Commentary
2359
Autophagy and cell growth – the yin and yang of
nutrient responses
Thomas P. Neufeld
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA
[email protected]
Journal of Cell Science
Journal of Cell Science 125, 2359–2368
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.103333
Summary
As a response to nutrient deprivation and other cell stresses, autophagy is often induced in the context of reduced or arrested cell growth.
A plethora of signaling molecules and pathways have been shown to have opposing effects on cell growth and autophagy, and results of
recent functional screens on a genomic scale support the idea that these processes might represent mutually exclusive cell fates.
Understanding the ways in which autophagy and cell growth relate to one another is becoming increasingly important, as new roles for
autophagy in tumorigenesis and other growth-related phenomena are uncovered. This Commentary highlights recent findings that link
autophagy and cell growth, and explores the mechanisms underlying these connections and their implications for cell physiology and
survival. Autophagy and cell growth can inhibit one another through a variety of direct and indirect mechanisms, and can be
independently regulated by common signaling pathways. The central role of the mammalian target of rapamycin (mTOR) pathway in
regulating both autophagy and cell growth exemplifies one such mechanism. In addition, mTOR-independent signaling and other more
direct connections between autophagy and cell growth will also be discussed.
This article is part of a Minifocus on Autophagy. For further reading, please see related articles: ‘Ubiquitin-like proteins and autophagy at a glance’ by Tomer Shpilka et al.
(J. Cell Sci. 125, 2343-2348) and ‘Autophagy and cancer – issues we need to digest’ by Emma Liu and Kevin Ryan (J. Cell Sci. 125, 2349-2358).
Key words: Autophagy, Cell growth, Mammalian target of rapamycin (mTOR), Protein synthesis, Lysosome, Cell cycle
Introduction
Cells have an intrinsic drive to increase their mass, which is
illustrated by the ravenous growth of embryos and juveniles
during development, and the logarithmic proliferation of cells in
culture. Even in adult organisms, whose size has reached a steady
state, continued growth and proliferation of differentiated and
stem cells allows replacement of damaged and senescent cells,
and many cells and organs are capable of considerable growth
through hypertrophy. Cell growth can thus be considered a
default state for many cell types, requiring only the presence of
permissive factors such as nutrients and appropriate hormonal
signals. The process of cell growth, however, has enormous
energetic requirements and is rapidly abandoned when conditions
become unfavorable. In response to nutrient limitation, DNA
damage, excessive oxidation, viral infection and other cell
stresses, the ATP-consuming processes that underlie cell
growth are rapidly turned off, allowing the cell to conserve or
marshal its resources to deal with the stressor.
In addition to switching off energetically demanding growth
processes, cells induce a variety of responses to stress that enable
them to survive in suboptimal conditions. Central among these is
macroautophagy (herein referred to as autophagy), whereby
portions of cytoplasm are sequestered into vesicles known as
autophagosomes and degraded by hydrolytic enzymes following
fusion of autophagosomes with the lysosome. This process
supports cell survival by eliminating damaged and potentially
harmful cellular structures, and by releasing the breakdown
products as nutrients that can be re-used by the cell or exported
for use by other cells.
On a fundamental level, autophagy and cell growth are mirror
images of one another. If cell growth is defined as the process of
mass accumulation through the net uptake and conversion of
nutrients into macromolecules, autophagy can be considered to
act in opposition to these biosynthetic processes through the
catabolic breakdown of biomolecules. Thus, under conditions
conducive to cell growth, the coupling of high rates of
biosynthesis with low rates of autophagy allows cellular
resources to be channeled towards growth with maximal
efficiency. This synthetic flow is reversed in response to
nutrient limitation or other stresses that halt cell growth and
induce autophagy. This inverse correlation between autophagy
and growth is widely observed under a variety of environmental
conditions. For example, growth inhibition and autophagy
induction are coordinated responses to nutrient starvation,
growth factor withdrawal, cellular stresses such as oxidative
damage, protein misfolding, viral infection, and substrate
detachment or contact inhibition of cultured cells (Fig. 1)
(reviewed by Neufeld, 2004).
Although the coupling of autophagy and growth might appear
intuitive and logical, the potential cause-and-effect relationships
between these two processes and the underlying mechanisms that
link them are just beginning to be unraveled. In this Commentary,
I highlight key regulatory steps and signaling pathways involved
in autophagy and growth control, and discuss the regulatory
relationships by which these processes are coordinated in the cell.
Understanding the physiological benefits of this coordination and
the consequences of its disruption should provide insight into
potential autophagy-based therapeutic approaches.
2360
Journal of Cell Science 125 (10)
A
Journal of Cell Science
B
C
Autophagy and the cell growth machinery
Formation of autophagosomes and their fusion with lysosomes
are the defining events of autophagy. Whereas autophagosome–
lysosome fusion largely exploits general factors of the endocytic
pathway, autophagosome formation is driven by the coordinated
activity of distinct sets of components that are dedicated to this
process. Many of these components were first identified through
pioneering genetic screens in yeast, and have clear homologs in
metazoan cells. These include: (1) two ubiquitin-like molecules,
Atg12 and Atg8 [microtubule-associated protein 1 light chain 3
alpha (LC3) and additional family members in mammals], and
the E1-, E2- and E3-like processing machinery that regulates
their conjugation to control autophagosome formation and size;
(2) a multi-component protein complex containing the class III
phosphatidylinositol 3-kinase Vps34 (PIK3C3 in mammals),
which promotes autophagosome nucleation from cytosolic
membranes; and (3) a complex containing the serine/threonine
protein kinase Atg1 (ULK1 and ULK2 in mammals), which
integrates signals from multiple upstream regulators to control
the localization or activity of Vps34 and other autophagy
components. One such regulator, the protein kinase target of
rapamycin [Tor1 and Tor2 in yeast; mammalian TOR (mTOR) in
mammals], has a central role in controlling Atg1 and ULK1
activity in response to nutrients and growth conditions. Together,
these three systems act in concert, along with a number of
accessory proteins, to form autophagosomes at a rate that is
Fig. 1. Correlation between cell size and autophagy.
(A) Bax2/2, Bak2/2 mouse bone marrow-derived cells that were
cultured in the presence (left) or absence (right) of the growth
factor interleukin 3 for six weeks. Of note are the reduced
cytoplasmic volume and abundant autophagosomes (arrows).
Image reprinted from Lum et al., 2005 with permission from
Elsevier (Lum et al., 2005). (B) Camera lucida drawings of
sectioned larval fat body cells from fed (left) or three-day starved
(right) Drosophila larvae. Dark circular structures represent lipid
droplets. Image reprinted from Butterworth et al., 1965 with
permission from John Wiley and Sons (Butterworth et al., 1965).
(C) Histogram indicating cell size (forward light scatter) of
Atg52/2 mouse embryonic fibroblasts carrying a doxycyclinerepressible Atg5 transgene. Cells were starved (ST+, right) in
amino-acid-free DMEM or incubated in normal cell culture media
(ST-, left), in the presence or absence of doxycycline (DOX).
Repression of Atg5 leads to increased cell size under starvation
conditions. Image reprinted from Hosokawa et al., 2006 with
permission from Elsevier (Hosokawa et al., 2006).
appropriate for specific environmental conditions (reviewed by
Mizushima et al., 2011).
The biosynthetic pathways that promote the accumulation of
mass to drive cell growth are also highly coordinated. The best
understood of these processes is the control of protein synthesis,
which is generally rate limiting for cell growth and sufficient to
drive cell transformation (reviewed by Proud, 2007). The activity
of translation initiation and elongation factors [e.g. eukaryotic
translation initiation factors (eIF) 2, 3 and 4 and eukaryotic
translation elongation factor 2 (eEF2)] are tightly regulated by
protein kinases such as mTOR and Gcn2 (for general control nonrepressed 2, also known as EIF2AK4) in response to nutrient and
growth factor levels. The production of ribosomes is similarly
sensitive to growth conditions, and this regulation encompasses all
three RNA polymerases, as well as post-transcriptional controls, to
ensure a balanced production of ribosomal proteins and RNAs.
More recently, we have begun to define the signals that link
cell growth to lipid synthesis and organelle biogenesis, and
these appear to involve a complex interplay of transcriptional,
translational and post-translational control (reviewed by Zoncu
et al., 2011b). It is crucial that the synthesis rates of proteins, lipids
and other biomolecules are regulated in parallel, and that these, in
turn, are linked to the rates of nutrient uptake and, finally, to the
overall growth and division rates of the cell and its organelles.
