Download The regulation and function of Class III PI3Ks: novel roles for Vps34

Biochem. J. (2008) 410, 1–17 (Printed in Great Britain)
1
doi:10.1042/BJ20071427
REVIEW ARTICLE
The regulation and function of Class III PI3Ks: novel roles for Vps34
Jonathan M. BACKER1
Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.
The Class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar
protein sorting 34), was first described as a component of the
vacuolar sorting system in Saccharomyces cerevisiae and is
the sole PI3K in yeast. The homologue in mammalian cells,
hVps34, has been studied extensively in the context of endocytic
sorting. However, hVps34 also plays an important role in the
ability of cells to respond to changes in nutrient conditions. Recent
studies have shown that mammalian hVps34 is required for the
activation of the mTOR (mammalian target of rapamycin)/S6K1
(S6 kinase 1) pathway, which regulates protein synthesis in
response to nutrient availability. In both yeast and mammalian
cells, Class III PI3Ks are also required for the induction
of autophagy during nutrient deprivation. Finally, mammalian
hVps34 is itself regulated by nutrients. Thus Class III PI3Ks
are implicated in the regulation of both autophagy and, through
the mTOR pathway, protein synthesis, and thus contribute to the
integration of cellular responses to changing nutritional status.
INTRODUCTION
cerevisiae [10]. The sequence of Vps34p did not give a clue
as to its function until the cloning of the mammalian Class IA
PI3K, p110α, revealed it to be a lipid kinase [11]. The homology
between VPS34 and p110α led to the direct demonstration
that Vps34p has PI3K activity [12]. Vps34 homologues have
been identified in unicellular organisms (Schizosaccharomyces
pombe, Candida albicans, Dictyostelium discoideum), plants,
Caenorhabditis elegans, Drosophila melanogaster, as well as
vertebrates [13–19]. In mammals, hVps34 (mammalian Vps34
homologue) is ubiquitously expressed [19].
PI3Ks are classified by substrate specificity and subunit organization [1,2]. Class I enzymes, which produce PtdIns(3,4,5)P3
in vivo, all contain homologous 110 kDa catalytic subunits
(p110α, β, δ and γ ). The regulatory subunits for Class IA
enzymes (p85α, β, p55α and p55γ ) and Class IB enzymes
(p101) are not structurally related. Class II PI3Ks do not
produce PtdIns(3,4,5)P3 , but appear to produce both PtdIns(3,4)P2
and PtdIns3P in vivo [20,21]. The three isoforms of Class II
PI3K (commonly called PI3K C2α, β and γ ) are monomeric
and are notable for the presence of a C-terminal C2 domain not
found in other PI3Ks. Finally, the sole Class III PI3K, Vps34,
only produces PtdIns3P. The enzyme is closely associated with
a protein kinase, Vps15, which has sometimes been described as
a Vps34 regulatory subunit; the functional relationship between
these two proteins is discussed below.
Vps34 exhibits considerable homology with the catalytic
subunits of other PI3Ks, particularly at the level of domain
Vps34 (vacuolar protein sorting 34) is a member of the PI3K
(phosphoinositide 3-kinase) family of lipid kinases, all of which
phosphorylate the 3 hydroxy position of the phosphatidylinositol
ring. In the accepted nomenclature for PI3Ks [1,2], Vps34 is
classified as the sole Class III enzyme, whose substrate specificity
is limited to phosphatidylinositol. Thus its product in cells is
PtdIns3P. This distinguishes it from the more numerous and better
studied Class I and Class II enzymes, which can produce PtdIns3P,
PtdIns(3,4)P2 or PtdIns(3,4,5)P3 , depending on the isoform [1,2].
The first known functions of Vps34 were in the regulation of
vesicular trafficking in the endosomal/lysosomal system, where it
is involved in the recruitment of proteins containing PtdIns3Pbinding domains to intracellular membranes [3,4]. However,
Vps34 has also been implicated in other signalling processes,
including nutrient sensing in the mTOR [mammalian TOR (target
of rapamycin)] pathway in mammalian cells, trimeric G-protein
signalling to MAPK (mitogen-activated protein kinase) in yeast,
and autophagy in both yeast and higher organisms [5–9]. Given
the widening interest in Vps34, the present review will provide a
focused discussion of its biochemistry, regulation and function in
eukaryotic cells.
STRUCTURE AND CATALYTIC ACTIVITY OF Vps34
Vps34 was first discovered and cloned by Emr and co-workers
in 1990, as part of a screen for vps mutants in Saccharomyces
Key words: hVps34, hVps15, phosphoinositide, phosphoinositide
3-kinase (PI3K), target of rapamycin (TOR), vacuolar protein
sorting 34 (Vps34).
Abbreviations used: AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated kinase; CPY, carboxypeptidase Y; CSF-1, colonystimulating factor 1; Cvt, cytosol-to-vacuole; ECD, evolutionarily conserved domain; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; EEA1, early
endosomal antigen 1; EGF, epidermal growth factor; eGFP, enhanced green fluorecent protein; ESCRT, endosomal sorting complex required for transport;
GAP, GTPase-activating protein; GFP, green fluorescent protein; Hrs, hepatocyte-growth-factor regulated tyrosine kinase substrate; MAPK, mitogenactivated protein kinase; MTM, myotubularin; mTOR, mammalian target of rapamycin; PAS, pre-autophagosomal structure; PH, pleckstrin homology; PI3K,
phosphoinositide 3-kinase; PX, phox homology; siRNA, small interfering RNA; S6K1, S6 kinase 1; dS6K, Drosophila S6K; SARA, Smad anchor for receptor
activation; SH3, Src homology 3; SNARE, soluble N -ethylmaleimide-sensitive fusion protein-attachment protein receptor; TOR, target of rapamycin;
TORC1/2, TOR complex 1/2; TSC, tuberous sclerosis complex; UVRAG, UV radiation resistance-associated gene; Vps, vacuolar protein sorting; hVps,
mammalian Vps homologue.
To avoid confusion, species-independent references to Class III PI3K use the term Vps34. Specific references to the Class III PI3K from yeast use the
term Vps34p. The mammalian Class III PI3K was first cloned from human cells [19], and has been previously referred to as hVps34. Although this term is
inaccurate, we have retained the usage in the present review in the interest of continuity with the pre-existing literature. A similar nomenclature is used for
yeast Vps15p and mammalian hVps15 (formerly called p150 [29]).
1
[email protected]
c The Authors Journal compilation c 2008 Biochemical Society
2
Figure 1
J. M. Backer
Proteins associated with Vps34 in yeast and mammals
The domain structure of Vps34-associated proteins is shown. All domain borders are taken from the mammalian enzymes, with the exception of Atg14p and Vps38p, which occur only in yeast. BH,
Bcl2-homology; C/C, coiled-coil.
organization. Currently, the only existing structure of a PI3K
catalytic subunit is the Class I PI3Kγ structure solved by Williams
and co-workers [22]. As compared with PI3Kγ , Vps34 has
a structurally uncharacterized N-terminal region of approx. 50
amino acids, followed by C2, helical and kinase domains that
are approx. 30 % homologous with PI3Kγ (Figure 1). The C2
domain fold in the PI3Ks is related to that of PLCγ (phospholipase
Cγ ), and is likely to be involved with interactions with acidic
phospholipids rather than Ca2+ [22]. The helical domain has an
important regulatory role in Class IA PI3K catalytic domains,
where it mediates inhibitory contacts with the Class IA regulatory
subunit p85 [23]. A parallel role for the Vps34 helical domain
has not been studied. The kinase domain shows the classic twolobed structure characteristic of protein kinases. Budovskaya
et al. [24] showed that the extreme C-terminal 11 residues of
yeast Vps34p are required for lipid kinase activity, independently
of any effects on binding to Vps15p (the putative serine kinase
that associates with Vps34p, discussed below). Consistent with
this finding, antibodies targeted to the C-terminus of mammalian
hVps34 are potent inhibitors of the enzyme [25].
Vps34 enzymes are unique among PI3Ks in that they will
only utilize phosphatidylinositol as a substrate. The reason
for this selectivity is thought to lie in the make-up of the
substrate recognition loop which, in Class I or II PI3K, contains
multiple basic residues that accommodate the negative charges
in PtdIns4P and PtdIns(4,5)P2 [26]. In contrast, this region
of Vps34 is relatively uncharged, limiting Vps34 substrates to
phosphatidylinositol itself. Vps34 is also distinct from Class I
enzymes in that it is more active in the presence of Mn2+ than
Mg2+ [19]. Mammalian hVps34 will also utilize Ca2+ -ATP, and
reaches approx. 50 % of maximal activity with this cation (M.R.
Lau and M.J. Fry, personal communication). In this regard, it is
similar to the C2β isoform of Class II PI3K, which is also active
in the presence of Ca2+ -ATP [27].
c The Authors Journal compilation c 2008 Biochemical Society
In addition to lipid kinase activity, PI3Ks also possess activity
towards protein substrates. Yeast Vps34p is known to undergo
autophosphorylation [28], although the sites of phosphorylation
have not been identified. Panaretou et al. [29] showed that a
complex of mammalian hVps34 with mammalian hVps15 showed
significant kinase activity towards generic substrates such as
myelin basic protein, but the relative contributions of the two
proteins was not determined [29]. Although several potential
substrates have been identified for Class I enzymes [30–32],
authentic in vivo substrates for Vps34 enzymes have not yet been
identified. A functional role for Vps34 protein kinase activity
was suggested by differences in the phenotypes of C. albicans
strains that were Vps34-null compared with strains expressing
a lipid-kinase-deficient/protein-kinase-active mutant Vps34 [33],
although these results could also reflect scaffolding functions of
Vps34.
