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Biochimie 90 (2008) 313e323

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Research paper

Regulation of macroautophagy by mTOR and Beclin 1 complexes

Sophie Pattingre a,b, Lucile Espert c, Martine Biard-Piechaczyk c, Patrice Codogno a,b,*
a
INSERM U756, 5 rue Jean-Baptiste Clement, 92296 Chatenay-Malabry, France
b
Universite Paris-Sud 11, Faculte de Pharmacie, 92296 Chatenay-Malabry, France
c
CPBS, UM1, UM2, CNRS, Institut de Biologie, 34965 Montpellier, France
Received 15 June 2007; accepted 31 August 2007
Available online 8 September 2007

Abstract

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic
vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast
are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell
function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell
growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important
role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations,
including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome for-
mation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in reg-
ulating autophagosome formation.
2007 Elsevier Masson SAS. All rights reserved.

Keywords: Autophagy; Beclin 1; mTOR; Lysosome; Signal transduction

1. Introduction a process that degrades macromolecules, and also eliminates

Cell homeostasis depends on the balance between the bio-
synthesis and catabolism of macromolecules. There are two
major systems in eukaryotic cells that degrade cellular compo-
nents: the proteasome and the lysosome [1]. The proteasomal
degradative pathway is selective for proteins. The lysosomal
system is responsible for the degradation of several classes of
macromolecules, and for the turnover of organelles by several
mechanisms collectively known as autophagy. This term em-
braces several different mechanisms: microautophagy, macro-
autophagy, crinophagy and chaperone-mediated autophagy
[2]. This review will focus, except where otherwise indicated,
on macroautophagy (hereafter referred to as autophagy),
* Corresponding author. INSERM U756, 5 rue Jean-Baptiste Clement,
92296 Chatenay-Malabry, France. Tel.: 33 1 46835720; fax: 33 1
46835844.
E-mail address: patrice.codogno@u-psud.fr (P. Codogno).
organelles and unwanted structures. Autophagy is a mechanism
conserved among eukaryotic cells that starts with the formation
of a double membrane-bound vacuole, known as an autophago-
some, which ultimately fuses with the lysosomal compartment
to degrade the sequestered material. The discovery of ATG
genes in the yeast Saccharomyces cerevisiae that govern auto-
phagosome formation has boosted research on autophagy in the
last decade [3e5]. Most of these genes are conserved from
yeast to human and several reviews have been dedicated to
the role of Atg proteins in the different steps of the formation
of autophagosomes [3, 6, 7]. Autophagy occurs at a basal
rate in most cells, where it acts as a cytoplasmic quality control
mechanism to eliminate protein aggregates and damaged or-
ganelles [8]. The physiological importance of basal autophagy
in maintaining tissue homeostasis has been recently demon-
strated in conditional brain and liver ATG knockout mouse
models [9e11]. These studies have also demonstrated the role
of autophagy in preventing the deposition of aggregate-prone

0300-9084/$ - see front matter 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2007.08.014
314 S. Pattingre et al. / Biochimie 90 (2008) 313e323

proteins in the cytoplasm, and the non-selective contribution
of autophagy to the elimination of ubiquitinated proteins that
are efficient substrates for the proteasome [12e14]. The anti-
aging role of autophagy probably depends, at least in part, on
its quality control function that limits the deposition of
aggregate-prone proteins and the formation of damaging
reactive oxygen species by mitochondria [15]. On the other
hand, when the supply of nutrients is limited, stimulating au-
tophagy contributes to the lysosomal recycling of nutrients to
maintain protein synthesis and glucose synthesis from amino
acids (in the liver), and substrates for oxidation and ATP pro-
duction in the mitochondria [8, 16] and inhibition of the de-
fault apoptotic pathway [17]. In vivo, a recent study showed
that at birth the sudden interruption of the supply of nutrients
via the placenta triggers autophagy in newborn mouse tissues
to maintain energy homeostasis and survival [18].
Autophagy has also been recently recognized as a mecha-
nism contributing to the innate immune response toward path-
ogens [19e24], and to antigen presentation by MHC class II
[25e27]. It is also implicated in numerous diseases, including,
cancer, neurodegenerative disease, muscle and liver disorders.
Several reviews have discussed the beneficial or harmful role
of autophagy in these diseases [28e34].
Although autophagy is recognized as being a cytoprotective
process, for some years it has also been suspected of being in-
volved in type-2 Programmed Cell Death (PCD) or autophagic
cell death (as distinct from type-1 PCD or apoptosis) (re-
viewed in [35]). Only recently, genetic studies have shown
that cells can be killed by autophagy when apoptosis is
inhibited [36e38]. Moreover, in a different context it has
been shown that autophagy contributes with apoptotic signals
to the killing of cells [39e43] and elimination of apoptotic
cells by phagocytic cells [44].
It is now important to elucidate the regulation of autophagy
in order to clarify its dual effects in cell survival and cell
death. The knowledge of the relationship between signaling
pathways and the molecular machinery involved in the forma-
tion of autophagosomes will be essential. Recent studies sug-
gest that the regulation of autophagy overlaps with that of
apoptosis, and that signaling molecules may modulate several
different players in the autophagic molecular machinery [43,
45]. The aim of the present review is to describe the roles of
two major protein complexes (mTOR complex 1 and the Be-
clin 1 complex) in the regulation of autophagy and their con-
sequences for the outcome of autophagy.
2. The morphology of autophagy and the autophagic
machinery: an overview
The aim of this section is to briefly summarize what we
know about the anatomy of autophagy, and the role of Atg
and other proteins involved in the formation and maturation
of autophagosomes. For more detailed discussions, readers
should consult recent reviews dedicated to the morphology of
autophagy [46, 47], the origin of the membrane source for au-
tophagy [48e50], the role of Atg proteins during the formation
of autophagosomes [6, 7] and the molecular events that govern
the maturation of autophagosomes [46] (Fig. 1). Autophagy