Together, these regulatory steps represent nodes of control at
which interactions between cell growth and autophagy can occur.
Cell growth and autophagy
Coordination of these processes can thus be achieved either
through common signaling pathways that intersect at multiple
independent control points, or through autophagy- or growthdependent effects and signals (Fig. 2). Here, each of these models
is considered in turn.
Coordinated regulation of growth and autophagy
by common signals
An efficient means of achieving coordination between autophagy
and cell growth rates is through the joint regulation of these
processes by shared upstream signaling pathways. Although the
molecules that act directly on autophagy and biosynthesis are
distinct, a number of regulators have been shown to control both
processes in parallel (Fig. 3).
Journal of Cell Science
mTOR-dependent regulation of autophagy and growth
As alluded to above, the kinase mTOR has a central role in
controlling both cell growth and autophagy. mTOR controls the
translation initiation step through direct phosphorylation of two
key targets, EIF4E-binding protein 1 (EIF4EBP1) and S6 kinase
(S6K, also known as RPS6KB1), which, in turn, regulate eIF3and eIF4-dependent interactions between the mRNA 59 cap, the
poly(A)-tail and the 40S and 60S ribosomal subunits (Zoncu et al.,
2011b). mTOR also has a major impact on protein synthesis and
cell growth by promoting ribosome biogenesis. In yeast, this
involves inhibitory phosphorylation of three transcription factors,
Stb3, Dot6 and Tod6, which act as repressors of ribosome
biogenesis (RiBi) and ribosomal protein (RP) gene expression
(Huber et al., 2011). This phosphorylation is mediated by the
protein kinase Sch9, a yeast homolog of S6K and AKT, and a
direct TOR substrate. mTOR also contributes to ribosome
biogenesis by promoting expression of ribosomal RNA. Recent
studies indicate that there is a nucleolar role for mTOR
complexes directly at the promoters of rRNA and tRNA genes,
and have identified the RNA polymerase I and III factors TIF1A
(also known as TRIM24), TFIIIC (also known as GTF3) and
MAF1 as downstream targets (Kantidakis et al., 2010; Mayer
et al., 2004; Vazquez-Martin et al., 2011).
Independent of these effects on protein synthesis, mTOR
regulates the autophagic machinery and suppresses
autophagosome formation under conditions that are favorable
for growth. Autophagy inhibition by mTOR largely occurs
independently of the targets known to be involved in growth
A
Signal
B
Signal
C
Signal
Integrator
Autophagy
Cell growth
Autophagy Cell growth
Cell growth
Autophagy
Fig. 2. Potential signaling relationships between autophagy and cell
growth. The inverse correlation between rates of cell growth and autophagy
can be accomplished through several distinct mechanisms. Common upstream
signaling pathways (A) can provide a coordinated effect on autophagy and
cell growth. Such pathways bifurcate at integration points that independently
control the autophagy and growth machinery downstream. Inhibitory effects
of autophagy on cell growth (B) and of cell growth on autophagy (C) can
provide a direct mechanism for linking these processes. Please refer to the text
for additional details.
2361
regulation, such as EIF4EBP1 or S6K. Instead, mTOR directly
interacts with and phosphorylates components of the Atg1 protein
kinase complex, including Atg1 (ULK1), Atg13 and FIP200 (also
known as RB1CC1) (reviewed by Mizushima, 2010). In yeast,
Atg13 is phosphorylated on multiple serine and threonine
residues under growth conditions, and this disrupts its
association with Atg1. Ohsumi and co-workers recently found
that Atg13 is phosphorylated directly by TOR in vitro, and
showed that Atg13 lacking these phosphorylation sites stably
associates with, and activates, Atg1 and is sufficient to promote
autophagy under growth conditions, independent of TOR activity
(Kamada et al., 2010). This ability of non-phosphorylatable
Atg13 to bypass TOR signaling highlights the Atg1 complex as
the crucial downstream target mediating the effects of TOR on
autophagy.
How TOR-dependent phosphorylation of Atg13 promotes
Atg1 activation and induction of autophagy remains poorly
understood. Interestingly, Herman and colleagues have
demonstrated that Atg1 forms dimers or oligomers under
conditions that favor autophagy, and that this self-association is
promoted by Atg13 (Yeh et al., 2011). Forced dimerization
of Atg1 increases its kinase activity and the level of
autophosphorylation, which is a prerequisite for inducing
autophagy (Kijanska et al., 2010; Yeh et al., 2010). However,
this is not sufficient to induce autophagy, suggesting that Atg13
has additional functions. Regulation of metazoan Atg1 (ULK1)
appears to differ from that in yeast, as mTOR inhibits ULK1
without affecting its interaction with Atg13. A functional role
for Atg13 phosphorylation has not yet been demonstrated
in mammals, and mTOR-mediated phosphorylation of other
components of the ULK1 complex might have important roles.
Recently, Ser757 of ULK1 was identified as an mTORdependent phosphorylation site that influences the association
of ULK1 with the AMP-activated protein kinase (AMPK), which
is an important activator of autophagy in response to cellular
energy levels (Kim et al., 2011a; Shang et al., 2011) (see below).
The potential role of mTOR to regulate later steps of
autophagosome maturation, movement and lysosomal fusion is
less well characterized. At least in some cell types, mTOR
activation can promote autophagosome–lysosome fusion, in part
by facilitating the interaction of Rab7 with its lysosomal effector
Rab-interacting lysosomal protein (RILP) (Bains et al., 2009;
Bains et al., 2011; Yamamoto et al., 2006). Furthermore,
reactivation of mTOR during sustained nutrient starvation is
required to allow re-formation and recycling of lysosomes
following the massive delivery of autophagic membrane and
cargo. This recovery of mTOR activity and lysosomal integrity is
dependent on autophagic degradation and efflux, suggesting that
nutrients derived from the autolysosome are able to promote
mTOR activation (Rong et al., 2011; Shin and Huh, 2011; Yu
et al., 2010). Nutrient efflux from autolysosomes also appears
to promote TOR signaling required for secretory activity in
senescent cells (Narita et al., 2011).
Recent investigations into the nutrient-dependent regulation
and localization of mTOR are consistent with this model. In
response to an increase in nutrient levels, mTOR is recruited to
the surface of lysosomes by members of the Rag family of
heterodimeric GTPases (Sancak et al., 2010). The lysosomal
translocation of mTOR requires nutrient-dependent GTP loading
of the Rag complex, and this recruitment is sufficient to bypass
the nutrient input to mTOR activation. Interestingly, nucleotide
2362
Journal of Cell Science 125 (10)
Amino acids
Gl
Glucose
Δψ, Π, pH
tRNAs
Energy
AMPK
TOR
CKIs
Lysosome
CDKs
Gcn2
Atg1
Vps34
Actively translating
ribosome
Endoplasmic reticulum
Journal of Cell Science
Gcn4
pRb E2F
Autophagy gene expression
Fig. 3. Effects of cell growth on autophagy. Growth-dependent import of nutrients, such as amino acids and glucose, generates multiple signals that influence
the induction of autophagy through their effects on pH, osmotic pressure (P) and the electrochemical gradient (DY), as well as through several key signaling
molecules such as Gcn2 (EIF2AK4 in mammals), TOR (or mTOR) and AMPK. The eIF2a kinase Gcn2 senses reduced amino acid levels through the
accumulation of uncharged tRNAs, and activates autophagy-related gene expression through the transcription factor Gcn4 (ATG4 in mammals). Amino acids
activate the mTOR pathway, in part, by recruiting mTOR complexes to the lysosomal surface through factors such as the Rag GTPases and p62, and through
effects on intracellular lysosomal positioning. The AMP-activated protein kinase (AMPK) responds to glucose uptake, and regulates multiple downstream targets
that have effects on autophagy, including mTOR, Atg1 (ULK1 in mammals) and CDKs (through cyclin kinase inhibitors; CKIs). The induction of autophagy can
also be suppressed by the presence of actively translating ribosomes on the surface of the endoplasmic reticulum, which might limit the availability of sites for
autophagosome nucleation. Additional components of TOR, Atg1 and Vps34 complexes are indicated. Please refer to the text for details.
loading of Rag GTPases is sensitive to the amino acid
concentrations within the lysosome, and subunits of the
vacuolar ATPase protein pump are required for this signaling
(Zoncu et al., 2011a). The microtubule-dependent positioning of
lysosomes within the cell has also been shown to influence
mTOR activity, and this appears to be mediated by nutrientdependent changes in intracellular pH (Korolchuk et al., 2011).