METHODS FOR STUDYING Vps34
Studies of hVps34 have been hampered by the lack of specific inhibitors. Interestingly, both yeast and human Vps34 share a lysine
residue that, in human Class I PI3Ks, is the site of covalent modification by the pan-PI3K inhibitor wortmannin (Lys833 in PI3Kγ )
[34,35]. However, whereas Class I enzymes and mammalian
hVps34 are extremely sensitive to wortmannin (IC50 = 5–15 nM),
yeast Vps34p is relatively resistant (IC50 = 3 µM) [28]. The
reason for this disparity in inhibitor sensitivity is not understood.
Yeast Vps34p is also inhibited by LY294002 (IC50 = 50 µm) in
a range similar to that for other PI3Ks and PI3K-related protein
kinases such as mTOR [28]. At the present time, specific smallmolecule inhibitors of hVps34 have not been identified, although
a recent upsurge in pharmaceutical company interest in hVps34
as a potential drug target should alleviate this problem. Although
The regulation and function of Class III PI3Ks
3-methyladenine has been suggested to be a specific inhibitor of
hVps34 [9], it in fact inhibits Class I and II PI3Ks as well [36].
Courtneidge and co-workers demonstrated that antibodies to
the C-terminus of Class IA PI3Ks are specific inhibitors of these
enzymes [37,38]. This approach has been successfully applied to
mammalian hVps34 [25], and microinjection of inhibitory antihVps34 antibodies has been a useful tool for the identification
of hVps34-dependent functions in mammalian cells (see below)
[5,39–46].
Methods for measuring Vps34 activity in cells and in vitro
are similar to methods used for other PI3K isoforms, and share
some of the same limitations; this area has been comprehensively
reviewed recently [47]. PtdIns3P, the product of Vps34, can be
extracted from [32 P]orthophosphate- or [3 H]myo-inositol-labelled
cells, and the deacylated lipids can be separated by ion-exchange
HPLC [48]. This method does not, of course, provide information
as to the spatial localization of the lipids. MS-based methods are
also being developed for analysis of phosphoinositide abundance
in cells and tissues [47]. A blotting method, in which lipid extracts
from cells are probed with recombinant FYVE domains from
SARA (Smad anchor for receptor activation), has been used to
measure PtdIns3P in intact cells [49].
A widely used alternative method, analogous to the use
of GFP (green fluorescent protein)–PH (pleckstrin homology)
domains for detection of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 , is
the expression of GFP-linked FYVE or PX (phox homology)
domains, which are specific for PtdIns3P; tandem domains are
sometimes used to increase binding affinity [50,51]. Although
these constructs do provide information about spatial localization
of PtdIns3P, they cannot be used to accurately quantify the net
production of PtdIns3P in cells. The threshold for detection of
cytosolic eGFP (enhanced GFP) by fluorescence microscopy
is approx. 200 nM [52], meaning that small vesicles labelled
with an eGFP reporter may not be visible above background
autofluorescence. Thus a change in the size of eGFP–FYVElabelled membranous structures (e.g. through the fusion or
aggregation of pre-existing eGFP-labelled vesicles) will cause
an increase in the fluorescence signal independently of any
change in PtdIns3P production. This is a particular problem
in the quantification of PtdIns3P in autophagosomal structures,
which tend to be detected as large cytosolic vesicular aggregates.
In contrast, detection of eGFP-labelled probes in the plasma
membrane can be accurately quantified using TIRF (total internal
reflection fluorescence) microscopy (reviewed in [53]).
Even as a probe of the sites of PtdIns3P production, there
are a number of caveats involved in the use of FYVE and PX
domain constructs. First, as is the case with PH-domain probes
[54], FYVE-domain probes tend to identify sites of PtdIns3P
abundance, such as endosomes, but may fail to detect PtdIns3P in
membranes with lower, but still physiologically important, concentrations of the lipid. Secondly, lipid-binding domains with similar lipid specificities may localize differently in cells [54]. For example, TAFF1 (tandem FYVE fingers 1) contains two PtdIns3Pspecific FYVE domains yet localizes to the Golgi, rather than to
endosomes [56]. A third problem is that FYVE-domain probes
can perturb PtdIns3P-dependent processes. Thus overexpression
of a 2X-FYVE construct blocks endosomal fusion and EGF (epidermal growth factor) receptor sorting [57]. As an alternative to
FYVE-domain-based probes, antibodies specific for PtdIns3P are
commercially available, but they have not been as well characterized as antibodies for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 [58–61].
Except in yeast, where Vps34p is the sole PI3K, methods for
evaluation of intracellular PtdIns3P levels are not necessarily
indicative of Vps34 activity. For example, previous studies have
shown that Class II PI3Ks also produce PtdIns3P in vivo [21,62],
3
although this seems to be primarily in the plasma membrane.
In mammalian cells, the dephosphorylation of PtdIns(3,4,5)P3
by endosomal PtdIns 5- and 4-phosphatases provides another
source of endosomal PtdIns3P [63]. Alternatively, the Type Iα
inositol polyphosphate 4-phosphatase localizes to endosomes,
where it produces PtdIns3P through the dephosphorylation of
PtdIns(3,4)P2 [64]. The existence of Vps34-independent sources
of PtdIns3P in vivo was directly demonstrated in C. elegans,
where the phenotype of a Vps34 null mutant (let-512) is
rescued by siRNA (small interfering RNA) knockdown of MTM
(myotubularin) 1 and MTM6 [65]. Myotubularins are PtdIns3Pspecific lipid phosphatases (see below), and their ability to rescue
a null allele of Vps34 means that additional sources of PtdIns3P
must exist in C. elegans. Consistent with this, siRNA knockdown
of the C. elegans Class II PI3K, F39B1.1, reduces the rescue of
let-512 mutants by the myotubularin knockdown.
The enzymatic activity of recombinant Vps34 is readily
assayed in vitro [19], and the activity of endogenous Vps34
can be measured in immunoprecipitates of Vps34 or its binding
partners [5,66]. As antibodies to Vps34 can be inhibitory,
care must be taken to test antibodies for inhibition using
recombinant Vps34, and to elute Vps34 from the antibody
prior to assay if necessary [5]. Although lipid kinase activity
can be measured in immunoprecipitates from cells expressing
epitope-tagged Vps34, we have found that the specific activity
of overexpressed mammalian hVps34 is in fact approx. 5 % of
that of endogenous hVps34 (Y. Yan and J.M. Backer, unpublished
work). This presumably reflects the requirement for hVps15 and
other hVps34-associated proteins for full activity (see below).
Thus studies on the regulation of hVps34 that depend on its
overexpression in the absence of hVps15, and perhaps other
binding partners, may not accurately reflect the physiological
regulation of the enzyme.
The hVps34 gene has not yet been disrupted in mice. Genetic
approaches to studying Vps34 in metazoans has been useful
in C. elegans, where mutations of the let-512 gene lead to
L3/L4 embryonic lethality, disruption of endocytic uptake and an
expansion of the outer nuclear membrane [17]. In Drosophila,
mutations in ird1 (the Drosophila homologue of the Vps34related kinase Vps15; see below), which would presumably also
inhibit Drosophila dVps34, lead to a loss of starvation-induced
induction of antimicrobial peptides, suggesting a link between
Vps34 and innate immunity [67]. However, ird1 mutants also
show constitutive activation of the Toll pathway, in contrast
with results in RAW264.7 macrophages, where expression of
kinase-dead hVps34 blocks TLR9 (Toll-like receptor 9)-mediated
responses [68]. These could reflect differences in flies compared
with mammals, or differences between the phenotype of inhibition
of Vps34 compared with its presumed upstream regulator Vps15
(see below).
REGULATION OF Vps34 BY Vps15
Like Vps34p, Vps15p was cloned from S. cerevisiae by the
Emr laboratory [69]. Although Vps15 has been described as
a Vps34 regulatory protein, the sequence of Vps15 suggests
that it functions as a protein kinase. Homologues have been
described in mammals and Drosophila [29,67], and a share similar
domain organization (Figure 1). Vps15 contains an N-terminal
myristoylation consensus sequence, and the yeast and mammalian
enzymes are myristoylated [29,70]. Mutation of the Glu2 acceptor
site has no effect on partitioning of Vps15p into particulate
fractions, but does partially reduce Vps15p phosphorylation and
is additive with other Vps15p mutations for temperature-sensitive
c The Authors Journal compilation c 2008 Biochemical Society
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J. M. Backer
growth [70]. Vps15 consists of a predicted protein kinase domain
followed by a central region containing multiple HEAT repeats
and a series of C-terminal WD40 domains. It was suggested by
Murray et al. [71] that these WD40 repeats might form a βpropeller-like structure, similar to the C-terminus of Gβ subunits
[71]; in fact recent studies from Dohlman and co-workers suggest
that Vps15p binds to yeast Gα subunits (see below) [7].
The relationship between the Vps34 and Vps15 proteins was
first suggested by the similar phenotypes caused by their deletion
in S. cerevisiae, which do not cleanly fall into any of the five
classes (A–E) of vps mutants defined by Stevens, Emr and
their co-workers [72–74]. vps34 and vps15 stains resemble
Class D vps mutants [72], with defects in the sorting of newly
synthesized hydrolases to the yeast vacuole and in vacuolar
segregation during mitosis, but with a relatively normal vacuole
morphology. However, vps15 and vps34 strains also show
sensitivity to growth at 37 ◦C and to osmotic stress, and have
abnormal cytoplasmic membranous structures, which are more
characteristic of Class C mutants [10,73,74]. Thus Vps15p and
Vps34p appear to form a distinct subset of vps gene products [75].