Fig. 1. Atg proteins and the autophagic pathway. The production of PtIns3P by the Beclin 1/hVps34 complex is an early event in autophagosome formation. This
first step is essential for the recruitment of other Atg proteins at the isolation membrane or phagophore. Thereafter, sequential recruitment of Atg12eAtg5 and LC3
(Atg8) conjugates occurs at the isolation membrane. The LC3-I form is covalently bound to phosphatidylethanolamine (PE) a lipid in the autophagosomal mem-
brane to form LC3-II. After completion, most of the Atg proteins, except a fraction of LC3-II bound to the luminal side of the autophagosomal membrane are
recycled to the cytosol. The Atg1 complex may be implicated in different steps of the formation of autophagosomes to regulate the transport of Atg proteins
to and from the isolation membrane. The last step in this process is the fusion between autophagosomes and lysosomes, and the degradation of the sequestered
material, including LC3-II.
 S. Pattingre et al. / Biochimie 90 (2008) 313e323
begins with a pre-autophagosomal structure of unknown origin
that gives rise to an isolation membrane or phagophore [7,
47]. This membrane elongates to form the autophagosome,
which is a double membrane-bound structure in the 0.5e
1.5 mm range in mammalian cells. Once this has been formed,
the autophagosomes can receive input from the endocytic path-
way to form a hybrid organelle known as the amphisome [47,
51]. However, similarly to what we observe in yeast, the auto-
phagosome can directly merge with the lysosomal compart-
ment [52]. Atg proteins involved in the formation of
autophagosomes are almost entirely conserved in eukaryotic
cells [3]. These proteins can be divided into four functional
groups: (1) the Atg1 complex that integrates signaling from
the TOR (Target of Rapamycin) kinase. This aspect will be dis-
cussed in greater detail in Section 3.2.1. This complex is in-
volved in several steps during the initiation, nucleation and
expansion steps of autophagosome formation. (2) The Beclin
1 complex (the ortholog of the yeast protein Atg6)/class III
PI3K (hVps34) is involved in the nucleation phase (see Section
4). (3) Two ubiquitin-like conjugation systems, the Atg12 and
the LC3 systems (LC3 the ortholog of the yeast protein Atg8)
act sequentially during autophagosome formation [53]. Atg12
conjugates Atg5 during the initiation step, and promotes the
conjugation of LC3 to phosphatidylethanolamine that is re-
quired during the elongation step. (4) A recycling pathway con-
trolled by Atg1 and Atg9 that mediates the shuttling of Atg
proteins in and out of the autophagosomal membranes during
the formation of autophagosomes [54, 55]. The maturation of
autophagosomes requires the activity of Rab GTPases (Rab 7
and Rab 24) [56e58], and of other factors that are involved
in fusion events during vesicular trafficking in the endocytic
pathway [59e61].
3. mTOR complexes
TOR is a conserved Ser/Thr protein kinase that regulates
cell growth, cell cycle progression, nutrient import, protein
synthesis and autophagy [62e66]. The discovery of this kinase
is rooted in a soil sample from Easter Island containing a bac-
terium (Streptomyces hygroscopicus) that produces the anti-
fungal metabolite, rapamycin (from Rapa Nui, the local
name for Easter Island). Rapamycin binds to the FKBP12 pro-
tein to form a complex that interacts and inhibits several func-
tions regulated by TOR [66]. Apart from yeast, where two
TOR genes, TOR1 and TOR2, have been identified, all eukary-
otic genomes examined so far contain a single TOR gene. TOR
is a large protein (280 kDa) that belongs to a group of kinases
known as the phosphatidylinositol-related kinases (PIKK) [63,