Importantly, lysosome-mediated regulation of mTOR influences
multiple, if not all, aspects of mTOR function, including its effects
on targets involved in cell growth and autophagy. This implies that
mTOR might retain its activation state for some time after its
departure from the lysosomal surface and diffusion or transport to
other cellular compartments. Indeed, activation of mTOR might be
required for its release from the lysosome (Ohsaki et al., 2010).
Compartmentalization of mTOR from its targets, such as ULK1,
has been suggested to promote autophagy during senescence, but
the extent to which similar mechanisms contribute more generally
to mTOR signaling is unclear (Narita et al., 2011). Live imaging
and fluorescence recovery after photobleaching (FRAP)-based
analysis of mTOR complexes might help to clarify this issue. This
centralized control of mTOR activation status probably makes a
substantial contribution to coordinating its diverse functions in
autophagy and cell growth.
mTOR-independent control of autophagy and growth
Despite the multiple inputs to mTOR signaling and its pervasive
effects on autophagy and cell growth, a surprisingly large number
of signaling pathways and regulators have been shown to affect
these processes independently of changes in mTOR activity.
Small-molecule screens have identified several compounds that
induce autophagy without affecting mTOR. These chemicals
inhibit a variety of intracellular targets and processes, including
ion channels, Ca2+ homeostasis and G-protein signaling
(Williams et al., 2008; Zhang et al., 2007). A strong correlation
has been observed between the ability of these compounds to
promote long-lived protein degradation and to increase cellular
levels of phosphatidylinositol 3-phosphate [PtdIns(3)P], the
product of Vps34, thus implicating this kinase as an ultimate
autophagic effector. Despite some reports linking Vps34 to
mTOR activation (Byfield et al., 2005; Nobukuni et al., 2005), no
effect on the phosphorylation of mTOR substrates has been found
for these compounds (Williams et al., 2008; Zhang et al., 2007).
Similarly, a genomic siRNA screen for autophagy regulatory
genes has found little correlation between autophagy induction
and mTOR activity, whereas nearly half of the 236 identified hits
had a substantial effect on PtdIns(3)P levels (Lipinski et al.,
2010). Gene ontology analysis of these hits has revealed a
Journal of Cell Science
Cell growth and autophagy
substantial enrichment in signaling molecules and transcription
factors, including multiple genes involved in growth factor and
cytokine signaling that have well-characterized effects on cell
growth. Taken together, these results indicate that the ability of
signaling pathways to regulate autophagy and cell growth in
parallel is not confined to the mTOR pathway and might be
widespread.
The oncogenic Ras–RAF–MAPK cascade stimulates cell
growth through multiple transcription factors including MYC,
JUN (also known as transcription factor AP-1) and ETS1, which
target genes that regulate cell cycle progression, ribosome
biogenesis and cytokine signaling (McCubrey et al., 2007).
Ballabio and colleagues recently identified the mammalian
transcription factor EB (TFEB) as a master regulator of a large
number of autophagy and lysosomal genes that facilitate
the coordination of autophagosome formation, fusion and
degradation (Settembre et al., 2011). Furthermore, this group
showed that TFEB can be negatively regulated through direct
phosphorylation by extracellular-signal-regulated kinase 2
(ERK2), which is independent of mTOR activity. Similarly in
yeast, Ras and its downstream target cAMP-dependent protein
kinase (PKA) inhibit the starvation-induced expression of
autophagy genes, such as ATG8, independently of TOR, and
PKA promotes expression of growth gene networks such as the
RiBi regulon through inactivation of the repressor Tod6 (Graef
and Nunnari, 2011; Lippman and Broach, 2009). Thus both
mammalian and yeast Ras signaling pathways use a coordinated
transcriptional mechanism to link growth stimulation and
autophagy inhibition in response to nutrients.
This transcriptional response is probably shared by many other
factors that regulate growth in response to multiple stimuli.
Botstein and colleagues found that 25% of all yeast genes
display a similar transcriptional response to changes in nutrient
conditions regardless of carbon source, a pattern they termed a
‘universal’ growth rate response (GRR) (Slavov and Botstein,
2011). Growth-related genes involved in ribosome biogenesis and
translation display a positive universal GRR (i.e. increased
transcription in rich medium), whereas autophagy- and vacuolarassociated genes have a negative GRR. Although such
widespread responses certainly reflect the activity of multiple
transcriptional regulators, individual transcription factors can
also generate coordinated effects on growth and autophagy. For
example, the yeast transcriptional regulator Gcn4 responds to
amino acid starvation by promoting expression of autophagy
genes and repressing genes encoding ribosome proteins and
translation factors (Natarajan et al., 2001). The synthesis of Gcn4
protein itself increases under conditions of general translation
inhibition, providing an additional layer of connection between
autophagy and cell growth (Hinnebusch, 1997).
In metazoans, the FOXO family of transcription factors
suppresses cell growth through the expression of inhibitors of
translation and proliferation (Jünger et al., 2003; Stahl et al.,
2002). Concurrently to this, they stimulate autophagy by
activating the expression of a large number of autophagyassociated genes (Mammucari et al., 2007; Zhao et al., 2007).
Interestingly, FOXO1 also has a transcription-independent
capacity to promote autophagy. This process involves
deacetylation of cytoplasmic FOXO1 in response to starvation
or oxidative stress, which promotes its association with ATG7
and leads to autophagy through an, as yet, unclear mechanism
(Zhao et al., 2010). Dual nuclear and cytoplasmic effects have
2363
also been described for p53, which induces autophagy in
response to DNA damage through the activation of target genes
such as those encoding the lysosomal protein DNA-damage
regulated autophagy modulator 1 (DRAM1) and ULK1, as well
as factors, such as sestrin 2, phosphatase and tensin homolog
(PTEN) and tuberous sclerosis 2 (TSC2), that inhibit mTOR
signaling (Crighton et al., 2006; Feng et al., 2005; Gao et al.,
2011; Maiuri et al., 2009). These inductive effects on autophagy
through p53-mediated transcription are opposed by a cytoplasmic
function of p53, which increases mTOR activity and suppresses
autophagy under basal conditions (Tasdemir et al., 2008).
Other factors also regulate cell growth and autophagy
through a combination of mTOR-dependent and -independent
mechanisms. For example, in addition to its transcriptional
effects noted above, PKA also inhibits autophagy in yeast by
directly phosphorylating Atg1 and Atg13, which prevents their
localization to the sites of autophagosome assembly (Stephan
et al., 2009). At the same time, PKA activation increases the
sensitivity of cells to rapamycin and exacerbates the growth
defects of tor mutants, which indicates that PKA has antagonistic
effects on TOR (Ramachandran and Herman, 2011). Taken
together, these results suggest that PKA sends both positive and
negative signals to the autophagy network, and that the inhibitory
effect is dominant under most conditions. Similarly, the energysensing AMPK regulates autophagy both upstream and
downstream of mTOR. By inhibiting mTOR activity under
conditions of low energy, AMPK indirectly promotes activation
of the ULK1 complex. In addition, AMPK can directly
phosphorylate ULK1, in this case leading to further activation
and induction of autophagy (Egan et al., 2011; Kim et al., 2011b;
Shang et al., 2011).
Regulation of autophagy by cell growth
As discussed above, the inverse relationship between cell growth
and autophagy can result in part from their co-regulation.
However, this close correlation is also consistent with a causeand-effect connection between these processes, and suggests
that autophagy and cell growth might inhibit each other
independently of shared upstream signaling components.
We can examine this issue from the viewpoint of protein
synthesis and cell cycle progression, two processes that are
central to cell growth and proliferation. The first obligate step in
increasing cellular protein mass is the import of amino acids. The
regulation of this process in metazoan cells is poorly defined, but
it is clear that growth factor signaling promotes the expression of
a variety of nutrient transporters on the cell surface, in part by
blocking their endocytic degradation (Edinger, 2007; Edinger and
Thompson, 2002; Hennig et al., 2006). Amino acid uptake in
growing cells might directly lead to mTOR activation and hence
suppression of autophagy. However, rapid incorporation of
amino acids into growing peptide chains might limit their free
intracellular concentration and thus mTOR activation. Transport
of amino acids into the cell can also have secondary
consequences, such as changes in osmolarity and cell
membrane polarization, which have been shown to influence
autophagic signaling (Häussinger et al., 1990). In addition, there
is evidence that some amino acid transporters might act as
‘transceptors’ with dual transport and signaling functions, and
mTOR and PKA have been implicated as targets of such
molecules (Taylor, 2009).
