
Lysosomes play a central role in the degradation of
intracellular cargo. Disruptions in lysosomal functions
have been described in various human lysosomal storage
diseases, such as Niemann-Pick disease types A-C,
neuronal ceroid lipofuscinoses, and Krabbe disease. Over
a decade of research implicate lysosomal malfunction in
neurodegenerative conditions such as Parkinson’s and
Alzheimer’s disease (AD) [101]. Interestingly, lysosomal
inhibition has been observed to precede the well-known
AD molecular hallmarks, such as increased β-amyloid
cellular load and hyperphosphorylated tau. Indeed,
dystrophic neurites characteristic for AD are filled with
cathepsin-positive auto-lysosomes, aggregated Tau
proteins [102], and multivesicular bodies (MVBs)/late
endosomes enriched in amyloid precursor protein (APP)
derivatives [103, 104]. It has been proposed that Aβ42
localized to MVBs [104] is released from neurons via
exosomes [105] to form extracellular amyloid deposits
[106]. More recent studies have shown accumulation
of autophagic vacuoles in hippocampal neurons
[107], increased rate of endocytosis, and high cellular
cholesterol content in AD models [108] potentially
caused by lysosomal dysfunction. Furthermore, certain
polymorphisms in the lysosomal protease cathepsin
D increase risk for AD [109]. As discussed in the field,
it is plausible that dysregulated molecular trafficking
and defective autophagosomal clearance initiate
amyloidogenesis and subsequently phospho-Tau
aggregation [110–112]. Therefore, understanding the
mechanisms regulating lysosomal function is expected to
greatly aid in deciphering neurodegenerative disorders,
and potentially provide therapeutic means of combating
these diseases.
The efforts of our scientific community over the
last decade revealed the complexity of the mTORC1
signaling pathway, which is consistent with its function
in integrating diverse amino acid and cellular signaling
at the lysosome, a key metabolic hub. As we continue
to study and refine our understanding of the function of
each participating cellular protein regulating mTORC1, it
appears that multiple layers of signal integration exist. In
addition to mTORC1, several other protein complexes are
regulated by cellular signaling and directly impact amino
acid sensing. Two good examples of such bi-functional
proteins are the sestrin protein family and the FNIPs.
Sestrins are regulated by p53 and AMPK and exhibit GDI
functions towards the mTORC1-tethering Rags; FNIPs
function with FLCN to impart GAP activity on Rag C/D
and tether mTORC1 to lysosomal membranes, possibly
in an AMPK-dependent manner. Thus, AMPK and p53
signaling may be conveyed via sestrins and FNIPs to
directly modulate the tethering and activation of mTORC1.
This hypothesis, however, remains to be tested in future
studies.
Advances in our understanding of normal and
pathological cellular stress, energy/nutrient management,
and related signaling have supported the development
of more specific mTOR inhibitors. The ability of these
new compounds to fight neurodegeneration and cancer
is currently being tested in clinical trials [113, 114].
Alternatively, drugs targeting mTORC1-interacting
proteins, rather than mTORC1, such as the Ragulator
components, sestrins, and FLCN, may be a more specific
and therefore potentially more efficacious strategy.
Relatedly, it will be important to investigate the role
of various proteins in the association of mTORC1 and
mTORC2 to non-lysosomal organelles, as deeper insight
into the functions of the complexes localized to different
organelles will help clarify drug development strategies
[94]. Despite the enormous increase in knowledge about
the lysosomal mTOR signaling over the last few years,
it seems that much remains to be discovered about its
implications in human pathophysiology and maintenance
of cellular homeostasis.
Acknowledgments: We would like to thank the members
of our laboratory for their helpful comments during our
meetings. We would also like to thank the NIH for the
MBRS fellowship (to I.C.N., grant # R25GM096161) and
the American Federation for Aging Research (AFAR, grant
# RAG13447) for the generous support of our studies (to
R.D.).
Conflict of interest statement: Authors state no conflict
of interest.
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