66]. Members of the PIKK family contain a catalytic carboxy-
terminal domain that has similarities with the catalytic
domains of phosphatidylinositol-3 and phosphatidylinositol-4
kinases. Four functional domains are conserved in TOR pro-
teins including the central FAT (FRAP, ATM, TTRAP) domain,
the FRB (FKBP12-rapamycin binding domain) domain, the ki-
nase domain and at the most C-terminal part of the protein the
FATC domain. It has been suggested that these two domains
may interact to expose the catalytic domain. The FATC domain
315
is absolutely essential for TOR kinase activity. TOR exists in
two different complexes TORC1 and TORC2 [63, 65, 66]. In
yeast, only TOR2 can form TORC2 [66] and TORC1 is sen-
sitive to rapamycin [67]. In other species, these two com-
plexes are structurally and functionally conserved [65, 66].
Mammalian TORC1 (mTORC1) is composed of mLST8
(GbL) and raptor, and is sensitive to rapamycin [68e70].
The rapamycin-insensitive complex mTORC2 is composed
of mTOR, mLST8 (GbL), rictor, SIN1 and protor [71e74].
mTORC1 controls protein synthesis, nutrient import and
autophagy [45, 62, 65, 66, 75]. Studies in Drosophila and
mammalian cells have shown that two proteins, S6 kinase
and 4E-BP1, link mTORC1 to the control of mRNA transla-
tion [62, 75] and two proteins, Atg1 and S6K, to the control
of autophagy [45, 62]. The downstream functions of
mTORC2 are less known than those of mTORC1. mTORC2
appears to be involved in actin cytoskeleton regulation [72,
74] and Akt/PKB regulation [76]. Activation of Akt/PKB
depends not only on its phosphorylation at position T308
by PDK1 but also at position S473 [77]. Recently, it has
been demonstrated that mTORC2 is the PDK2 that phosphor-
ylates Akt/PKB at position S473 [76]. As Akt/PKB acts up-
stream of mTORC1, it has been suggested that mTORC2
could be located upstream of mTORC1 (reviewed in [63,
64]). However, recent studies have shown that in vivo the
phosphorylation at S473 is not required for the phosphoryla-
tion at T308, and the phosphorylation of TSC2, an Akt/PKB
target in the mTORC1 signaling pathway [64]. These find-
ings strongly suggest that mTORC2 is not in fact located
upstream of mTORC1. Interestingly, recent knockout studies
of raptor [78] and rictor [78, 79] have shown that the contri-
butions of mTORC1 and mTORC2 are critical in the early
stages of embryogenesis and at midgestation, respectively.
3.1. mTOR complex regulation
The mTORC1 complex is regulated by the heterodimer
TSC1 (tuberous sclerosis complex 1)/TSC2, which acts as
a brake on mTOR-dependent signaling [64, 80]. TSC2 is
a GTPase-activating protein for the small G-protein Rheb,
which, in its GTP-form binds to and activates mTOR. The
mTOR signaling network receives input from growth factors
signaling via the class I PI3K signaling pathway [64, 80].
When TSC2 is phosphorylated by Akt/PKB, the TSC1/TSC2
complex becomes inactive, and mTORC1 signaling is activated.
The energy sensor AMPK (AMP-activated kinase), which is
activated when the AMP/ATP ratio increases, and the inducible
stress sensors REDD1 and REDD2 inactivate mTORC1 by ac-
tivating TSC2 [64]. Activation of the tumor suppressor p53 and
calcium signaling can also inhibit mTOR by activating AMPK
[81, 82]. mTORC1 activity is also regulated by lipid signaling
molecules [83], and the redox status of the cell [84]. Amino
acids upregulate mTORC1 [85e88]. Recent studies have shown
that amino acids mediate mTOR/raptor-dependent signaling by
activating hVps34 [89, 90]. Moreover, in one of these studies,
the activity of the Beclin 1-associated hVps34 was inhibited
by amino acid starvation [89]. These findings are difficult to
316 S. Pattingre et al. / Biochimie 90 (2008) 313e323