Journal of Cell Science
2364
Journal of Cell Science 125 (10)
Subsequent steps in protein synthesis can also have substantial
regulatory effects on autophagy. Following their transport into
the cell, amino acids are ligated to their cognate tRNAs by
aminoacyl-tRNA synthetases. Disruption of this process causes
accumulation of uncharged tRNAs, which directly activates the
eIF2a kinase Gcn2 (also known as EIF2AK4 in mammals),
leading to translation arrest, upregulation of GCN4 and increased
expression of autophagy genes (Hinnebusch, 1994). In addition,
export of tRNA from the nucleus is inhibited by starvation, and
depletion of the tRNA nuclear exporter XPOT can activate
autophagy in the presence of abundant nutrients (Huynh et al.,
2010). In this case, autophagy correlates with reduced mTOR
activity, which indicates that tRNAs can influence multiple
signaling pathways.
In rapidly growing cells, the elongation of polypeptide chains
might also inhibit autophagosome formation. Recent studies have
implied that PtdIns(3)P-rich domains of the rough endoplasmic
reticulum (ER) are central staging areas of autophagosome
initiation (Axe et al., 2008). The tight juxtaposition of the
growing autophagic membrane with the ER surface (HayashiNishino et al., 2009; Ylä-Anttila et al., 2009) suggests that
engagement of active ribosomes with the ER translocon might be
incompatible with this initiation process. In rapidly growing
cells, high occupancy of the surface of the ER with translating
ribosomes might therefore limit its availability as a platform for
autophagosome formation (Blommaart et al., 1995).
The cell division cycle of proliferating cells provides another
stage for the interaction between autophagy and growth control.
It has been known for some time that cells in mitosis are resistant
to a variety of autophagic stimuli, including starvation and
mTOR inhibition (Eskelinen et al., 2002), and this might
provide a crucial barrier to autophagic degradation of spindle
components, genetic material and other exposed cellular
structures. A study using multiple cell cycle markers and a
variety of autophagy inducers found that each stimulus had
maximal effects in the G1 and S phases of the cell cycle, with
little activity in the G2 and M phases (Tasdemir et al., 2007).
This autophagy timecourse corresponds inversely with activity of
the mitotic protein cyclin-dependent kinase 1 (CDK1). Yuan and
colleagues recently identified Vps34 as a substrate of CDK1–
cyclin-B during mitosis in human cells (Furuya et al., 2010). That
study found that CDK1-mediated phosphorylation of Vps34 on
Thr159 disrupts its association with beclin 1, an essential core
component of Vps34 complexes, thereby reducing the lipid kinase
activity of Vps34. Thr159 phosphorylation of Vps34 increases
during mitosis, correlating with a reduction in autophagy. These
findings suggest that suppression of autophagy during mitosis can
result from CDK1-dependent inhibition of Vps34 activity. By
contrast, yeast Cdk1 appears to have a positive function in
autophagy because loss of Cdk1 activity leads to G1 arrest and
autophagy inhibition (Yang et al., 2010). The region of Vps34
that surrounds Thr159 is not well conserved in yeast, which
indicates that other Cdk1 substrates probably mediate its effects
on autophagy.
Regulation of autophagy by CDKs might be a general
phenomenon. The CDKN1B (also known as p27 and KIP1) and
CDKN2A (also known as p16 and INK4) families of G1 CDK
inhibitors are important regulators of cell cycle and growth in
response to stress, hormonal and developmental signals. Recent
studies have revealed that these factors have a similarly crucial
role in regulating autophagy by integrating signals from multiple
upstream pathways. Liang and co-workers have shown that
autophagy is inhibited in mouse embryonic fibroblasts that lack
p27, and that overexpression of p27 or depletion of CDK2 or
CDK4 induces autophagy in breast adenocarcinoma cells (Liang
et al., 2007). Both p27 and p16 are linked to autophagy through
the tumor suppressor retinoblastoma 1 (RB1, also known as pRb),
which is a central downstream target of CDK4 in cell cycle
regulation. RB1 is required for the induction of autophagy by p27
and p16, and can trigger autophagy when overexpressed (Jiang
et al., 2010). RB1 controls cell cycle progression, cell growth and
survival by binding to the transcription factor E2F and inhibiting
expression of E2F target genes. Similarly, induction of autophagy
by RB1 requires binding of RB1 to E2F, and this process can be
antagonized by overexpression of E2F. The effects of RB1 and
E2F are complex, however, and might be influenced by cell type
and stress conditions. For example, E2F is required for autophagy
induction by DNA-damaging agents in U2OS cells, and
overexpression or activation of E2F can promote autophagy
and increase expression of Atg1 (ULK1), ATG5, LC3 and
DRAM1 in these cells (Polager et al., 2008). In addition, RB1–
E2F complexes have a negative effect on autophagy in response
to hypoxia, in this case by inhibiting expression of BNIP3 (for
BCL2/adenovirus E1B 19kDa interacting protein 3), an activator
of VPS34–beclin-1 complexes and inhibitor of mTOR (Tracy
et al., 2007).
Even a single CDK can have both positive and negative effects
on autophagy. Klionsky and co-workers have found that the yeast
CDK Pho85 has both positive and negative effects on autophagy
induction, depending on the cyclin partner it is associated with
(Yang et al., 2010). The relative expression levels of these cyclins
vary with cell cycle phase and in response to environmental
signals, and each phase probably directs Pho85 to different
cellular compartments or downstream targets, thereby allowing
Pho85 to provide an integrated autophagic response to multiple
growth and cell cycle cues.
In addition to these molecular links, processes that are intrinsic
to cell growth and proliferation might themselves be incompatible
with autophagy. For example, periodic reorganization of
microtubules into a mitotic spindle in dividing cells would be
expected to disrupt transport of autophagic vesicles or of
components required for their formation. Similarly, dedicating
vesicular trafficking pathways towards active synthesis and
secretion might preclude their use in autophagy. Growth and
autophagy might also compete for raw materials, such as
membrane lipids, or for regulators that function in both
processes. For example, Vps34 can assemble into at least three
complexes with distinct subunits and functions, but only one them
(which is defined by the presence of Atg14) is dedicated to
autophagosome formation (Simonsen and Tooze, 2009). Such
competition-based mechanisms could prevent autophagy from
causing damage during vulnerable cell cycle phases.
Regulation of cell growth by autophagy
Bone marrow cells derived from mice that are deficient for the
proapoptotic BCL2 family proteins BAX and BAK are able to
survive in culture for several weeks in the absence of growth
factors. This survival is autophagy-dependent and coincides with
a dramatic reduction in cell mass (Lum et al., 2005). In vivo,
starvation of Drosophila can result in a 90% decrease in the size
of larval fat body cells, which is accompanied by a massive
induction of autophagy (Butterworth et al., 1965). Genetic
Journal of Cell Science
Cell growth and autophagy
disruption of autophagy in this system suppresses the observed
reduction in growth, and overexpression of Atg1, which induces
autophagy, is sufficient to reduce cell size in the fat body (Scott
et al., 2007; Scott et al., 2004). In starved mice, the intracellular
protein degradation rate of hepatocytes approaches 40% per day in
vivo, and starvation-induced size reduction of mouse embryonic
fibroblasts can be substantially inhibited by disrupting Atg5
(Conde and Scornik, 1976; Hosokawa et al., 2006). Together,
these examples illustrate the intrinsic ability of autophagy to
directly reduce cellular mass through the degradation of bulk
cytoplasm (Fig. 4A).