The similarities between the Vps34p and Vps15p phenotypes
led to experiments showing that overexpression of Vps34p
complements for growth at elevated temperatures in Vps15pmutant strains containing a single mutation in the Vps15p
kinase domain, but not Vps15p-null strains or strains containing
triple mutants in the kinase domain [76]. Production of the
Vps34p product, PtdIns3P, is abolished in vps15 strains
or in strains expressing predicted kinase-dead mutants of
Vps15p, and strains expressing a temperature-sensitive allele
of Vps15p are temperature-sensitive for PtdIns3P production
[77]. These results clearly show that a functional Vps15p
with an intact kinase domain is required for Vps34p activity in vivo. Vps15p and Vps34p are both membrane-associated
in yeast [76]. They also co-immunoprecipitate, and the deletion
of Vps15p leads to a loss of Vps34p membrane association
[76]. Interestingly, a native kinase domain of Vps15p is also
required for its binding to Vps34p; point mutations at residues
predicted to abolish kinase activity, based on homology with other
serine/threonine kinases, led to a marked inhibition of Vps15p–
Vps34p co-immunoprecipitation [77]. More recent analysis of the
Vps34p–Vps15p interaction identified residues 837–864 in the Cterminus of Vps34p as sufficient to bind to the kinase and HEAT
domains of Vps15p [24].
Although Vps15p is a protein kinase that binds Vps34p and
whose putative kinase activity is required for Vps34p activity, Vps15p does not appear to be a Vps34p kinase. Vps34p
is phosphorylated in yeast, but the preponderance of this
phosphorylation reflects autophosphorylation rather than phosphorylation by Vps15p [28]. Thus Vps34p phosphorylation is
similar in vps15 compared with wild-type strains, and disabling
mutations in the kinase domain of Vps34p eliminate most of the
in vivo phosphate incorporation into Vps34p. Additional results
question whether Vps34p is regulated by phosphorylation, as
its activity is minimally affected by partial dephosphorylation
with potato acid phosphatase [28]. However, this experiment
was performed with Vps34p overexpressed in vps15 yeast, and
it is not clear how dephosphorylation would affect the activity
of Vps34p from wild-type strains. Overall, the contribution of
phosphorylation to the regulation of Vps34p activity is unclear.
If Vps15 does not activate Vps34p by phosphorylation, why
is an intact Vps15p kinase domain required for Vps34p activity?
Vps15p-mediated autophosphorylation could be required for an
allosteric activation of Vps34p. However, deletion of 30 amino
acids from the Vps15p C-terminus (residues 1426–1455) but not a
nearby internal deletion (residues 1412–1427) abolishes Vps15p
c The Authors Journal compilation c 2008 Biochemical Society
phosphorylation at 30 ◦C, but only inhibits vacuolar protein sorting
at elevated temperatures [70]. It is not clear whether the effect
of the C-terminal deletion on Vps15p phosphorylation is due to
an inhibition of Vps15p kinase activity, or to the removal of its
major sites of autophosphorylation. Nonetheless, the C-terminal
truncation mutant supports vacuolar sorting, and therefore Vps34p
activity, at permissive temperatures in the absence of Vps15p
phosphorylation.
An alternative hypothesis is that Vps15p acts on Vps34p
primarily by recruiting it to cellular membranes. Consistent
with this, overexpression of Vps34p in both vps15 strains and
strains expressing putative kinase-dead Vps15p (E220R) causes
a significant increase in production of PtdIns3P. However, this
lipid production correlates with a partial restoration of vacuolar
sorting only in the mutant vps15 strain and not in the vps15 strain
[77]. Rescue of sorting in the mutant Vps15p yeast presumably
reflects the fact that the E200R mutation weakens but does not
abolish Vps15p–Vps34p binding [76,77]. The authors note that
PtdIns3P levels are higher in the kinase-dead vps15 strain than
in the vps15 strain, and suggest that the different phenotypes
may reflect a threshold level of PtdIns(3)P required for sorting.
Alternatively, these results may suggest that the critical role of
Vps15p involves targeting of Vps34p as opposed to its activation.
Indeed, the in vitro lipid kinase activity of Vps34p extracted
from wild-type and vps15 yeast has not been compared, and
the activity of mammalian hVps34 was increased only 2-fold by
complex formation with hVps15 [29]. On the other hand, siRNA
knockdown of hVps15 in mammalian cells leads to a decrease
in hVps34 protein levels (Y. Yan and J.M. Backer, unpublished
work), suggesting that hVps15 may be required for hVps34
stability. The precise role of Vps15 in the regulation of Vps34
is still uncertain.
It is not clear why the same mutations that disrupt the
protein kinase activity of Vps15p (as measured by its autophosphorylation) also weaken or abolish its binding to Vps34p
[77]. It is possible that binding of Vps15p and Vps34p in
yeast requires Vps15p-mediated phosphorylation of an as yet
unidentified accessory protein. Vps34p and Vps15p do exist
in complexes with other proteins (discussed below), but the
deletion of these accessory proteins does not disrupt the Vps34p–
Vps15p association in vivo [78]. Furthermore, direct
Vps34–Vps15 binding was detected in a two-hybrid assay [24].
Similarly, recombinant mammalian hVps34 and hVps15 do directly associate in vitro [29], although the requirement for an intact
hVps15 kinase domain for this interaction has not been tested.
Interestingly, Vps15 has not been formally proven to possess
protein kinase activity. A comparison of the hVps15 sequence
with that of other protein kinases shows significant differences;
hVps15 lacks, for example, the canonical GXGXXG motif
involved in ATP binding [79] (the sequence is GSTRFF
in hVps15). The major evidence that Vps15p is a protein
kinase comes from experiments in which point mutations
predicted to disrupt kinase function also abolish Vps15p
autophosphorylation [28,69]. However, since the Vps15p mutants
were immunoprecipitated from yeast, and given that the same
point mutations are known to abolish interactions between
Vps15p and other proteins (e.g. Vps34p [77]), it remains possible
that in vitro phosphorylation of Vps15p is in fact due to a copurifying kinase. This kinase would presumably be present in
sub-stoichiometric amounts, and might not have been detected
in parallel immunoprecipitations from [35 S]methionine-labelled
yeast [69]. Similarly, the kinase activity of yeast Vps15p towards
exogenous substrates has never been demonstrated, and the
kinase activity of mammalian hVps15 has only been assayed
in a complex with hVps34 [29]. Like yeast Vps34p, mammalian
The regulation and function of Class III PI3Ks
hVps34 has protein as well as lipid kinase activity [19], so the
identity of the active kinase(s) in this latter experiment is not clear.
SIGNALLING BY Vps34: PtdIns3P -BINDING DOMAINS
Vps34 signalling is presumed to be mediated by the membrane
recruitment of proteins containing modular domains that bind to
PtdIns3P. These domains included FYVE domains, zinc-finger
domains named for the first four proteins known to contain
the domain [Fab1p, YOTB, Vac1p, EEA1 (early endosomal
antigen 1)] [80] and PX domains, named for the Phox homology
domain of the p47phox and p40phox subunits of the phagocyte
NADPH oxidase [81]. FYVE and PX domain proteins have been
extensively reviewed [82–86] and will not be discussed at length
here. FYVE-domain containing proteins that regulate scaffolding
and/or sorting steps in the endosomal system include EEA1 [87–
89] and Hrs (hepatocyte-growth-factor-regulated tyrosine kinase
substrate)/Vps27 [90–92]. The PtdIns3P 5-kinase Fab1/PIKfyve,
which contains a FYVE domain, is involved in endosome/Golgi
sorting and late-endosomal trafficking [93–98]. Whereas most of
these proteins have been localized to endosome-related structures,
Alfy, a FYVE-domain-containing protein involved in autophagy,
is localized in the nuclear envelope rather than in endocytic
vesicles [99]. This may be related to the observation that
disruption of the Vps34 gene in C. elegans (let-512) alters
the morphology of the nuclear membrane [17]. PX-domaincontaining proteins regulated by PtdIns3P include the phagocyte
NADPH oxidase, via its p47phox and p40phox subunits [100–102],
the sorting nexins SNX2, SNX3, SNX4 and SNX16 [103–
106], the t- (target) SNARE (soluble N-ethylmaleimide-sensitive
fusion protein-attachment protein receptor) Vam7 [107], the CISK
(cytokine-independent survival kinase) protein kinase, which
is related to the Akt kinase [108,109], and the PX-domaincontaining RGS (regulator of G-protein signalling) protein, RGSPX1, a Gαs -specific GAP (GTPase-activating protein) that inhibits
EGF receptor degradation when overexpressed [110].
Vps34 IN THE ENDOCYTIC SYSTEM
Sites of Vps34 action in the endosomal system
The discovery that yeast Vps34p is a PtdIns-specific PI3K [11,12]
led to numerous papers showing inhibition of vesicular trafficking
in mammalian cells by PI3K inhibitors such as wortmannin
(reviewed in [111]). However, it was not clear from these studies
which PI3K isoform was responsible. The late 1990s saw the
identification of EEA1 as a wortmannin-sensitive component of
endocytic vesicles [87], and the identification of FYVE domains
as PtdIns3P-specific lipid-binding domains that direct EEA1 to
early endosomes [80,112,113]. Given that GFP–FYVE domain
targeting to endosomes requires Vps34p in yeast [112], it seemed
likely that hVps34 would be responsible for many of the PI3Kdependent steps in the endocytic system.
In fact, different experimental approaches to perturbing hVps34
signalling have yielded somewhat different assignments of
hVps34 function in the endosomal system of mammalian cells.