reconcile with the stimulatory role of the hVps34eBeclin 1
complex during starvation-induced autophagy (see Section
4.1.1) [91e93]. However, the existence of different pools of
hVps34eBeclin 1 complexes is an intriguing possibility that
cannot be excluded. A further degree of complexity in the role
of amino acid signaling is suggested by studies that report
mTOR-independent control of autophagy during leucine re-
striction (see Section 3.2.2) [94, 95].
invagination of the lysosomal membrane. These findings sug-
gest that TOR may be involved in balancing macroautophagic
and microautophagic activities.
Following on from these data, and some of the findings dis-
cussed in the preceding section, several studies have shown
that signaling pathways that activate TOR also inhibit autoph-
agy, whereas signaling pathways that inhibit TOR stimulate
autophagy [45, 62] (Fig. 2).

3.2. mTOR in autophagy 3.2.1. Downstream targets of mTOR in the

The first evidence that TOR has a role in regulating autoph-
agy came from experiments involving rat hepatocytes that
showed that rapamycin partially reverses the inhibitory effects
of amino acids on autophagic proteolysis [96]. The stimulatory
effect of rapamycin on autophagy has been confirmed in dif-
ferent models [45]. This would give credence to the observa-
tion that TORC2 is not directly involved in the regulation of
autophagy.
However, the possible relevance of this complex in autoph-
agy remains to be established because TORC2 is sensitized to
long-term treatment with rapamycin [97].
The first genetic evidence for the role of TOR in autophagy
came from studies in yeast demonstrating that a temperature-
sensitive TOR mutant induces autophagy at a permissive tem-
perature [98]. In the yeast S. cerevisiae, TOR and the EGO
complex positively regulate microautophagy [99], an autopha-
gic process by which cytoplasmic fractions are engulfed by
regulation of autophagy
In yeast, the Atg1 Ser/Thr protein kinase functions down-
stream of TOR to regulate different steps in autophagosome
formation [7, 62, 100]. Atg1 is contained in a dynamic protein
complex with Atg17, Cvt9, Atg13 and Vac8, the composition
of which depends upon the phosphorylation status of Atg1 and
Atg13 [101, 102]. Under nutrient-rich conditions, in which
Atg1 and Atg13 are hyperphosphorylated, Atg1 interacts
with Atg17 and Cvt9, and Atg13 is associated with Vac8. Af-
ter starvation, the dephosphorylation of Atg13 leads it to asso-
ciate with Atg1 and other partners, resulting in an Atg17
dependent autophagy. Similarly, rapamycin favors the dephos-
phorylation of Atg13 and the activation of Atg1 [101, 103].
However, the role of Atg1 kinase activity in autophagy is
not clear [100, 104]. Homologs of Atg1 have been shown to
be involved in autophagy in multicellular organisms [55,
105e108]. However, the precise role of the Atg1 complex dur-
ing the formation of the autophagosome in metazoans is not