In addition to these presumably non-selective effects in
growth-arrested cells, autophagy might further reduce the
growth rate of actively growing cells through targeted
elimination of growth-promoting molecules, complexes and
organelles. The protein p62 (also known as sequestosome 1) is
an intriguing candidate for such a factor. p62 is a multifunctional
adapter protein that binds LC3 and other Atg8 family members
through its LC3-interacting region (LIR), and thereby becomes
incorporated into autophagosomes (Bjørkøy et al., 2005). This
results in efficient autophagy-dependent degradation of p62, and,
indeed, p62 protein levels are widely used as indicators of
autophagic flux (Klionsky et al., 2008). Recently, an important
role for p62 in mTOR activation has been described. In cells
A
Ribophagy
Mitophagy
-Ub-Ub-
Bulk
autophagy
p62-mediated
selective autophagy
B
ULK1
mTOR
WIPI1
P-
Key
Mitochondrion
Fig. 4. Negative effects of autophagy on cell growth. (A) Multiple forms of
autophagy can contribute to growth suppression. From left to right: bulk
autophagy of cytoplasmic contents including cytosol and organelles;
mitophagy, which utilizes adapter molecules such as Atg32, Nix and p62;
ribophagy, which requires the ubiquitin protease Ubp3 and its activator Bre5;
and p62-dependent degradation of ubiquitylated (Ub) signaling molecules and
of p62 itself, which can promote mTOR signaling. (B) Activation of
autophagy factors such as ULK1 and WIPI1 might lead to downregulation of
mTOR through multiple mechanisms, possibly including ULK1-mediated
phosphorylation (P) of Raptor (indicated by the blue oval) and WIPI1dependent effects on maturation of endocytic vesicles.
2365
where p62 has been depleted or knocked out, mTOR fails to be
activated in response to amino acids, which leads to a decrease in
cell growth. Remarkably, p62 has been shown to stimulate
mTOR signaling by supporting the interaction between Rag
GTPases, the mTOR partner Raptor and the lysosomal surface,
which promotes amino-acid-dependent recruitment of mTOR to
the lysosome and its subsequent activation (Duran et al., 2011).
These results imply that autophagy can impair mTOR-dependent
cell growth through selective degradation of p62. In addition, p62
contains a C-terminal ubiquitin-associated (UBA) domain and
can promote selective autophagy of ubiquitylated soluble or
aggregated proteins (Bjørkøy et al., 2005; Kim et al., 2008),
which potentially include other factors involved in cell growth
regulation. In a proteomic study tracing the progressive
autophagic degradation of proteins in breast cancer cells, it has
been found that proteins involved in translation, including
mTOR, ribosomal protein S6 and tRNA synthetases, are among
the most rapidly depleted factors (Kristensen et al., 2008).
Whether these and other growth-promoting factors are selectively
targeted for autophagy in a p62-dependent manner remains to be
shown.
Autophagy can also be employed by cells to selectively
degrade larger complexes and organelles that are required for cell
growth, such as ribosomes and mitochondria. In yeast, starvation
results in rapid sequestration and digestion of ribosomes in the
vacuole. This process is essential for survival under starvation
conditions, is highly selective and involves the ubiquitin protease
Ubp3, its activator Bre5, and ubiquitylation of specific ribosomal
proteins (Kraft et al., 2008). Interestingly, the mammalian
orthologs of Ubp3 and Bre5 interact with ULK1, which is
required for the clearance of ribosomes from the cytoplasm
of reticulocytes (Behrends et al., 2010; Kundu et al., 2008).
Ribosomal degradation represents a potent mechanism to
directly reduce cellular growth capacity. In addition, large
macromolecular complexes such as P granules in C. elegans
and midbody rings of dividing cell populations can indirectly
promote growth by acting as stem cell factors, and these
complexes have also been shown to be selective autophagy
substrates (Kuo et al., 2011; Pohl and Jentsch, 2009; Zhang et al.,
2009).
Mitochondria can be selectively degraded by autophagy in a
process referred to as mitophagy recently (reviewed in Youle and
Narendra, 2011). Both starvation and mitochondrial damage
can trigger mitophagy, and this requires LIR-motif-containing
proteins [Atg32 in yeast, Nix (also known as BNIP3L) and
possibly p62 in mammalian cells], which are thought to guide
expansion of autophagosomal membranes over the mitochondrial
surface through interaction with Atg8 family members. In yeast,
this interaction is regulated by phosphorylation of Atg32 (Aoki
et al., 2011). It was recently demonstrated that mitochondria can
be protected from starvation-induced mitophagy by a PKAdependent process of fusion and elongation, which somehow
spares mitochondria from engulfment into autophagosomes
(Gomes et al., 2011; Rambold et al., 2011). Similarly, in yeast
that are grown under conditions in which mitochondria are
required for metabolism, mitophagy is not induced, not
even by severe starvation (Kanki and Klionsky, 2008). In
starved mammalian cells, degradation of mitochondria occurs
subsequent to degradation of cytosolic proteins and ribosomes
(Kristensen et al., 2008). Thus, mitochondria appear to be the last
Journal of Cell Science
2366
Journal of Cell Science 125 (10)
resort for nutrients, which probably reflects their essential role in
both growing and stationary cells.
Several components of the autophagic machinery have
been shown to have important autophagy-independent roles in
regulating apoptotic cell death, and it is interesting to speculate
that similar dual functions might be at work in controlling cell
growth. One possible example of this is a feedback signaling loop
from ULK1 to mTOR, which involves mTOR inhibition by its
downstream kinase (Fig. 4B). In Drosophila and mammalian
cells, Tor (or mTOR)-dependent phosphorylation of its substrates
S6K and EIF4EBP1 is increased in Atg1 (or ULK1)-depleted
cells, and decreased in response to the overexpression of Atg1
(Lee et al., 2007; Scott et al., 2007). These effects correlate with
changes in the cytoplasmic localization and trafficking of TOR,
and with ULK1-dependent phosphorylation of the TORC1
component Raptor (Chang and Neufeld, 2009; Dunlop et al.,
2011). Feedback signaling from Atg1 to mTOR might help to
amplify weak or variable signals into a stable biphasic switch,
and might allow pathways that regulate ATG1 and autophagy
independently of mTOR to influence growth in an mTORdependent manner. Other autophagy regulators can also affect
mTOR signaling independently of their effects on autophagy. A
recent study identified the Atg18 ortholog WD repeat domain,
phosphoinositide interacting 1 (WIPI1) as an inhibitor of mTOR
in human melanocytes (Ho et al., 2011). WIPI1 has an
autophagy-independent role in the biogenesis of melanosomes,
which are lysosome-related organelles that develop through an
endosome-like maturation process. WIPI1 has been shown to
function in part by suppressing mTOR activity, which negatively
regulates transcription of melanogenic proteins in these cells.
As mTOR activation is sensitive to disruption of endocytic
maturation (Flinn et al., 2010; Li et al., 2010), it will be
interesting to see whether WIPI1 also regulates TOR in other
contexts through its effects on endocytic trafficking.
Conclusions
The separation of cell growth and autophagy into nonoverlapping states appears to provide several advantages,
including maximal resource efficiency and protection against
cellular damage. However, although autophagy and cell growth
tend to display an inverse response to many stimuli, it should
be noted that this correlation is not absolute. High levels of
autophagy can be observed in some rapidly growing cells, and in
some cases this might actually contribute to cell growth
and biosynthesis. Deeper investigation into these apparent
‘exceptions to the rule’ might prove particularly fruitful. For
example, exercise induces both autophagy and cell growth of
muscle fibers (Ogura et al., 2011), and this might involve a
temporal separation into distinct phases of fiber breakdown and
rebuilding. Similarly, senescent cells display high levels of
autophagy despite showing highly active mTOR signaling and
protein synthesis. In this case, these processes are separated
spatially within the cell, with autophagy components being
insulated within an ‘mTOR–autophagy spatial coupling
compartment’ that protects them from mTOR-mediated
downregulation (Narita et al., 2011). In other cases, such as in
Ras-transformed cells, autophagy might provide intermediate
metabolites that are specifically required to fuel the altered
metabolic pathways of these cells and to facilitate their rapid
growth (Guo et al., 2010; Kim et al., 2011b; Lock et al., 2011).
As new roles of autophagy continue to be revealed, the view
that emerges is that this ancient process is deeply entwined with
the basic cellular functions of growth, proliferation and survival.
Disentangling these connections will be important to our
understanding of the molecular regulation of each of these
processes. Furthermore, as autophagy-based therapies against a
number of diseases are pursued, it will be important to consider
how potential treatments might impact other processes such as
cell growth that are linked to autophagy. This will be aided by a
better understanding of growth-dependent and -independent
mechanisms that regulate autophagy.
Funding
The work of our laboratory is supported by the National Institutes of
Health [grant number GM62509]. Deposited in PMC for release
after 12 months.
References
Aoki, Y., Kanki, T., Hirota, Y., Kurihara, Y., Saigusa, T., Uchiumi, T. and Kang, D.