The first studies specifically addressing the role of hVps34
in mammalian cells used inhibitory antibodies, and showed
that specific inhibition of hVps34 activity delays passage of
internalized PDGF (platelet-derived growth factor) receptors
through the early endosome, slows the recycling of transferrin
receptors, inhibits EEA1 recruitment to early endosomes [25],
inhibits homotypic early endosomal fusion [39] and inhibits
the formation of internal vesicles in multivesicular bodies [43].
5
hVps34 is also implicated in the movement of endosomes on
microtubule tracks [40], through the recruitment of KIF16B, a
PX domain-containing kinesin that is targeted to endosomes in a
PI3K-dependent manner [114]. Anti-hVps34 antibodies inhibit
both apical and basolateral endocytic trafficking in polarized
hepatocytes [42]. Expression of a kinase-dead hVps34 inhibits
maturation of procathepsin D in lysosomes, consistent with a role
for hVps34 in endosomal sorting [115]. In contrast, inhibition
of PtdIns3P signalling by overexpression of FYVE domains
specifically inhibits EGF receptor degradation but does not affect
fluid-phase transport or formation of endosomal carrier vesicles
from donor early endosomes [57]. Similarly, stable knockdown
of hVps34 blocks multivesicular body formation and slows the
degradation of the EGF receptor, but does not affect fluid-phase
endocytosis or EEA1 binding to early endosomes [116]. Finally,
inhibition of PtdIns3P production in the early endosome via
specific targeting of the PtdIns3P phosphatase MTM1 causes
pronounced tubulation of the early endosome, with impaired
transferrin receptor egress but only slight effects on EEA1
targeting or EGF-receptor degradation [117].
The differential methodologies used in these studies have
distinct experimental drawbacks. Methods aimed at globally
blocking PtdIns3P signalling through expression of FYVE
domains [57] will also inhibit signalling by Class II PI3Ks, which
produce PtdIns3P in the plasma membrane [21,62]. Specific
targeting of MTM1 to the early endosome [117] may be less
affected by this problem. Inhibition of PtdIns3P production
using hVps34 inhibitors (antibodies, kinase-dead mutants or
drugs, when available) and hVps34 knockdown will block both
PtdIns3P signalling as well as any signalling by the protein
kinase activity of hVps34. The knockdown strategy also alters
the composition of multiprotein complexes containing beclin-1
and hVps15 (see below), which could have additional effects on
hVps34-independent functions of these proteins. An additional
complication is that PI3K inhibitors increase the activation level
of Rab5 in phagosomes and only block a subset of Rab5dependent events [118]. Secondary effects of hVps34 inhibition
due to enhanced Rab5 signalling in the early endosome might be
expected to spare early endosomal trafficking events relative to
events in the late endosome or multivesicular body.
Finally, it should be noted that some effects of hVps34
inhibition might be due to a decrease in the availability of
PtdIns3P as a substrate for PIKfyve/Fab1, a PtdIns3P-5-kinase
[94,119,120]. PIKfyve/Fab1 has been independently implicated in
the maintenance of endosome and lysosome/vacuole morphology
[119,121,122], the regulation of endosome-to-Golgi trafficking
[95] and delivery of ubiquitinated cargo to the lysosome/vacuole
lumen [98,119,123,124].
Mechanism of Vps34 action in the endosomal system
A model for the regulation of Vps34 in the early endosome
came from the identification of mammalian hVps34 and hVps15
in the eluate from a RAB5–GTP affinity column [39]. In vitro
experiments showed direct GTP-dependent binding of Rab5 to
human hVps15 and hVps15–hVps34 dimers, but not to hVps34
itself. Rab5 binding requires the HEAT and WD40 domains of
human hVps15 [71]. These experiments suggest that hVps34
is recruited to Rab-positive early endosomes through Rab5–
Vps15 interactions. This model was validated in cell culture,
where constitutively active Rab5 leads to the recruitment of
endogenous or overexpressed hVps34 and hVps15 to enlarged
EEA1-positive endosomes in HeLa cells [71]. The finding that
overexpressed hVps34 is recruited to endosomes without coexpression of hVps15 was explained by the fact that hVps15
c The Authors Journal compilation c 2008 Biochemical Society
6
Figure 2
J. M. Backer
Rab5 regulation of hVps34–hVps15 in the early endosome
Activated Rab5 binds to hVps15, recruiting the hVps15–hVps34 complex to the early endosome. Rab5 effectors bind simultaneously to PtdIns3P and to Rab5, thereby increasing the specificity of
targeting. C/C, coiled-coil; PI[3]P, PtdIns3P ; RBD, Rab5-binding domain.
is in excess compared with hVps34 in HeLa cells. Interestingly, a
net movement of hVps34 or hVps15 from cytosolic to membrane
compartments was not observed in cell fractionation studies [71],
suggesting that the Rab5 induces a redistribution of hVps34–
hVps15 between membrane compartments, rather than a direct
recruitment from cytosol to membrane.
Overall, the model provides an attractive explanation for the
specificity of Rab5 signalling in the early endosome, since Rab5
effectors such as EEA1 are recruited both directly, by binding
to GTP–Rab5, and indirectly, by recruitment of hVps34–hVps15
and subsequent production of PtdIns3P [39,71,89] (Figure 2).
Two other early endosomal regulatory proteins, Rabenosyn-5
and Rabankyrin-5, share the ability to bivalently interact with
both Rab5 and, via FYVE domains, with PtdIns3P [125,126].
The assembly of these Rab- and PtdIns3P-binding proteins is
thought to regulate early endosomal docking and fusion. Thus
EEA1 interacts with syntaxin 6 and syntaxin 13, endosomal
SNARE proteins that regulate vesicle docking/priming [127,128].
Rabenosyn-5 binds to hVps45 [126], which is related to Sec1
proteins that are negative regulators of SNARE pairing [129].
The endosomal role of Rabankyrin-5 is not clear, and it may play
a more important role in the regulation of pinocytosis [125].
hVps34 and hVps15 also interact with Rab7 in late endosomes
[130]. Unlike Rab5, interactions with Rab7 are strongest with
wild-type or nucleotide-free (N125I) forms, as opposed to
activated (Q76L) or inhibitory (T22N) forms. Accumulation of
PtdIns3P in late endosomes, based on GFP–FYVE staining, is
decreased in cells expressing dominant-negative Rab7 mutants,
suggesting a role for Rab7 in maintaining hVps34 activity in the
late endosome.
The last few years have seen rapid progress in dissecting
the role of PtdIns3P signalling in ubiquitin-dependent sorting
of internalized membrane proteins in the multivesicular body;
this work has been extensively reviewed (see for example
[131]) and will be briefly summarized here. The importance of
ubiquitination during endocytic sorting was first discovered in
yeast [132,133], and the ubiquitin-dependent endocytic sorting
of the EGF, CSF-1 (colony-stimulating factor 1) and other
receptors have been reported in mammalian cells (reviewed in
[134–136]). The degradative sorting of internalized ubiquitinated
cargo requires the sequential engagement of the ESCRT
(endosomal sorting complex required for transport) complexes
I, II and III [4,137,138], and the eventual invagination of the
ubiquitinated membrane proteins into an intraluminal vesicle. A
key regulator of this process is Vps27/Hrs, which contains both
c The Authors Journal compilation c 2008 Biochemical Society
a FYVE domain and an UIM (ubiquitin-interacting motif) and
is itself ubiquitinated [139]. Hrs/Vps27 interacts with endosomal
PtdIns3P, with ubiquitinated cargo, and with ESCRT I, thereby
initiating the assembly of the ESCRT complex on to endosomal
membranes [92,140,141]. In addition, one of the components of
ESCRT III, Vps24, binds to PtdIns(3,5)P2 [142]. Thus Vps34
plays a key role in the initiation of the ESCRT-mediated
processing of ubiquitinated cargo.
Finally, Vps34p is required for retrograde endosome-to-Golgi
transport, a process that requires the assembly of Vps34p, Vps29p,
Vps26p, Vps17p and Vps5p into what is termed the retromer
complex [143]. This pathway is required for the retrieval from
endosomes to the Golgi of Vps10p, the yeast homologue of
the mammalian mannose 6-phosphate receptor [144]. Another
protein required for Vps10p retrieval is Vps30p [145], which
was subsequently shown to be present in a Vps34p–Vps15p–
Vps30p–Vps38p complex involved in vesicular trafficking ([8];
discussed below). This led to the finding that the Vps34p–
Vps15p–Vps30p–Vps38p complex was required for retromer
assembly, presumably by the PtdIns3P-mediated recruitment of
the PX-domain-containing proteins Vps5p and Vps17p [146].
Results from mammalian cells suggests that hVps34 might also
influence the endosome-to-Golgi sorting by providing substrate
for PIKfyve/Fab1, a PtdIns3P 5-kinase [94,119,120], which has
been shown to regulate this pathway [95].
Vps34 AND AUTOPHAGY
Vps34 is essential for macroautophagy, a process by which
cells degrade cytosolic content by the formation of a doublewalled vesicular structure that eventually fuses with lysosomes
[147,148]. Macroautophagy is a physiological response to nutrient
deprivation, and has also been implicated in innate immunity,
development, tumour suppression and clearance of neuronal
aggregates [149–151]. Whereas autophagy is activated by nutrient
deficiency, it is inhibited by nutrient sufficiency. In yeast, this is
mediated by the nutrient-stimulated activation of the TOR protein
kinase, which leads to the phosphorylation and inactivation of
components of the autophagy pathway [147,148]. TOR is also an
important regulator of cell growth, via its effects on transcription,
protein synthesis and ribosome biogenesis (discussed below).