Fig. 2. Control of the autophagic machinery by signaling pathways. Autophagy is regulated by at least two complexes: mTORC1 and Beclin 1/hVps34. Both com-
plexes receive input from intra- and extra-cellular stimuli. mTORC1 integrates nutrients, energy, growth factors, calcium and amino acid signaling. Once activated,
mTORC1 inhibits autophagy by acting on the Atg1 kinase complex. The precise role of S6K, which acts downstream from mTORC1, on autophagy remains to be
elucidated. It has recently been shown that amino acids stimulate the activity of the mTORC1 complex via hVps34. However, the mechanism by which hVps34
activates mTOR is unknown, and its relationship with the Beclin 1/hVps34 complex needs to be clarified. Actually hVps34 activates mTOR, which is an inhibitory
signal of autophagy, but the Beclin 1/hVps34 complex is essential for initiating autophagy. The interaction between Beclin 1 and hVps34 is reinforced by UVRAG,
which upregulates autophagy, but is inhibited by Bcl-2, an inhibitor of autophagy. The interaction between Beclin 1 and Bcl-2 is inhibited by nutrient withdrawal
and the activation of signaling pathways (JNK1). The positive role of the Beclin 1 complex in autophagy may be explained by the production of PtIns3P (that is
associated with WIPI-1a, the mammalian homolog of Atg18). The Beclin 1 complex regulates, at least partially, the H2O2-dependent activity of Atg4, an enzyme
that controls the conjugation and deconjugation of LC3 in starvation-induced autophagy (see Fig. 1). The possibility that signaling pathways regulate the activity of
other Atg proteins cannot be excluded.
 S. Pattingre et al. / Biochimie 90 (2008) 313e323
known, in part because several components of the Atg1 com-
plex are not evolutionarily conserved (Atg11, Atg13, Atg17)
[3], and in part because the physiological target of the kinase
activity of Atg1 remains to be identified [100]. A further de-
gree of complexity results from the fact that Atg1 and its as-
sociation with the pre-autophagosomal structure in yeast is
also regulated by its phosphorylation by cAMP-dependent
protein kinase A [109, 110]. A recent study in Drosophila
demonstrates that the kinase activity of Atg1 is required to
stimulate autophagy [111]. Moreover, this study shows that
Atg1 exercises negative feedback control on dTOR. Interest-
ingly, cells with a high level of Atg1-dependent autophagy
are eliminated by apoptosis. This study and others [39, 41e
43] show that autophagy is an alternative way of triggering
apoptosis.
S6K, another downstream target of mTOR, is involved in
the regulation of autophagy. In Drosophila, S6K has been
shown to contribute to the stimulation of autophagy, although
it is not mandatory for its initiation [108]. It has been sug-
gested that in mammalian cells S6K may contribute to the
basal activity of autophagy via its feedback inhibition of the
class I, PI3K-dependent insulin signaling pathway [45, 112].
Moreover, Atg1 has recently been shown to inhibit cell growth
in Drosophila and mammalian cells by down-regulating S6K
[113]. These studies indicate that a crosstalk exists between
autophagy and cell growth regulation. However, the role of
S6K in autophagy needs further clarification.
3.2.2. mTOR-independent regulation of autophagy
As mentioned in the preceding section, the bypassing of
mTOR in the regulation of autophagy by amino acids has
been reported in muscle-derived cells and hepatocytes [94,
95]. Moreover, mTOR-independent stimulation of autophagy
has also been observed in response to trehalose [114] and
LiCl treatment [115]. After LiCl treatment, autophagy is in-
duced via the inhibition of inositol monophosphatase indepen-
dently of mTOR inhibition. The depletion of free inositol and
reduced levels of myo-inositol-1,4,5 phosphate (IP3) stimulate
autophagy. According to these findings, inhibition of the endo-
plasmic reticulum IP3 receptor stimulates autophagy [116]. In-
terestingly, enhancing the IP3 level inhibits autophagy induced
by nutrient depletion. These results suggest that inositol and
IP3 regulate autophagy via a signaling pathway parallel to
mTOR, or impinge on the molecular machinery of autophagy
downstream of mTOR.
4. Beclin 1 complex
Beclin 1 was originally discovered from a mouse brain li-
brary using a yeast two-system hybrid with the antiapoptotic
protein Bcl-2 as bait [117]. It is a 60-kDa protein containing
450 amino acids that comprise four specific domains: (1)
a Bcl-2 binding domain extending from amino acids 88 to
150; (2) a coil-coiled domain from amino acids 150 to 244;
(3) an evolutionarily-conserved domain from amino acids
244 to 337; and (4) a nuclear export signal from amino acids
180 to 190. This last domain is responsible for transporting of
317
Beclin 1 from the nucleus to the cytosol, and it is only the cy-
tosolic form that regulates autophagy [118]. Beclin 1 has been
identified in the trans-Golgi Network [92], at the endoplasmic
reticulum, at the mitochondria and the perinuclear membrane
[118, 119].
The yeast homolog of Beclin 1 is Atg6. Structural analysis