(2011). Phosphorylation of Serine 114 on Atg32 mediates mitophagy. Mol. Biol. Cell
22, 3206-3217.
Axe, E. L., Walker, S. A., Manifava, M., Chandra, P., Roderick, H. L., Habermann, A.,
Griffiths, G. and Ktistakis, N. T. (2008). Autophagosome formation from membrane
compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected
to the endoplasmic reticulum. J. Cell Biol. 182, 685-701.
Bains, M., Florez-McClure, M. L. and Heidenreich, K. A. (2009). Insulin-like growth
factor-I prevents the accumulation of autophagic vesicles and cell death in Purkinje
neurons by increasing the rate of autophagosome-to-lysosome fusion and degradation.
J. Biol. Chem. 284, 20398-20407.
Bains, M., Zaegel, V., Mize-Berge, J. and Heidenreich, K. A. (2011). IGF-I stimulates
Rab7-RILP interaction during neuronal autophagy. Neurosci. Lett. 488, 112-117.
Behrends, C., Sowa, M. E., Gygi, S. P. and Harper, J. W. (2010). Network
organization of the human autophagy system. Nature 466, 68-76.
Bjørkøy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A.,
Stenmark, H. and Johansen, T. (2005). p62/SQSTM1 forms protein aggregates
degraded by autophagy and has a protective effect on huntingtin-induced cell death. J.
Cell Biol. 171, 603-614.
Blommaart, E. F. C., Luiken, J. J. F. P., Blommaart, P. J. E., van Woerkom, G. M.
and Meijer, A. J. (1995). Phosphorylation of ribosomal protein S6 is inhibitory for
autophagy in isolated rat hepatocytes. J. Biol. Chem. 270, 2320-2326.
Butterworth, F. M., Bodenstein, D. and King, R. C. (1965). Adipose tissue of
Drosophila melanogaster. I. An experimental study of larval fat body. J. Exp. Zool.
158, 141-153.
Byfield, M. P., Murray, J. T. and Backer, J. M. (2005). hVps34 is a nutrient-regulated
lipid kinase required for activation of p70 S6 kinase. J. Biol. Chem. 280, 3307633082.
Chang, Y. Y. and Neufeld, T. P. (2009). An Atg1/Atg13 complex with multiple roles in
TOR-mediated autophagy regulation. Mol. Biol. Cell 20, 2004-2014.
Conde, R. D. and Scornik, O. A. (1976). Role of protein degradation in the growth of
livers after a nutritional shift. Biochem. J. 158, 385-390.
Crighton, D., Wilkinson, S., O’Prey, J., Syed, N., Smith, P., Harrison, P. R., Gasco, M.,
Garrone, O., Crook, T. and Ryan, K. M. (2006). DRAM, a p53-induced modulator of
autophagy, is critical for apoptosis. Cell 126, 121-134.
Dunlop, E. A., Hunt, D. K., Acosta-Jaquez, H. A., Fingar, D. C. and Tee, A. R. (2011).
ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and
hinders substrate binding. Autophagy 7, 737-747.
Duran, A., Amanchy, R., Linares, J. F., Joshi, J., Abu-Baker, S., Porollo, A.,
Hansen, M., Moscat, J. and Diaz-Meco, M. T. (2011). p62 is a key regulator of
nutrient sensing in the mTORC1 pathway. Mol. Cell 44, 134-146.
Edinger, A. L. (2007). Controlling cell growth and survival through regulated nutrient
transporter expression. Biochem. J. 406, 1-12.
Edinger, A. L. and Thompson, C. B. (2002). Akt maintains cell size and survival by
increasing mTOR-dependent nutrient uptake. Mol. Biol. Cell 13, 2276-2288.
Egan, D. F., Shackelford, D. B., Mihaylova, M. M., Gelino, S., Kohnz, R. A.,
Mair, W., Vasquez, D. S., Joshi, A., Gwinn, D. M., Taylor, R. et al. (2011).
Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects
energy sensing to mitophagy. Science 331, 456-461.
Eskelinen, E. L., Prescott, A. R., Cooper, J., Brachmann, S. M., Wang, L., Tang, X.,
Backer, J. M. and Lucocq, J. M. (2002). Inhibition of autophagy in mitotic animal
cells. Traffic 3, 878-893.
Feng, Z., Zhang, H., Levine, A. J. and Jin, S. (2005). The coordinate regulation of the
p53 and mTOR pathways in cells. Proc. Natl. Acad. Sci. USA 102, 8204-8209.
Flinn, R. J., Yan, Y., Goswami, S., Parker, P. J. and Backer, J. M. (2010). The late
endosome is essential for mTORC1 signaling. Mol. Biol. Cell 21, 833-841.
Furuya, T., Kim, M., Lipinski, M., Li, J., Kim, D., Lu, T., Shen, Y., Rameh, L.,
Yankner, B., Tsai, L. H. et al. (2010). Negative regulation of Vps34 by Cdk
mediated phosphorylation. Mol. Cell 38, 500-511.
Journal of Cell Science
Cell growth and autophagy
Gao, W., Shen, Z., Shang, L. and Wang, X. (2011). Upregulation of human autophagyinitiation kinase ULK1 by tumor suppressor p53 contributes to DNA-damage-induced
cell death. Cell Death Differ. 18, 1598-1607.
Gomes, L. C., Benedetto, G. D. and Scorrano, L. (2011). During autophagy
mitochondria elongate, are spared from degradation and sustain cell viability. Nat.
Cell Biol. 13, 589-598.
Graef, M. and Nunnari, J. (2011). Mitochondria regulate autophagy by conserved
signalling pathways. EMBO J. 30, 2101-2114.
Guo, J. Y., Chen, H. Y., Mathew, R., Fan, J., Strohecker, A. M., Karsli-Uzunbas, G.,
Kamphorst, J. J., Chen, G., Lemons, J. M., Karantza, V. et al. (2011). Activated
Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes
Dev. 25, 460-470.
Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F. and Gerok, W. (1990). Cell
swelling inhibits proteolysis in perfused rat liver. Biochem. J. 272, 239-242.
Hayashi-Nishino, M., Fujita, N., Noda, T., Yamaguchi, A., Yoshimori, T. and
Yamamoto, A. (2009). A subdomain of the endoplasmic reticulum forms a cradle for
autophagosome formation. Nat. Cell Biol. 11, 1433-1437.
Hennig, K. M., Colombani, J. and Neufeld, T. P. (2006). TOR coordinates bulk and
targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth.
J. Cell Biol. 173, 963-974.
Hinnebusch, A. G. (1994). Translational control of GCN4: an in vivo barometer of
initiation-factor activity. Trends Biochem. Sci. 19, 409-414.
Hinnebusch, A. G. (1997). Translational regulation of yeast GCN4. A window on
factors that control initiator-trna binding to the ribosome. J. Biol. Chem. 272, 2166121664.
Ho, H., Kapadia, R., Al-Tahan, S., Ahmad, S. and Ganesan, A. K. (2011). WIPI1
coordinates melanogenic gene transcription and melanosome formation via TORC1
inhibition. J. Biol. Chem. 286, 12509-12523.
Hosokawa, N., Hara, Y. and Mizushima, N. (2006). Generation of cell lines with
tetracycline-regulated autophagy and a role for autophagy in controlling cell size.
FEBS Lett. 580, 2623-2629.
Huber, A., French, S. L., Tekotte, H., Yerlikaya, S., Stahl, M., Perepelkina, M. P.,
Tyers, M., Rougemont, J., Beyer, A. L. and Loewith, R. (2011). Sch9 regulates
ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex
RPD3L. EMBO J. 30, 3052-3064.
Huynh, L. N., Thangavel, M., Chen, T., Cottrell, R., Mitchell, J. M. and PraetoriusIbba, M. (2010). Linking tRNA localization with activation of nutritional stress
responses. Cell Cycle 9, 3112-3118.
Jiang, H., Martin, V., Gomez-Manzano, C., Johnson, D. G., Alonso, M., White, E.,
Xu, J., McDonnell, T. J., Shinojima, N. and Fueyo, J. (2010). The RB-E2F1
pathway regulates autophagy. Cancer Res. 70, 7882-7893.
Jünger, M. A., Rintelen, F., Stocker, H., Wasserman, J. D., Végh, M., Radimerski, T.,
Greenberg, M. E. and Hafen, E. (2003). The Drosophila forkhead transcription factor
FOXO mediates the reduction in cell number associated with reduced insulin signaling.