Autophagy has been most intensively studied in yeast, where
mutants of autophagy-related Atg genes have provided important
experimental tools [152]. A number of yeast vps genes are also
required for autophagy, including VPS34 and VPS15 [8]. In
The regulation and function of Class III PI3Ks
Figure 3
7
Vps34p signalling complexes in yeast
Tetrameric complexes containing Vps15p, Vps34p, Vps30p/Atg6p and either Atg14p or Vps38p have been identified in yeast, and regulate vacuolar protein sorting and autophagy. Deletion of VPS34
or VPS15 causes additional trafficking phenotypes not seen with deletion of VPS30 , ATG14 or VPS38 , suggesting the existence of additional complexes. The Vps34p–Vps15p–Gpa1 complex
involved in pheromone signalling may also include Atg14p, which would function as a Gγ protein.
mammalian cells, hVps34 is also involved in nutrient signalling
to mTOR [5,6]; this is discussed below.
Tetrameric Vps34 complexes in yeast
A major advance in Vps34 signalling was the identification
by Ohsumi and co-workers of two distinct Vps34p–Vps15pcontaining complexes in yeast [8]. Both contain Vps34p, Vps15p
and a third protein, Vps30p/Atg6p, which had previously been
identified as being required for autophagy [153]. The complexes
were in fact identified by MS analysis of proteins isolated from
an anti-Vps30p antibody column. Additional analysis revealed
the presence of two other proteins, Vps38p and Atg14p; these
proteins are present in mutually exclusive complexes, based on
their ability to co-immunoprecipitate with Vps34p, Vps15p and
Vps30p but not with each other. Interestingly, the complexes are
functionally distinct. Deletion of VPS38 inhibits sorting of the
lysosomal hydrolase CPY (carboxypeptidase Y), but has no affect
on starvation-induced autophagy, whereas deletion of ATG14
inhibits autophagy, but has no effect on CPY sorting. Deletion
of VPS30 causes a loss of both CPY sorting and autophagy, but
does not affect the processing of newly synthesized protease
A or protease B (which occurs upon lysosomal delivery). In
contrast, deletion of VPS34 or VPS15 blocks autophagy, CPY and
protease A/B processing, and also leads to decreased growth at
37 ◦C. These results were the first indication of multiple Vps34pcontaining complexes that have distinct intracellular functions,
presumably by the differential regulation and/or targeting of
Vps34p (Figure 3).
The organization of the complex was deduced by co-immunoprecipitation experiments in deletion strains lacking each component [8]. Co-immunoprecipitation of Vps30p with Vps34p–
Vps15p is markedly decreased in a vps38 strain, whereas coimmunoprecipitation of Vps30p with Vps38p is unaffected by
deletion of VPS34 or VPS15. These results suggest that Vps38p
serves as a bridge between Vps34p–Vps15p and Vps30p. The reciprocal experiment with Atg14p was less informative, as Atg14p
is expressed at low levels relative to Vps38p, making it difficult
to detect the effects of its deletion on co-immunoprecipitation
of Vps30p with Vps34p–Vps15p. However, it was proposed that
Atg14p interacts with both Vps34p–Vps34p and Vps30p, since
deletion of any of these proteins resulted in decreased expression
of Atg14p, presumably due to decreased stability.
In yeast, deletion of either ATG6/VPS30 or ATG14 blocks the
recruitment of Atg5p and Atg8p to a PAS (pre-autophagosomal
structure) that also contains Atg1p, Atg2p and Atg16p [154].
The presumed mechanism of this inhibition is either a defect in
vesicular trafficking leading to the missorting of PAS components
or a loss of PtdIns3P production in the nascent PAS, with
subsequent defects in the recruitment of later autophagic effector
proteins. These may include Etf1, which is involved in the Cvt
(cytosol-to-vacuole) degradative pathway in yeast [155]; Etf1
lacks FYVE or PX domains yet interacts with PtdIns3P via a basic
motif. Two additional components of the Cvt pathway, Cvt13 and
Cvt20, contain PtdIns3P-binding PX domains that are required for
their association with the pre-autophagosome [156]. However, the
mechanisms by which Atg6/Vps30–Vps34p complexes and
the production of PtdIns3P regulate autophagy are still
incompletely understood.
Regulation of hVps34 in mammalian autophagy: beclin, Bcl2 and
UVRAG
Of the Vps34p–Vps15p-associated proteins discovered in yeast,
mammalian homologues have not been identified for either
Atg14p or Vps38p. The two proteins contain coiled-coil regions
but do not contain other structurally defined domains, and it is not
clear whether these proteins are needed for hVps34-dependent
functions in higher eukaryotes.
The mammalian homologue of Atg6p/Vps30p, beclin-1,
was first isolated as a Bcl2-interacting tumour suppressor in
mammalian cells [157,158]. The 450-amino-acid mammalian
protein contains a nuclear export signal [159], distinct Bcl2binding and coiled-coil domains, and a structurally uncharacterized region [the ECD (evolutionarily conserved domain)]
that is highly conserved among beclin zoologues (Figure 1)
[66]. In cells overexpressing both epitope-tagged beclin-1 and
mammalian hVps34, co-immunoprecipitation of the two species
is readily observed [66]. The overexpressed proteins greatly
exceed the amount of endogenous hVps15 or any potential
Vps38p homologue; these results suggest that, unlike yeast
Vps30p/Atg6p, beclin-1 can bind directly to mammalian hVps34.
This binding is mediated by the coiled-coil domain and ECD of
beclin, binding to the C2 domain of hVps34 [66,161]. Crosslinking studies have suggested that 50 % of mammalian hVps34
c The Authors Journal compilation c 2008 Biochemical Society
8
J. M. Backer
is bound to beclin-1, as compared with 20 % of Vps34p bound to
Vps30/Atg6 in yeast [162].
As mentioned above, Atg6p/Vps30p plays distinct roles in
vesicular trafficking compared with autophagy in yeast [8].
Beclin-1 is also required for normal vesicular trafficking in
C. elegans, where its knockout leads to a loss of PtdIns3Prich vesicular structures and inhibition of GFP–vitellogenin
uptake by oocytes [17]. Although beclin-1 and hVps34 were
shown to co-localize in the trans-Golgi network in HeLa cells
[162], the role of beclin-1 in vesicular trafficking in mammalian
cells may be limited. Recent studies suggest that processing
of newly synthesized cathepsin D in the mammalian lysosome
is normal in cell lines that express little beclin, or in beclinknockdown cells [66,164]. Fluid-phase endocytosis, EGF receptor
endocytosis and degradation, and maintenance of normal Golgi
and endosome/lysosome morphology are also normal in beclin-1knockdown cells [164]. In contrast, stable knockdown of hVps34
inhibits EGF receptor trafficking and cathepsin D processing
[116]. Thus the trafficking functions of Vps30p/Atg6p/Beclin-1
in yeast and C. elegans may not be retained in higher organisms.
In contrast, beclin-1 is required for autophagy in both C.
elegans and mammals [165–168]. Beclin-dependent autophagy
is inhibited by overexpression of Bcl2, which also causes a
decrease in beclin-1–hVps34 binding [169]. Thus the inhibition
of autophagy by Bcl2 may be in part via a disruption of
hVps34–beclin-1 interactions. The authors propose that the
relative balance of beclin–Bcl2 compared with beclin–hVps34
complexes defines a continuum between apoptotic cell death,
survival with normal levels of autophagy, and cell death due
to hyperactive autophagy [169]. Bcl2 may also be involved
in the regulation of autophagy by nutrients, as Bcl2–beclin-1
binding is inhibited by starvation [169]. A decrease in Bcl2–
beclin-1 binding in starved cells would presumably lead to
an increase in beclin-1–hVps34 binding, thereby promoting
autophagy. Liang et al. [161] identified the UVRAG (UV radiation
resistance-associated gene) tumour suppressor as another beclin1-binding partner that regulates hVps34 [161]. UVRAG is a 699amino-acid protein that is frequently mutated in human colon
cancer and contains an N-terminal proline-rich domain as well
as C2 and coiled-coil domains [170–173] (Figure 1). Crossimmunoprecipitation experiments demonstrated the presence
of beclin-1–Bcl2–hVps34–UVRAG complexes; expression of
isolated domains were used to show that the coiled-coil domains
of beclin-1 and UVRAG mediates their binding, independently of
beclin-1 binding to Bcl2. Expression of the isolated coiled-coil
domain of UVRAG inhibits autophagy, presumably by disrupting
beclin-1–UVRAG complexes. Similarly, autophagy is reduced by
UVRAG knockdown.
Whereas Bcl2 binding to beclin is decreased in nutrientstarved cells [169], UVRAG binding to beclin is unaffected by
starvation [161]. Moreover, increased UVRAG expression leads to
an increase in the amount of beclin-1-associated hVps34, and also
leads to an increase in the lipid kinase activity of overexpressed
hVps34 in cells co-transfected with beclin-1. This activation
requires UVRAG–beclin binding, as it is not observed with a
binding-defected UVRAG mutant. These findings led to a model
in which beclin-1-associated hVps34 is negatively regulated by
Bcl2, and positively regulated by UVRAG. Under nutrient-replete
conditions, Bcl2 binding would balance the effects of UVRAG
and reduce the amount of beclin-associated hVps34 activity.
Under conditions of nutrient starvation, dissociation of Bcl2
would cause a loss of its inhibitory input on beclin-1-associated
hVps34 activity. In this case, the continued tonic positive input
from UVRAG would result in an increase in beclin-1-associated
hVps34 activity. It is not yet clear whether Bcl2 acts primarily to
c The Authors Journal compilation c 2008 Biochemical Society
inhibit hVps34 binding to beclin-1 [169] or to inhibit the activity
of the hVps34–beclin-1–UVRAG complex [161].