has revealed that Atg6 is identical to Vps30, a protein involved
in the vacuolar protein sorting pathway [120]. Atg6/Vps30
forms a complex with Vps34 and its adaptator Vps15. When
this complex is associated with Vps14 (complex I), it regulates
autophagy. In contrast, when this complex is bound to Vps38
(complex II), it regulates the vacuolar protein sorting of car-
boxypeptidase Y [121]. Complex I contributes to autophago-
some formation by allowing other Atg proteins to relocate to
the pre-autophagosomal structure [122]. Interestingly, in
Atg6-defective yeast, Beclin 1 is only able to restore the au-
tophagy function of this mutant, suggesting that Beclin 1
does not regulate other lysosomal trafficking pathways [91,
93]. No mammalian homologs of Atg14 and Vps38 have yet
been identified. However, the interaction between Beclin 1
and Vps34 is conserved in mammals, and the Beclin 1/
hVps34 complex is also able to bind to other partners, as we
shall see below.
4.1. Partners of Beclin 1
Beclin 1 is part of a multiprotein complex, and acts as
a platform, recruiting activator or repressor of Beclin 1/
hVps34-dependent autophagy.
4.1.1. Beclin 1 and hVps34
Beclin 1 binds to the mammalian homolog of Vps34. This
interaction is evolutionarily conserved. By indirect immunoflu-
orescence, it has been shown that the Beclin 1/hVps34 complex
functions at the TGN (trans-Golgi network) [92]. The hVps34
binding domain of Beclin 1 has been identified as the evolu-
tionarily conserved domain (ECD) of Beclin 1. Using a struc-
tural-function approach, it has been shown that the ECD of
Beclin 1 is essential for its binding to hVps34, and its function
in autophagy and tumor suppression [91, 93]. In yeast, Vps30/
Atg6-Vps34 complex is implicated in vacuolar protein sorting
[121]. However, in mammals, no role of Beclin 1 in hVps34-
dependent protein trafficking has been identified [91, 93].
Recent studies have shown that the Beclin 1/hVps34 inter-
action can be modulated and autophagic levels modified as
a result. Indeed, Bcl-2, an inhibitor of autophagy, is able to
disrupt the Beclin 1/hVps34 interaction in the colon cancer
cell line HT-29 [119]. It is interesting to note that Bcl-2 and
hVps34 interact with two different domains of Beclin 1. UV-
RAG, as we will see below, stimulates autophagy and potenti-
ates Beclin 1/hVps34 interaction [123]. As seen in Section 3.1,
amino acids stimulate mTOR by activating hVps34 [89, 90].
This activation of hVps34 by amino acids is hard to reconcile
with the positive role of hVps34 in Beclin 1 dependent autoph-
agy. In addition, two studies of the role of amino acids in
Beclin 1-associated hVps34 activity reached contradictory
conclusions. In MCF.7 cells that stably overexpress Beclin 1,
318 S. Pattingre et al. / Biochimie 90 (2008) 313e323
it has been demonstrated that amino acid starvation inhibits
Beclin 1-associated hVps34 activity [89]. In contrast, in
C2C12 myotubes, amino acids decrease Beclin 1-associated
hVps34 activity [124]. The role of hVps34 in regulating au-
tophagy is complex and unclear. One hypothesis is that
hVps34 may act in different subcellular compartments to reg-
ulate autophagy or mTOR activity [85, 86].
4.1.2. Beclin 1 and UVRAG
UVRAG is a tumor suppressor candidate that frequently ex-
hibits a monoallele mutation in human colon cancer [125]. Its
role in autophagy has been recently demonstrated thanks to its
interaction with viral Bcl-2 [123]. UVRAG is part of the Be-
clin 1 complex, and it stimulates autophagy through a direct
interaction with the coiled-coil domain of Beclin 1. UVRAG
increases the interaction between Beclin 1 and hVps34. The
UVRAG/Beclin 1/hVps34 complex promotes autophagy, and
suppresses proliferation and tumorogenicity in human colon
cancer cells. UVRAG is also responsible for a congenital mal-
formation, known as situs inversus, that involves a reversal of
the positioning of some or all of the visceral organs [126]. The
role of autophagy in this developmental process remains to be
elucidated.
4.1.3. Beclin 1 and Bcl-2
Originally, Beclin 1 was discovered through its interaction
with Bcl-2 using a yeast two-system hybrid. This interaction is
conserved in antiapoptotic family members, since Beclin 1 is
also able to interact with Bcl-xL and viral Bcl-2 [117, 119,
127]. The inhibition of autophagy by Bcl-2 has been demon-
strated in yeast, in an ex vivo model, and in vivo in mice.
This inhibition is dependent on the Beclin 1/Bcl-2 interaction.
Interestingly, this interaction is modulated by conditions
known to regulate autophagy. Under nutrient-rich conditions,
when autophagy is inhibited, Beclin 1 and Bcl-2 interact
strongly. In contrast, in starvation, when autophagic rates are
high, the interaction between Beclin 1 and Bcl-2 is weak.
This suggests that Bcl-2 acts as a rheostat that turns autophagy
on or off when required. Under these conditions, it may be
possible to regulate Bcl-2 autophagic activity quickly. Recent
data show that Bcl-2 phosphorylation by the kinase JNK1 is
essential. Under starvation conditions, JNK1 phosphorylates