J. Biol. 2, 20.
Kamada, Y., Yoshino, K., Kondo, C., Kawamata, T., Oshiro, N., Yonezawa, K. and
Ohsumi, Y. (2010). Tor directly controls the Atg1 kinase complex to regulate
autophagy. Mol. Cell. Biol. 30, 1049-1058.
Kanki, T. and Klionsky, D. J. (2008). Mitophagy in yeast occurs through a selective
mechanism. J. Biol. Chem. 283, 32386-32393.
Kantidakis, T., Ramsbottom, B. A., Birch, J. L., Dowding, S. N. and White, R. J.
(2010). mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and
targets their repressor Maf1. Proc. Natl. Acad. Sci. USA 107, 11823-11828.
Kijanska, M., Dohnal, I., Reiter, W., Kaspar, S., Stoffel, I., Ammerer, G., Kraft, C.
and Peter, M. (2010). Activation of Atg1 kinase in autophagy by regulated
phosphorylation. Autophagy 6, 1168-1178.
Kim, J., Kundu, M., Viollet, B. and Guan, K. L. (2011a). AMPK and mTOR regulate
autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132-141.
Kim, M. J., Woo, S. J., Yoon, C. H., Lee, J. S., An, S., Choi, Y. H., Hwang, S. G.,
Yoon, G. and Lee, S. J. (2011b). Involvement of autophagy in oncogenic K-Rasinduced malignant cell transformation. J. Biol. Chem. 286, 12924-12932.
Kim, P. K., Hailey, D. W., Mullen, R. T. and Lippincott-Schwartz, J. (2008).
Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc.
Natl. Acad. Sci. USA 105, 20567-20574.
Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew,
D. S., Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A. et al. (2008).
Guidelines for the use and interpretation of assays for monitoring autophagy in higher
eukaryotes. Autophagy 4, 151-175.
Korolchuk, V. I., Saiki, S., Lichtenberg, M., Siddiqi, F. H., Roberts, E. A., Imarisio, S.,
Jahreiss, L., Sarkar, S., Futter, M., Menzies, F. M. et al. (2011). Lysosomal
positioning coordinates cellular nutrient responses. Nat. Cell Biol. 13, 453-460.
Kraft, C., Deplazes, A., Sohrmann, M. and Peter, M. (2008). Mature ribosomes are
selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/
Bre5p ubiquitin protease. Nat. Cell Biol. 10, 602-610.
Kristensen, A. R., Schandorff, S., Høyer-Hansen, M., Nielsen, M. O., Jäättelä, M.,
Dengjel, J. and Andersen, J. S. (2008). Ordered organelle degradation during
starvation-induced autophagy. Mol. Cell. Proteomics 7, 2419-2428.
Kundu, M., Lindsten, T., Yang, C. Y., Wu, J., Zhao, F., Zhang, J., Selak, M. A., Ney,
P. A. and Thompson, C. B. (2008). Ulk1 plays a critical role in the autophagic
clearance of mitochondria and ribosomes during reticulocyte maturation. Blood 112,
1493-1502.
Kuo, T. C., Chen, C. T., Baron, D., Onder, T. T., Loewer, S., Almeida, S.,
Weismann, C. M., Xu, P., Houghton, J. M., Gao, F. B. et al. (2011). Midbody
2367
accumulation through evasion of autophagy contributes to cellular reprogramming
and tumorigenicity. Nat. Cell Biol. 13, 1214-1223.
Lee, S. B., Kim, S., Lee, J., Park, J., Lee, G., Kim, Y., Kim, J. M. and Chung, J.
(2007). ATG1, an autophagy regulator, inhibits cell growth by negatively regulating
S6 kinase. EMBO Rep. 8, 360-365.
Li, L., Kim, E., Yuan, H., Inoki, K., Goraksha-Hicks, P., Schiesher, R. L., Neufeld,
T. P. and Guan, K. L. (2010). Regulation of mTORC1 by the Rab and Arf GTPases.
J. Biol. Chem. 285, 19705-19709.
Liang, J., Shao, S. H., Xu, Z. X., Hennessy, B., Ding, Z., Larrea, M., Kondo, S.,
Dumont, D. J., Gutterman, J. U., Walker, C. L. et al. (2007). The energy sensing
LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to
enter autophagy or apoptosis. Nat. Cell Biol. 9, 218-224.
Lipinski, M. M., Hoffman, G., Ng, A., Zhou, W., Py, B. F., Hsu, E., Liu, X.,
Eisenberg, J., Liu, J., Blenis, J. et al. (2010). A genome-wide siRNA screen reveals
multiple mTORC1 independent signaling pathways regulating autophagy under
normal nutritional conditions. Dev. Cell 18, 1041-1052.
Lippman, S. I. and Broach, J. R. (2009). Protein kinase A and TORC1 activate genes
for ribosomal biogenesis by inactivating repressors encoded by Dot6 and its homolog
Tod6. Proc. Natl. Acad. Sci. USA 106, 19928-19933.
Lock, R., Roy, S., Kenific, C. M., Su, J. S., Salas, E., Ronen, S. M. and Debnath, J.
(2011). Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation.
Mol. Biol. Cell 22, 165-178.
Lum, J. J., Bauer, D. E., Kong, M., Harris, M. H., Li, C., Lindsten, T. and
Thompson, C. B. (2005). Growth factor regulation of autophagy and cell survival in
the absence of apoptosis. Cell 120, 237-248.
Maiuri, M. C., Malik, S. A., Morselli, E., Kepp, O., Criollo, A., Mouchel, P. L.,
Carnuccio, R. and Kroemer, G. (2009). Stimulation of autophagy by the p53 target
gene Sestrin2. Cell Cycle 8, 1571-1576.
Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P.,
Burden, S. J., Di Lisi, R., Sandri, C., Zhao, J. et al. (2007). FoxO3 controls
autophagy in skeletal muscle in vivo. Cell Metab. 6, 458-471
Mayer, C., Zhao, J., Yuan, X. and Grummt, I. (2004). mTOR-dependent activation of
the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes
Dev. 18, 423-434.
McCubrey, J. A., Steelman, L. S., Chappell, W. H., Abrams, S. L., Wong, E. W.,
Chang, F., Lehmann, B., Terrian, D. M., Milella, M., Tafuri, A. et al. (2007).
Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and
drug resistance. Biochim. Biophys. Acta 1773, 1263-1284.
Mizushima, N. (2010). The role of the Atg1/ULK1 complex in autophagy regulation.
Curr. Opin. Cell Biol. 22, 132-139.
Mizushima, N., Yoshimori, T. and Ohsumi, Y. (2011). The role of Atg proteins in
autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107-132.
Narita, M., Young, A. R., Arakawa, S., Samarajiwa, S. A., Nakashima, T., Yoshida, S.,
Hong, S., Berry, L. S., Reichelt, S., Ferreira, M. et al. (2011). Spatial coupling of
mTOR and autophagy augments secretory phenotypes. Science 332, 966-970.
Natarajan, K., Meyer, M. R., Jackson, B. M., Slade, D., Roberts, C., Hinnebusch,
A. G. and Marton, M. J. (2001). Transcriptional profiling shows that Gcn4p is a
master regulator of gene expression during amino acid starvation in yeast. Mol. Cell.
Biol. 21, 4347-4368.
Neufeld, T. P. (2004). Role of autophagy in developmental cell growth and death:
insights from Drosophila. In Autophagy (ed. D. J. Klionsky), pp. 224-232,
Georgetown: Landes Bioscience.
Nobukuni, T., Joaquin, M., Roccio, M., Dann, S. G., Kim, S. Y., Gulati, P., Byfield,
M. P., Backer, J. M., Natt, F., Bos, J. L. et al. (2005). Amino acids mediate mTOR/
raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc.
Natl. Acad. Sci. USA 102, 14238-14243.
Ogura, Y., Iemitsu, M., Naito, H., Kakigi, R., Kakehashi, C., Maeda, S. and Akema, T.
(2011). Single bout of running exercise changes LC3-II expression in rat cardiac muscle.
Biochem. Biophys. Res. Commun. 414, 756-760.
Ohsaki, Y., Suzuki, M., Shinohara, Y. and Fujimoto, T. (2010). Lysosomal
accumulation of mTOR is enhanced by rapamycin. Histochem. Cell Biol. 134, 537544.
Pohl, C. and Jentsch, S. (2009). Midbody ring disposal by autophagy is a postabscission event of cytokinesis. Nat. Cell Biol. 11, 65-70.