Recently, several other UVRAG-binding proteins have been
implicated as regulators of autophagy that modulate the hVps34–
beclin-1–UVRAG complex (Figure 1). The 1300-amino-acid
protein Ambra1, originally identified as a gene involved in
neural development, interacts with beclin-1 in a two-hybrid
screen, and overexpressed Ambra1 binds to overexpressed beclin1 via a structurally undefined central domain [174]. Moreover,
Ambra1–beclin-1 complexes can be co-immunoprecipitated with
endogenous hVps34. Embryos with homozygous defects in
Ambra1 expression show decreased autophagy, and Ambra1
knockdown in cultured cells inhibits autophagy and decreases
the amount of endogenous beclin-1-associated hVps34. Thus
Ambra1 may regulate autophagy at least in part through its effects
on hVps34–beclin-1 binding. In contrast with Ambra1, which
interacts directly with Beclin-1, the endophilin family member
Bif-1 binds directly to UVRAG [175]. The interaction was
demonstrated with endogenous proteins, and subsequent analysis
showed that the Bif-1 SH3 (Src homology 3) domain binds to
the N-terminal proline-rich domain of UVRAG. Autophagy is
inhibited in Bif-1−/− mouse embryonic fibroblasts, and isolated
SH3 domains from Bif-1 inhibit autophagy, presumably by
competitively inhibiting the formation of endogenous Bif-1–
UVRAG complexes. Bif-1 knockout also reduces the activity
of overexpressed hVps34 in both fed and starved cells, and
overexpression of the BIF-1 SH3 domain inhibits the activity
of hVps34 in starved cells. These results suggest that the role of
Bif-1 in autophagy is related in part to an enhancement of hVps34
activity in beclin–UVRAG complexes. Bif-1 may also act on the
formation of the PAS by inducing membrane curvature through
its BAR (Bin/amphiphysin/Rvs) domain [176].
The identification of UVRAG–Beclin-1–hVps34 complexes is
significant, and the results clearly show that these complexes
play important roles in autophagy (Figure 4). Taken together
[161,169,174,175], the studies suggest a general model in which
rates of autophagy can be controlled by the regulated assembly
of a UVRAG–Beclin-1–hVps34 complex, whose production of
PtdIns3P is critical for formation of the PAS in starved cells.
However, with regard to the regulation of hVps34, several aspects
of the model have not been tested. First, the model predicts
a net increase in beclin-associated hVps34 in nutrient-starved
cells. This has been examined in MCF-7 cells overexpressing
wild-type beclin, where starvation-induced changes in beclin–
hVps34 binding were not observed [5]; the effect of starvation on
endogenous hVps34–beclin-1 binding has not been examined.
Similarly, this model predicts an increase in the activity of
beclin-associated hVps34 activity in nutrient-starved cells. This
has been addressed in two studies. Byfield et al. [5] showed
a decrease in the specific activity of hVps34 in FLAG–beclin
immunoprecipitates from starved MCF-7 cells, consistent with
a decrease in total hVps34 activity; the decrease was apparent
within 15 min, and reached approx. 50 % after 4 h of starvation.
In contrast, Tassa et al. [177] concluded that beclin-associated
hVps34 activity increases transiently in starved C2C12 cells.
However, the specificity of the lipid kinase assay used in this
study is unclear, as the beclin-associated activity was insensitive to
50 nM wortmannin, a concentration of inhibitor that fully inhibits
hVps34 [19] and blocks starvation-induced proteolysis [177].
To fully evaluate the UVRAG model, it will be necessary to
determine what proportion of cellular beclin-1 and hVps34–
beclin-1 complexes are bound to UVRAG. The differential
regulation of a subset of beclin–hVps34 complexes through their
association with UVRAG could be critical for autophagy, and the
regulation of hVps34 activity in UVRAG–beclin-1 complexes
The regulation and function of Class III PI3Ks
Figure 4
9
hVps34 signalling complexes in mammalian cells
A summary of mammalian complexes involving hVps34; the intracellular locations of these complexes have not been determined and are likely to be distinct. Complexes of hVps34 with beclin-1,
UVRAG and Bif-1 or hVps34, beclin-1 and Ambra1 have been observed; it is not yet clear whether these are distinct complexes or variants of the UVRAG–beclin-1 complex. Bcl2 binding to beclin-1
is nutrient-regulated and may modulate the binding of hVps34 to the beclin-1–UVRAG complex. The presence of hVps15 in these complexes has not yet been confirmed. It is not yet known whether
signalling by hVps34–hVps15 to mTOR requires additional binding partners. Binding of hVps34–hVps15 to Rab proteins and MTM1 in endosomes is mutually exclusive.
could be different from that observed for total cellular or total
beclin-associated hVps34. Consistent with this possibility, siRNA
knockdown of UVRAG markedly inhibits beclin-dependent
autophagy in MCF-7 cells, suggesting that beclin-associated
hVps34 is not sufficient to effectively drive autophagy in the
absence of UVRAG [161]. Similarly, if the enhancement of
autophagy by UVRAG overexpression [161] is due to an increase
in hVps34 binding to beclin-1, this suggests that only a fraction
of beclin–Vps34 complexes are bound to endogenous UVRAG in
control fibroblasts. Alternatively, UVRAG could have effects on
autophagy independently of effects on hVps34, for instance by
recruiting Bif-1 to nascent autophagosomal structures.
It is also important to note that the effects of UVRAG,
Ambra1 and Bif-1 on hVps34 activity have only been studied
in the presence of overexpressed beclin and hVps34, but with
endogenous, and hence sub-stoichiometric, levels of hVps15
[161,174,175]. As mentioned above, we find that overexpressed
hVps34 is only 5 % active relative to the endogenous enzyme,
presumably due to the lack of hVps15 and other binding partners.
Thus the regulation of overexpressed hVps34 is likely to be
non-physiological. For example, Takahashi et al. [175] found
that starvation caused an increase in the activity of epitopetagged hVps34, overexpressed in the absence of hVps15; this
is in disagreement with experiments measuring the activity of
endogenous hVps34, which decreases in starved cells [5,6]. In
yeast, Vps34p activity clearly requires Vps15p. Although it is
not yet known whether this will be the case with the mammalian
enzyme, we have found that hVps34 protein levels are markedly
inhibited by siRNA knockdown of hVps15 (M. Byfield and J.M.
Backer, unpublished work). Thus the effect of UVRAG, Bif-1
and Ambra1 on the activity of overexpressed hVps34 might be
to stabilize the enzyme via an enhancement of hVps34–beclin1 binding. It remains to be seen whether these proteins actually
regulate the activity of endogenous hVps34 complexes.
REGULATION OF Vps34 BY NUTRIENTS AND ITS ROLE IN mTOR
SIGNALLING
The TOR protein kinase is a central regulator of protein synthesis
and cell growth in eukaryotes [178,179]. In both yeast and
mammalian cells, TOR is present in two functionally distinct
multiprotein complexes [180–182]. In mammalian cells, TORC1
(TOR complex 1) is sensitive to cellular nutritional state and
regulates protein synthesis via the activation of a downstream
kinase, S6K1 (S6 kinase 1), and the inhibition of an inhibitor of
cap-dependent translation, 4E-BP1 (eukaryotic initiation factor
4E-binding protein 1) [183–187]. TORC2 (TOR complex 2) does
not respond to changes in nutritional conditions, and has been
implicated in cytoskeletal regulation as well as the regulatory
phosphorylation of Akt at the Ser473 site [188,189]. The TORC1 is
regulated by insulin and by nutrients, including glucose and amino
acids, as well as a variety of cellular stresses [179,185,190,191].
The insulin-responsive inputs to mTOR include Akt-mediated
inhibition of the TSC1–TSC2 dimer (where TSC is tuberous
sclerosis complex), which is a GAP for the Rheb GTPase
[190,192–195]. Rheb is required for activation of mTOR by
insulin and directly binds to mTOR [185]. Nutrient inputs to
mTOR include AMPK (AMP-activated kinase), whose activation
by decreasing ATP/AMP ratios in glucose-starved cells leads to
inhibition of mTOR [195–197]. The mechanism of amino acid
regulation of mTOR is not known.
Recent studies have shown that hVps34 contributes to the
regulation of mTOR by nutrients (Figure 5). Inhibition of hVps34
by microinjection of inhibitory antibodies, overexpression of
FYVE domains to sequester PtdIns3P or siRNA-mediated
knockdown of hVps34 expression blocks insulin-stimulated
phosphorylation of both S6K1 and 4E-BP1 [5,6]. hVps34
knockdown also blocks amino acid stimulation of S6K1 [6].
Conversely, overexpression of hVps34 activates S6K1 in the
absence of insulin stimulation. Given that hVps34 is not inhibited
by the TORC1 inhibitor rapamycin [5], these results suggest that
hVps34 is upstream of mTOR.
If hVps34 is upstream of mTOR, which of the inputs to mTOR
does it regulate? hVps34 activity is not regulated by insulin, and
knockdown of hVps34 does not inhibit Akt or block insulinstimulated phosphorylation of TSC2 [5,6]. Consistent with a role
for hVps34 in the nutrient input to mTOR, Byfield et al. [5] showed
that mammalian hVps34 is inhibited by amino-acid deprivation.