Bcl-2, and as a result, inhibits its interaction with Beclin 1
and stimulates autophagy. In contrast, under nutrient-rich con-
ditions, Bcl-2 phosphorylation is inhibited, allowing Bcl-2 to
interact with Beclin 1 and thus decreasing autophagy [128].
Interestingly, under starvation conditions the interaction be-
tween Bcl-2 and Bad, a BH3-only protein that positively reg-
ulates autophagy (see Section 4.2.3), is increased. It is
essential to investigate the role of Bcl-2 phosphorylation in
Bad/Bcl-2 interaction and in the equilibrium between autoph-
agy and apoptosis.
4.1.4. Other partners
4.1.4.1. Ambra-1. Ambra-1 has recently been shown to be an-
other Beclin 1 interacting protein that acts as an activator of
autophagy. Ambra-1 is mainly expressed in the brain, where
it plays an essential role during development [129].
4.1.4.2. ICP34.5. ICP34.5 is a herpes simplex virus type-1
neurovirulence protein that interacts with Beclin 1 and inhibits
its autophagic function. An HSV-1 that contains a mutant of
ICP34.5 unable to interact with Beclin 1 fails to inhibit au-
tophagy in neurons, and demonstrates impaired ability to
cause encephalitis in mice. Thus, Beclin 1 is used by the viral
protein to inhibit autophagy and confer pathogenicity [130].
4.2. Role of Beclin 1
By itself, Beclin 1 does not have any enzymatic activity but
acts as a platform, recruiting an activator (UVRAG) and a re-
pressor (Bcl-2) of Beclin 1/hVps34-dependent autophagy. This
part of the review will focus on the consequences of the stim-
ulation of autophagy by Beclin 1 in other mechanisms, such as
tumor suppression or cell death.
4.2.1. Is the regulation of autophagy by Beclin 1
a general paradigm?
How the Beclin 1/hVps34 complex regulates autophagy is
still unclear. Data from yeast have shown that Beclin 1 complex
is involved in autophagosome formation at an early stage, rather
than in the expansion step. This complex is essential for the
recruitment of other Atg proteins to the pre-autophagosomal
structure [7], and for the production of PtdIns3P. Its localization
on the PAS is regulated by Atg14 [131]. In addition, the product
of hVps34, PtIns3P, seems to play a role in autophagosomal
membrane expansion. Recently, the human ortholog of
Atg18, hWIPI-1a has been identified. After amino acid starva-
tion, hWIPI-1a, that contains a phospholipid binding domain
colocalizes with autophagosomes [132]. In yeast, this domain
binds to PtIns3P, and in consequence it plays a role in the for-
mation of the PAS [133].
It has been recently shown in a different context that au-
tophagy is regulated by ROS (Reactive Oxygen Species)
[41, 134]. Thus, it appears that ROS production leads to the
down-regulation of hAtg4, which is responsible for cleaving
LC3-II from autophagosomes, and the inhibition of autophagy
[134]. Interestingly, ROS-induced autophagy is partially de-
pendent upon the Beclin 1/hVps34 complex.
It has been demonstrated recently that autophagy can occur
independently of Beclin 1 in other contexts. Treatment of
a neuronal cell line with the neurotoxin 1-methyl-4-phenylpyr-
idinium causes a mitochondrial injury and increases autoph-
agy, even when Beclin 1 expression has been knocked-down
by siRNA [135]. This study adds an additional level of com-
plexity to the regulation of autophagy, because it shows that
at least two independent mechanisms can induce autophagy.
In starvation-induced autophagy, the Beclin 1/hVps34 com-
plex mediates non-selective bulk degradation of cytoplasmic
constituents. However, injured mitochondria and organelles
may directly stimulate autophagy [136]. The mechanism of
Beclin 1-independent autophagy remains to be clarified.
 S. Pattingre et al. / Biochimie 90 (2008) 313e323
4.2.2. Beclin1, autophagy and tumor suppression
The mammary cancer cell line MCF-7 expresses low or un-
detectable levels of Beclin 1. When stably transfected, Beclin
1 restores the autophagic capacities, and decreases the tumor-
ogenicity of the cells [137]. The homozygotic deletion of
Beclin 1 is lethal. Data from two independent laboratories
demonstrate that heterozygotous deletion of Beclin 1 in
mice leads to spontaneous tumors in liver, lung and B-cells,
suggesting that Beclin 1 is a haploinsufficient tumor suppres-
sor gene [138, 139]. The ECD domain [91] and the nuclear
export sequence [118] are both essential for Beclin 1 tumor
suppression function. In addition, UVRAG and Ambra-1,
that stimulate autophagy, are both tumor suppressor genes,
and Bcl-2, which inhibits autophagy, has oncogenic capac-
ities. It would be interesting to find out whether Bcl-2 is on-
cogenic as a result of its anti-autophagic function as well as
of its well-known antiapoptotic function [140]. To date, Beclin
1 is the only ATG gene reported to be a tumor suppressor
gene. One major question remains to be answered: is the
Beclin 1 tumor suppression function attributable to its auto-
phagic function? Recent evidence for a tumor suppression
function of autophagy has been recently published. Atg4 is
a cystein protease that cleaves LC3-I before it binds to a phos-