Polager, S., Ofir, M. and Ginsberg, D. (2008). E2F1 regulates autophagy and the
transcription of autophagy genes. Oncogene 27, 4860-4864.
Proud, C. G. (2007). Signalling to translation: how signal transduction pathways control
the protein synthetic machinery. Biochem. J. 403, 217-234.
Ramachandran, V. and Herman, P. K. (2011). Antagonistic interactions between the
cAMP-dependent protein kinase and Tor signaling pathways modulate cell growth in
Saccharomyces cerevisiae. Genetics 187, 441-454.
Rambold, A. S., Kostelecky, B., Elia, N. and Lippincott-Schwartz, J. (2011). Tubular
network formation protects mitochondria from autophagosomal degradation during
nutrient starvation. Proc. Natl. Acad. Sci. USA 108, 10190-10195.
Rong, Y., McPhee, C. K., Deng, S., Huang, L., Chen, L., Liu, M., Tracy, K.,
Baehrecke, E. H., Yu, L. and Lenardo, M. J. (2011). Spinster is required for
autophagic lysosome reformation and mTOR reactivation following starvation. Proc.
Natl. Acad. Sci. USA 108, 7826-7831.
Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A. L., Nada, S. and Sabatini, D. M.
(2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is
necessary for its activation by amino acids. Cell 141, 290-303.
Scott, R. C., Schuldiner, O. and Neufeld, T. P. (2004). Role and regulation of
starvation-induced autophagy in the Drosophila fat body. Dev. Cell 7, 167-178.
Journal of Cell Science
2368
Journal of Cell Science 125 (10)
Scott, R. C., Juhász, G. and Neufeld, T. P. (2007). Direct induction of autophagy by
Atg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17, 1-11.
Settembre, C., Di Malta, C., Polito, V. A., Garcia Arencibia, M., Vetrini, F., Erdin, S.,
Erdin, S. U., Huynh, T., Medina, D., Colella, P. et al. (2011). TFEB links autophagy
to lysosomal biogenesis. Science 332, 1429-1433.
Shang, L., Chen, S., Du, F., Li, S., Zhao, L. and Wang, X. (2011). Nutrient starvation
elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its
subsequent dissociation from AMPK. Proc. Natl. Acad. Sci. USA 108, 4788-4793.
Shin, C. S. and Huh, W. K. (2011). Bidirectional regulation between TORC1 and
autophagy in Saccharomyces cerevisiae. Autophagy 7, 854-862.
Simonsen, A. and Tooze, S. A. (2009). Coordination of membrane events during
autophagy by multiple class III PI3-kinase complexes. J. Cell Biol. 186, 773-782.
Slavov, N. and Botstein, D. (2011). Coupling among growth rate response, metabolic
cycle, and cell division cycle in yeast. Mol. Biol. Cell 22, 1997-2009.
Stahl, M., Dijkers, P. F., Kops, G. J., Lens, S. M., Coffer, P. J., Burgering, B. M. and
Medema, R. H. (2002). The forkhead transcription factor FoxO regulates
transcription of p27Kip1 and Bim in response to IL-2. J. Immunol. 168, 5024-5031.
Stephan, J. S., Yeh, Y. Y., Ramachandran, V., Deminoff, S. J. and Herman, P. K.
(2009). The Tor and PKA signaling pathways independently target the Atg1/Atg13
protein kinase complex to control autophagy. Proc. Natl. Acad. Sci. USA 106, 1704917054.
Tasdemir, E., Maiuri, M. C., Tajeddine, N., Vitale, I., Criollo, A., Vicencio, J. M.,
Hickman, J. A., Geneste, O. and Kroemer, G. (2007). Cell cycle-dependent
induction of autophagy, mitophagy and reticulophagy. Cell Cycle 6, 2263-2267.
Tasdemir, E., Maiuri, M. C., Galluzzi, L., Vitale, I., Djavaheri-Mergny, M.,
D’Amelio, M., Criollo, A., Morselli, E., Zhu, C., Harper, F. et al. (2008).
Regulation of autophagy by cytoplasmic p53. Nat. Cell Biol. 10, 676-687.
Taylor, P. M. (2009). Amino acid transporters: éminences grises of nutrient signalling
mechanisms? Biochem. Soc. Trans. 37, 237-241.
Tracy, K., Dibling, B. C., Spike, B. T., Knabb, J. R., Schumacker, P. and Macleod,
K. F. (2007). BNIP3 is an RB/E2F target gene required for hypoxia-induced
autophagy. Mol. Cell. Biol. 27, 6229-6242.
Vazquez-Martin, A., Cufı́, S., Oliveras-Ferraros, C. and Menendez, J. A. (2011).
Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein
of the rDNA transcription machinery in nucleoli and chromosomal nucleolus
organizer regions (NORs). Cell Cycle 10, 3140-3152.
Williams, A., Sarkar, S., Cuddon, P., Ttofi, E. K., Saiki, S., Siddiqi, F. H., Jahreiss, L.,
Fleming, A., Pask, D., Goldsmith, P. et al. (2008). Novel targets for Huntington’s
disease in an mTOR-independent autophagy pathway. Nat. Chem. Biol. 4, 295-305.
Yamamoto, A., Cremona, M. L. and Rothman, J. E. (2006). Autophagy-mediated
clearance of huntingtin aggregates triggered by the insulin-signaling pathway. J. Cell
Biol. 172, 719-731.
Yang, Z., Geng, J., Yen, W. L., Wang, K. and Klionsky, D. J. (2010). Positive or
negative roles of different cyclin-dependent kinase Pho85-cyclin complexes
orchestrate induction of autophagy in Saccharomyces cerevisiae. Mol. Cell 38, 250264.
Yeh, Y. Y., Wrasman, K. and Herman, P. K. (2010). Autophosphorylation within the
Atg1 activation loop is required for both kinase activity and the induction of
autophagy in Saccharomyces cerevisiae. Genetics 185, 871-882.
Yeh, Y. Y., Shah, K. H. and Herman, P. K. (2011). An Atg13 protein-mediated selfassociation of the Atg1 protein kinase is important for the induction of autophagy. J.
Biol. Chem. 286, 28931-28939.
Ylä-Anttila, P., Vihinen, H., Jokitalo, E. and Eskelinen, E. L. (2009). 3D tomography
reveals connections between the phagophore and endoplasmic reticulum. Autophagy
5, 1180-1185.
Youle, R. J. and Narendra, D. P. (2011). Mechanisms of mitophagy. Nat. Rev. Mol.
Cell Biol. 12, 9-14.
Yu, L., McPhee, C. K., Zheng, L., Mardones, G. A., Rong, Y., Peng, J., Mi, N., Zhao, Y.,
Liu, Z., Wan, F. et al. (2010). Termination of autophagy and reformation of lysosomes
regulated by mTOR. Nature 465, 942-946.
Zhang, L., Yu, J., Pan, H., Hu, P., Hao, Y., Cai, W., Zhu, H., Yu, A. D., Xie, X., Ma, D.
et al. (2007). Small molecule regulators of autophagy identified by an image-based
high-throughput screen. Proc. Natl. Acad. Sci. USA 104, 19023-19028.
Zhang, Y., Yan, L., Zhou, Z., Yang, P., Tian, E., Zhang, K., Zhao, Y., Li, Z., Song, B.,
Han, J. et al. (2009). SEPA-1 mediates the specific recognition and degradation of P
granule components by autophagy in C. elegans. Cell 136, 308-321.
Zhao, J., Brault, J. J., Schild, A., Cao, P., Sandri, M., Schiaffino, S., Lecker, S. H.
and Goldberg, A. L. (2007). FoxO3 coordinately activates protein degradation by the
autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell
Metab. 6, 472-483.
Zhao, Y., Yang, J., Liao, W., Liu, X., Zhang, H., Wang, S., Wang, D., Feng, J.,
Yu, L. and Zhu, W. G. (2010). Cytosolic FoxO1 is essential for the induction of
autophagy and tumour suppressor activity. Nat. Cell Biol. 12, 665-675.
Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y. and Sabatini, D. M.
(2011a). mTORC1 senses lysosomal amino acids through an inside-out mechanism
that requires the vacuolar H(+)-ATPase. Science 334, 678-683.
Zoncu, R., Efeyan, A. and Sabatini, D. M. (2011b). mTOR: from growth signal
integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21-35.