Conversely, addition of amino acids to starved cells leads to an
increase in hVps34 activity in an immune complex assay, and an
increase in intracellular PtdIns3P as detected by anti-PtdIns3P
staining [6]. These studies are in disagreement with an earlier
study that measured total hVps34 activity in starved C2C12 cells
[177]. However, the methods used to measure hVps34 in this study
were indirect, and included (i) assays of total lipid kinase activity
c The Authors Journal compilation c 2008 Biochemical Society
10
Figure 5
J. M. Backer
hVps34 in the mTOR pathway
hVps34 is inhibited by glucose and amino acid starvation or by AMPK activation, and is required for insulin-stimulated mTOR activation under nutrient-replete conditions. These results suggest that
hVps34–hVps15 is upstream of mTOR and inhibited by AMPK. These results lead to the hVps34 paradox, shown in blue: hVps34 plays opposing roles in nutrient sensing, as a positive effector of
autophagy, and as a positive effector of mTOR, which inhibits autophagy.
remaining in p85/p110-depleted lysates, despite the fact that such
lysates still contain Class II PI3Ks and highly abundant PI4K, and
(ii) in vitro lipid kinase activity of hVps34 after purification with
an inhibitory antibody, but without elution of the enzyme prior to
assay.
Studies in GRC LR+73 cells {a CHO (Chinese-hamster ovary)
cell derivative [198]} also showed a decrease in hVps34 activity
after incubation in glucose-free medium, even in the presence
of amino acids [5]. Glucose starvation is known to activate
AMPK [196,197], and treatment of cells with pharmacological
activators of AMPK [the energy poison oligomycin and the AMP
analogue AICAR (5-amino-4-imidazolecarboxamide riboside)]
inhibit hVps34 activity [5]. This latter finding has been questioned
by Meley et al. [199] in a study showing that the previously
described inhibition of autophagy by AICAR [200] was probably
due to off-target effects of the drug; the authors suggest a
similar explanation for the effects of AICAR on hVps34.
However, preliminary results suggest that the inhibition of hVps34
activity in glucose-starved cells is blocked by infection with
a dominant-negative AMPK adenovirus (M. Byfield and J.M.
Backer, unpublished work), suggesting that AMPK is in fact a
negative regulator of hVps34.
Interestingly, the phenotypes of hVps34 siRNA knockdown
as opposed to mTOR inhibition by rapamycin are not identical
with regard to S6K1. Whereas Thr389 phosphorylation is
blocked in hVps34 siRNA-treated cells, other insulin-stimulated
phosphorylation sites are unaffected, as measured by a gelshift (slower electrophoretic migration) or by immunoblotting
with anti-(phospho-Thr421 /Ser424 ) antibodies [5]. In contrast, this
selectivity for the Thr389 site is seen after brief treatment of cells
with rapamycin, but longer treatment leads to a complete loss of
the insulin-stimulated gel shift and loss of other phosphorylation
sites [5,201]. Thus inhibition of mTOR has more pleiotropic
effects on S6K1 phosphorylation than does inhibition of hVps34.
Finally, it should be noted that to date the published results use
c The Authors Journal compilation c 2008 Biochemical Society
the phosphorylation of mTOR substrates as a readout for mTOR
activity [5,6]; a direct effect of hVps34 on mTOR kinase activity
has not been demonstrated.
The finding that hVps34 is a positive regulator of mTOR
signalling that is inhibited by starvation presents a paradox
(Figure 5). Vps34p and hVps34 are required for starvationinduced autophagy in yeast and mammalian cells [9,148,177,202],
whereas TOR is an inhibitor of autophagy in yeast and mammals
[203,204]. These results would predict that hVps34 activity should
be activated by starvation, and that its activity would be inversely
correlated with that of mTOR. In fact, hVps34 is inhibited by
starvation, and its activity is positively correlated with mTOR
signalling [5,6]. However, it is important to note that studies in
Drosophila show that dS6K (Drosophila S6K) plays a similarly
paradoxical role with regard to autophagy and mTOR signalling:
dS6K is positively regulated by mTOR, yet autophagy is blocked
in dS6K−/− larvae [205]. Thus two distinct proteins in the mTOR
pathway are required for autophagy, yet are inhibited by starvation
and activated under conditions that activate TOR and inhibit
autophagy. Neufeld and co-workers suggest that inhibition of
dS6K by starvation might serve to limit cellular damage due to
excessive autophagy [205]. Additional studies will be required
to determine if this rationale also applies to hVps34.
hVps34 AND PHAGOCYTOSIS
hVps34 plays an important role in the regulation of phagocytosis.
Studies from the groups of Grinstein [41] and Deretic [45]
showed that hVps34 is not required for the engulfment of
opsonized particles by macrophages, which in fact requires Class
IA PI3Ks [206], but is required for phagosomal maturation
and fusion with late endosomes/lysosomes. The accumulation
of PtdIns3P in nascent phagosomes is transient, beginning after
closure of the phagocytic cup and declining within 5–10 min;
additional waves of PtdIns3P accumulation are seen at later
The regulation and function of Class III PI3Ks
times [41,207]. Nascent phagosomes also contain activated Rab5,
which may mediate the recruitment of hVps34 to these structures
immediately after closure [118]. In contrast, the later waves of
PtdIns3P accumulation are blocked by inhibitors of calmodulin
and calmodulin kinase II (see below) [207]. It is not yet clear
whether these increases in PtdIns3P are caused by a translocation
of hVps34 as opposed to an increase in hVps34 specific activity.
Mycobacterium tuberculosis evades destruction by macrophages by inhibiting recruitment of EEA1 to phagosomes, thereby
inhibiting phagosome maturation and fusion with lysosomes.
[45]. Infection of macrophages with M. tuberculosis also
blocks increases in cytosolic calcium that normally accompany
phagocytosis of opsonized particles [208]; this inhibition has
been attributed to either the production of a toxin, LAM
(lipoarabinomannan) [209], or inhibition of sphingosine kinase
[210]. Interestingly, Deretic and co-workers suggested that
increases in cytosolic calcium might also regulate hVps34, since
hVps34–hVps15 complexes bind to Ca2+ /calmodulin beads, and
treatment of macrophages with the calmodulin inhibitor W7
blocks phagosomal accumulation of EEA1 and PtdIns3P [209].
However, later studies showed that M. tuberculosis secrete a lipid
phosphatase, SapM, that hydrolyses PtdIns3P in the phagosome
and may account for the loss of EEA1 binding in infected
macrophages [211].
The effects of Ca2+ /calmodulin inhibitors on PtdIns3P production in macrophages may to be due to modulation of hVps34
targeting rather than hVps34 activity, since treatment of
macrophages with W7 has no effect on hVps34 activity measured
in immunoprecipitates (M. Byfield and J.M. Backer, unpublished
work). These effects are also apparently cell-type-specific [207],
since calmodulin inhibitors have no effect on endosomal PtdIns3P
levels or hVps34 activity in COS-7 cells, but do inhibit EEA1
association with endosomes in vivo and EEA1 binding to
PtdIns3P-containing membranes in vitro [49]. This latter study
proposed that Ca2+ /calmodulin helps to stabilize the structure of
the EEA1 C-terminal FYVE domain, presumably via interactions
with the IQ domain of EEA1 [212]. hVps34 does in fact contain
two predicted non-IQ calmodulin-binding sites, at residues 320–
334 and 800–816 [as predicted using the calmodulin target database (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html)],
consistent with its binding to calmodulin–agarose [209].
However, recombinant hVps34 and endogenous hVps34–hVps15
complexes are active in the absence of added calcium and in
the presence of 1 mM BAPTA [1,2-bis-(o-aminophenoxy)ethaneN,N,N ,N -tetra-acetic acid] (Y. Yan and J.M. Backer, unpublished
work). The physiological significance of calmodulin–hVps34
interactions in cells other than macrophages is not currently clear.
Vps34 AND MAPK SIGNALLING: REGULATION BY TRIMERIC
G-PROTEINS
A number of studies suggest that mammalian hVps34 may be
regulated by interactions with trimeric G-proteins. In HT-29
colon cancer cells, autophagy is inhibited by overexpression of
an activated mutated form of Gαi3 but is restored by treatment
of cells with synthetic PtdIns3P [9]. In RBL-2H3 basophilic
leukaemia cells, antigen-stimulated degranulation is mediated
by Fcε-receptor signalling to Class I PI3Ks. However, in cells
overexpressing the M1 muscarinic receptor, carbachol-stimulated
degranulation is blocked by inhibitory antibodies to hVps34 [46],
suggesting a link between hVps34 and Gαq -coupled receptors.
Experiments in S. cerevisiae have provided a direct link
between trimeric G-proteins and Vps34p–Vps15p. Dohlman and
co-workers identified both VPS34 and VPS15 in a screen for
suppressors of GPA1-mediated transcriptional responses involved
11
in pheromone signalling [7]. Deletion of either VPS34 or VPS15
inhibits α-factor stimulation of transcriptional responses and
MAPK activation, and reduces mating efficiency. Importantly,
deletions of VPS30 or VPS38 failed to disrupt GPA1 signalling,
nor do other vps mutants, suggesting that Vps34p and Vps15p
have signalling functions independent of their role in trafficking
and autophagy. Both proteins were shown to bind to Gpa1p.
Vps34p bound in the manner of a Gpa1p effector: it exhibited
preferential binding to GTPase-deficient mutants (Gpa1pQ323L),
and expression of active Gpa1p increased endogenous production
of PtdIns3P and caused the recruitment of a PX domain–
GFP fusion protein to endosomes. In contrast, Vps15p bound
preferentially to GDP-loaded Gpa1p. The authors propose that
Vps15p functions as a Gβ protein, binding to the Gα Gpa1
through its C-terminal WD40 domains, which are predicted to
form a β-propeller [7,71]. The known Vps34p-associated protein
Atg14p is proposed to serve as a Gγ protein, based on sequence
homology with known Gγ proteins, and Atg14p does in fact bind
to Gpa1p in a GDP-dependent manner (H. Dohlman, personal
communication). Of note, a Gβγ pair composed of Vps15p–
Atg14p would be somewhat atypical, in that membrane targeting
is achieved via a myristoylated Vps15p (instead of a prenylated
Gγ ). Furthermore, Atg14p is not required for Vps15p stability
(unlike traditional Gβγ pairs), although Vps15p does stabilize
Atg14p [8].