pholipid in the autophagosomal membrane. Four isoforms of
Atg4 coexist in humans. In Atg4C/ mice, the developmental
phenotype is normal, but starvation-induced autophagy is
reduced, and an increase in 3-methylcholanthrene-induced
fibrosarcomas has been observed [141]. An elegant study
has recently shown that autophagy suppresses tumor progres-
sion by protecting cells against genomic instability during
metabolic stress [142]. These findings are consistent with a
tumor suppression role of autophagy. It is also interesting to
note that both the MAPLC3 and ATG7 genes are located in
a chromosomic region that is known to be frequently deleted
in cancer [143]. Finally, reinforcing the hypothesis that auto-
phagy is a tumor suppressor mechanism, it is interesting to
note that oncogenic signaling molecules suppress autophagy,
such as class I PI3K, akt/PKB and mTOR, whereas tumor sup-
pressors, such as PTEN, p53 and Dapk, stimulate autophagy
[140]. However, numerous studies have shown that autophagy
promotes cancer cell survival [144e146] by inhibiting cell
death after metabolic stress for example see Ref. [147].
Further studies on ATG genes are needed to clarify the role
of autophagy in tumor progression.
4.2.3. Beclin 1, autophagy and apoptosis
Recently, a BH3 domain was identified in the Beclin 1 se-
quence from amino acids 112 to 159 [127, 148]. BH3-only
proteins are a category of pro-apoptotic Bcl-2 related proteins
[149]. The BH3-only protein BAD and the pharmacological
BH3 mimetic ABT737 disrupt the Beclin 1eBcl-2 (or Bcl-xL)
complex and stimulate autophagy [127]. However and in
contrast to other BH3-only proteins, the pro-apoptotic role
of Beclin 1 is not clear. No defect in cell death was observed
in heterozygotous Beclin 1/ mice [138, 139]. Only one
study reports a pro-apoptotic function for Beclin 1 after over-
expression of Beclin 1 in MKN28 human gastric cancer cells
319
treated with cis-diamminedichloroplatinum [91]. In contrast,
Beclin 1 inactivation triggers apoptotic cell death in Caenorhab-
ditis elegans embryos [150]. It is also interesting to note that
a complex between a glutamate receptor, nPIST and Beclin 1
is implicated in neuronal cell death in Lurcher mice, a model
of neurodegenerative disease caused by a mutation in the d2
glutamate receptor gene [151]. Further studies are needed to
clarify the role of Beclin in apoptosis.
5. Concluding remarks
The signaling of autophagy depends on two tightly regu-
lated complexes. mTORC1 signaling inhibits autophagy, and
is also able to regulate other processes such as protein transla-
tion. In contrast, the only known function of the Beclin 1 com-
plex is to stimulate autophagy. However, in yeast, the Atg6/
Vps30 complex controls both autophagy and the trafficking
of lysosomal enzymes. One important aspect of extending
our knowledge of how Beclin 1 regulates autophagy will in-
volve identifying the mammalian homologs of Atg14 and
Vps38, proteins that are associated with Atg6/Vps30 com-
plexes in yeast.
Interestingly, analogs of rapamycin (CCI-779, RAD001,
AP23573) and small-molecule Bcl-2 family inhibitors (phar-
macological BH3 mimetics) are in clinical development for
antitumor therapy [152, 153]. As discussed in this review,
both these families of drugs stimulate autophagy by targeting
the mTORC1 complex and the Beclin 1 complex. It will be
important to investigate carefully whether stimulation of au-
tophagy by these compounds acts as an adjuvant to or a brake
on antitumor treatment, and whether autophagy has a beneficial
or harmful role during tumor progression [31]. Moreover, we
know that these drugs are not specific for autophagy, and so
developing specific inhibitors of autophagy will be an impor-
tant step toward assessing the therapeutic implications of ma-
nipulating autophagy [31, 33].
Another emerging concept is that signaling pathways have
entry points other than mTOR by which to modulate the activ-
ity of the molecular machinery of autophagy, and post-transla-
tional modifications other than the ubiquitin-like conjugation
systems Atg12 and Atg8(LC3) by which they can regulate au-
tophagy [53]. In attempting to elucidate the intricacies of the
plasticity of autophagy regulation, and thus to discover spe-
cific drugs that target autophagy, the regulation of Beclin 1/
hVps34 complex activity by the phosphorylation of Bcl-2,
and that of the activity of Atg4 by oxidation [134] are proba-
bly only the tip of the iceberg.
Acknowledgements
Work in P. Codognos laboratory is supported by institu-
tional funding from The Institut National de la Sante et de
la Recherche Medicale (INSERM) and grants from the Asso-
ciation pour la Recherche sur le Cancer to SP and PC. This
work was also supported by institutional funds from the Centre
National de la Recherche Scientifique (CNRS), Montpellier I
and II Universities and grants from SIDACTION and the
320 S. Pattingre et al. / Biochimie 90 (2008) 313e323
Agence Nationale de Recherches sur le SIDA (ANRS). LE
was the recipient of a fellowship from SIDACTION.
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