ANTAGONISTS OF Vps34 SIGNALLING: LIPID PHOSPHATASES
hVps34 signalling is terminated by degradation of PtdIns3P. Two
major pathways for PtdIns3P turnover have been elucidated.
The first involves the sequestration of PtdIns3P in the internal
vesicles of multivesicular bodies, where the lipids are presumably
degraded by subsequent fusion of the multivesicular bodies with
lysosomes [214]. However, hVps34 signalling is also antagonized
by the myotubularin family of lipid phosphatases, whose mutation
results in severe genetic diseases affecting skeletal muscle and
the nervous system [215,216]. Myotubularin family members
resemble tyrosine phosphatases but show activity toward PtdIns3P
and PtdIns(3,5)P2 [217–220]. Interestingly, six out of 14 family
members are catalytically inactive; the finding that mutations in
the inactive MTM5 and MTM13 cause human disease [221,222]
presumably involves the heterodimerization of inactive with active
myotubularin isoforms [223]. All myotubularins contain protein
tyrosine phosphatase and PH-GRAM (PH glucosyltransferase,
Rab-like GTPase activators and myotubularins) domains, and
most contain coiled-coil domains. Some isoforms also contain
SID (SET-interacting domain), PH and FYVE domains [216,224].
Interestingly, hVps34–hVps15 is found in a complex with
MTM1 [225]. Binding is mediated by the WD40 domain
of hVps15, which also binds to Rab5 and Rab7 [71,130].
Furthermore, co-immunoprecipitation experiments suggest that
hVps15 binding to Rab5 or Rab7 and MTM1 is mutually
exclusive. The binding of hVps15 to both activators (Rab5 and
Rab7) and inhibitors (MTM1) of hVps34 signalling suggest that
fine-tuning the magnitude or duration of endosomal PtdIns3P
levels is important for endosomal function.
hVps34 AND HUMAN DISEASE
With the exception of two studies suggesting a linkage between
mutations in the hVps34 promoter and schizophrenia [226,227],
disease-related mutations or changes in hVps34 expression have
not been identified in humans. In contrast, mutations in the
myotubularin family of PtdIns3P phosphatases are involved
c The Authors Journal compilation c 2008 Biochemical Society
12
J. M. Backer
in myotubular myopathy and Charcot–Marie–Tooth neuropathy
[215,217], presumably due to a deregulation of PtdIns3P
synthesis. Although it is not yet known what cellular functions
are disrupted in patients with these disorders, pharmacological
inhibition of hVps34 might be useful in restoring normal levels
of PtdIns3P.
Nonetheless, hVps34 is strongly implicated in a number of
cellular processes that are involved in human disease, which
could make hVps34 a candidate target for pharmacological
modulation. hVps34 is a key regulator of autophagy, whose
role in the immune system, in the clearance of pathological
protein aggregations in neurodegenerative disease and in tumour
suppression is increasingly clear [228]. For example, upregulation of autophagy by inhibition of mTOR reduces toxicity
in a Huntington’s disease model [229], whereas inhibition
of autophagy by ablation of Atg7 in Purkinje cells leads to
neurodegeneration [230]. Similarly, knockdown of either beclin1 or hVps34 blocked the IGF-1 (insulin-like growth factor 1)stimulated clearance of mutant huntingtin aggregates in HeLa
cells [230a]. If pharmacological up-regulation of hVps34 leads
to enhanced autophagy, then this might provide an alternative or
adjunct approach to the treatment of neurodegenerative disorders.
Alternatively, hyperactivation of the mTOR pathway by nutrient
excess has been implicated in the development of insulin
resistance [231] through the phosphorylation of IRS-1 (insulin
receptor substrate 1) [232,233]. hVps34 is required for signalling
through the mTOR pathway [5,6], and suppression of hVps34
activity might be useful in the management of insulin resistance
in obesity. It is a significant problem that treatment of neurodegeneration compared with obesity/insulin resistance would
require opposite pharmacological modulation of hVps34. This
is in some ways the same problem faced by drugs targeting the
Class IA PI3K p85/p110α: inhibition of the enzyme may be
useful in the treatment of cancer [234], but could exacerbate
insulin resistance or diabetes due to the role of p110α in
glucose homoeostasis [235,236]. However, in this latter case,
the development of diabetes is a temporary and treatable side
effect of what would presumably be a short-term treatment for a
life-threatening malignancy. In contrast, neurodegeneration and
insulin resistance are both long-term term problems requiring
chronic therapy, and it is not clear how the risks and benefits of
hVps34-targeted therapy would balance out.
UNANSWERED QUESTIONS IN Vps34 SIGNALLING
The present review has attempted to highlight aspects of Vps34
biology that remain unclear or controversial. First, at the
biochemical level, we know surprisingly little about Vps34, and
even less about Vps15. Does hVps34 activity in mammalian cells
require hVps15 as it does in yeast? Is the Vps34–Vps15 interaction
regulated, and if so by what? Is Vps15 a protein kinase, and,
if so, what are its substrates? How many Vps34 complexes are
there? Current evidence suggests at least three in S. cerevisiae
(Vps34p–Vps15p–Vps30p–Atg14p, Vps34p–Vps15p–Vps30p–
Vps38p and Vps34p–Vps15p–Gpa1p) [7,8]. Do these complexes
also exist in mammalian cells, and are there mammalian
homologues of ATG14 and VPS38? Are they related to the
UVRAG–beclin-1–hVps34–Vps15 (presumably)–Bif-1–Ambra1
complexes [66,161,174,175]? How do distinct Vps34 complexes confer differential signalling: by regulation of Vps34
activity or by differential targeting of Vps34? Granting that there
has been disagreement as to whether subsets of Vps34 show
increases or decreases in activity during starvation [5,6,177], what
is the mechanism of the nutrient regulation of hVps34 activity and
hVps34 binding to beclin-1?
c The Authors Journal compilation c 2008 Biochemical Society
Secondly, recent research has identified functions for Vps34
that extend beyond its well-established role in endocytic
trafficking. These include nutrient sensing in the mTOR pathway,
and GPCR (G-protein-coupled receptor)-mediated regulation of
the MAPK pathways [5–7]. Thus a lingering question is whether
these new functions are related to the old ones. That is to say,
are the novel functions secondary to Vps34-mediated vesicular
trafficking, or do they represent independent Vps34 activities? The
results are not yet clear. There are multiple systems in which
the endocytosis of signalling receptors plays a role in signalling
[237], either by down-regulation of a signalling input (for
example, in the CSF-1 receptor system [238]), or in some cases by
moving receptors to a compartment that contains a signalling cofactor {for example, the role of the endosomal SARA protein
in TGFβ (transforming growth factor β) signalling [239]}.
In this regard, inhibition of hVps34 does not block receptor
internalization or sorting to early endosomes [25], although it does
delay receptor degradation and might therefore enhance some
aspects of receptor signalling.
However, signalling in the mTOR system suggests a subtler
requirement for normal endomembrane trafficking. Both mTOR
and Rheb have been observed in Golgi and endoplasmic reticulum
membranes [240], and mTOR is activated by overexpression of
a Golgi-targeted Rheb, but not by Rheb targeted to other cellular
membranes [241]. Moreover, the Rheb-GAP TSC2 also has GAP
activity towards the early endosomal GTPase Rab5 [242], and
overexpression of Rheb leads to enlarged early endosomal vesicles
[243], a phenotype reminiscent of Rab5 hyperactivation. TOR
itself appears to regulate trafficking; Neufeld and co-workers have
shown that mTOR signalling inhibits the endocytic degradation
of amino acid transporters, but stimulates fluid phase uptake
of bulk nutrients [244]. Thus it will be important to determine
whether site-specific disruption of endosomal compartments, or
site-specific amplification or inhibition of hVps34, modulates
mTOR signalling.
Finally, there remains the apparently contradictory requirements for hVps34 during autophagy and mTOR signalling in
mammalian cells [5,6,9]. To date, the involvement of hVps34
in mTOR signalling has only been documented in tissue culture,
and it will be important to test whether it also holds true in animals.
Recent data from Neufeld and co-workers suggests that dVps34
is not required for mTOR signalling in Drosophila (T. Neufeld,
personal communication). In this case, the convergence of the
hVps34 and mTOR signalling pathways may have occurred only
in more complex organisms. The physiological role of hVps34 in
nutrient sensing in mammals, and in the control of both
autophagy and mTOR-regulated protein synthesis, will be an
important and exciting area for future study.
This work was supported by RO1-DK070679 and RO1-GM55692 (J. M. B.) and by a grant
from the Janey Fund. I thank Dr Erik Snapp (Albert Einstein College of Medicine, New
York, NY, U.S.A.) and Dr Henrik Dohlman (University of North Carolina, Chapel Hill, NC,
U.S.A.) for helpful discussion, and Dr Paul Herman (Ohio State University, Columbus,
OH, U.S.A.) for discussion and for critical reading of the manuscript.
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Received 17 October 2007/18 December 2007; accepted 18 December 2007
Published on the Internet 29 January 2008, doi:10.1042/BJ20071427
c The Authors Journal compilation c 2008 Biochemical Society