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

Biochem. J. (2008) 410, 1–17 (Printed in Great Britain) doi:10.1042/BJ20071427 1

REVIEW ARTICLEThe regulation and function of Class III PI3Ks: novel roles for Vps34Jonathan 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 (vacuolarprotein sorting 34), was first described as a component of thevacuolar sorting system in Saccharomyces cerevisiae and isthe sole PI3K in yeast. The homologue in mammalian cells,hVps34, has been studied extensively in the context of endocyticsorting. However, hVps34 also plays an important role in theability of cells to respond to changes in nutrient conditions. Recentstudies have shown that mammalian hVps34 is required for theactivation of the mTOR (mammalian target of rapamycin)/S6K1(S6 kinase 1) pathway, which regulates protein synthesis inresponse to nutrient availability. In both yeast and mammalian

cells, Class III PI3Ks are also required for the inductionof autophagy during nutrient deprivation. Finally, mammalianhVps34 is itself regulated by nutrients. Thus Class III PI3Ksare implicated in the regulation of both autophagy and, throughthe mTOR pathway, protein synthesis, and thus contribute to theintegration of cellular responses to changing nutritional status.

Key words: hVps34, hVps15, phosphoinositide, phosphoinositide3-kinase (PI3K), target of rapamycin (TOR), vacuolar proteinsorting 34 (Vps34).

INTRODUCTION

Vps34 (vacuolar protein sorting 34) is a member of the PI3K(phosphoinositide 3-kinase) family of lipid kinases, all of whichphosphorylate the 3′ hydroxy position of the phosphatidylinositolring. In the accepted nomenclature for PI3Ks [1,2], Vps34 isclassified as the sole Class III enzyme, whose substrate specificityis limited to phosphatidylinositol. Thus its product in cells isPtdIns3P. This distinguishes it from the more numerous and betterstudied 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 ofvesicular trafficking in the endosomal/lysosomal system, where itis involved in the recruitment of proteins containing PtdIns3P-binding domains to intracellular membranes [3,4]. However,Vps34 has also been implicated in other signalling processes,including nutrient sensing in the mTOR [mammalian TOR (targetof rapamycin)] pathway in mammalian cells, trimeric G-proteinsignalling to MAPK (mitogen-activated protein kinase) in yeast,and autophagy in both yeast and higher organisms [5–9]. Giventhe widening interest in Vps34, the present review will provide afocused discussion of its biochemistry, regulation and function ineukaryotic cells.

STRUCTURE AND CATALYTIC ACTIVITY OF Vps34

Vps34 was first discovered and cloned by Emr and co-workersin 1990, as part of a screen for vps mutants in Saccharomyces

cerevisiae [10]. The sequence of Vps34p did not give a clueas to its function until the cloning of the mammalian Class IAPI3K, p110α, revealed it to be a lipid kinase [11]. The homologybetween VPS34 and p110α led to the direct demonstrationthat Vps34p has PI3K activity [12]. Vps34 homologues havebeen identified in unicellular organisms (Schizosaccharomycespombe, Candida albicans, Dictyostelium discoideum), plants,Caenorhabditis elegans, Drosophila melanogaster, as well asvertebrates [13–19]. In mammals, hVps34 (mammalian Vps34homologue) is ubiquitously expressed [19].

PI3Ks are classified by substrate specificity and subunit organ-ization [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 IAenzymes (p85α, β, p55α and p55γ ) and Class IB enzymes(p101) are not structurally related. Class II PI3Ks do notproduce 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 IIPI3K (commonly called PI3K C2α, β and γ ) are monomericand are notable for the presence of a C-terminal C2 domain notfound in other PI3Ks. Finally, the sole Class III PI3K, Vps34,only produces PtdIns3P. The enzyme is closely associated witha protein kinase, Vps15, which has sometimes been described asa Vps34 regulatory subunit; the functional relationship betweenthese two proteins is discussed below.

Vps34 exhibits considerable homology with the catalyticsubunits of other PI3Ks, particularly at the level of domain

Abbreviations used: AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated kinase; CPY, carboxypeptidase Y; CSF-1, colony-stimulating factor 1; Cvt, cytosol-to-vacuole; ECD, evolutionarily conserved domain; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; EEA1, earlyendosomal 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, mitogen-activated 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 receptoractivation; 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 theterm Vps34p. The mammalian Class III PI3K was first cloned from human cells [19], and has been previously referred to as hVps34. Although this term isinaccurate, we have retained the usage in the present review in the interest of continuity with the pre-existing literature. A similar nomenclature is used foryeast Vps15p and mammalian hVps15 (formerly called p150 [29]).

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Figure 1 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 PI3Kcatalytic subunit is the Class I PI3Kγ structure solved by Williamsand co-workers [22]. As compared with PI3Kγ , Vps34 hasa structurally uncharacterized N-terminal region of approx. 50amino acids, followed by C2, helical and kinase domains thatare approx. 30% homologous with PI3Kγ (Figure 1). The C2domain fold in the PI3Ks is related to that of PLCγ (phospholipaseCγ ), and is likely to be involved with interactions with acidicphospholipids rather than Ca2+ [22]. The helical domain has animportant regulatory role in Class IA PI3K catalytic domains,where it mediates inhibitory contacts with the Class IA regulatorysubunit p85 [23]. A parallel role for the Vps34 helical domainhas not been studied. The kinase domain shows the classic two-lobed structure characteristic of protein kinases. Budovskayaet al. [24] showed that the extreme C-terminal 11 residues ofyeast Vps34p are required for lipid kinase activity, independentlyof any effects on binding to Vps15p (the putative serine kinasethat associates with Vps34p, discussed below). Consistent withthis finding, antibodies targeted to the C-terminus of mammalianhVps34 are potent inhibitors of the enzyme [25].

Vps34 enzymes are unique among PI3Ks in that they willonly utilize phosphatidylinositol as a substrate. The reasonfor this selectivity is thought to lie in the make-up of thesubstrate recognition loop which, in Class I or II PI3K, containsmultiple basic residues that accommodate the negative chargesin PtdIns4P and PtdIns(4,5)P2 [26]. In contrast, this regionof Vps34 is relatively uncharged, limiting Vps34 substrates tophosphatidylinositol itself. Vps34 is also distinct from Class Ienzymes in that it is more active in the presence of Mn2+ thanMg2+ [19]. Mammalian hVps34 will also utilize Ca2+-ATP, andreaches approx. 50% of maximal activity with this cation (M.R.Lau and M.J. Fry, personal communication). In this regard, it issimilar to the C2β isoform of Class II PI3K, which is also activein the presence of Ca2+-ATP [27].

In addition to lipid kinase activity, PI3Ks also possess activitytowards protein substrates. Yeast Vps34p is known to undergoautophosphorylation [28], although the sites of phosphorylationhave not been identified. Panaretou et al. [29] showed that acomplex of mammalian hVps34 with mammalian hVps15 showedsignificant kinase activity towards generic substrates such asmyelin basic protein, but the relative contributions of the twoproteins was not determined [29]. Although several potentialsubstrates have been identified for Class I enzymes [30–32],authentic in vivo substrates for Vps34 enzymes have not yet beenidentified. A functional role for Vps34 protein kinase activitywas suggested by differences in the phenotypes of C. albicansstrains that were Vps34-null compared with strains expressinga lipid-kinase-deficient/protein-kinase-active mutant Vps34 [33],although these results could also reflect scaffolding functions ofVps34.

METHODS FOR STUDYING Vps34

Studies of hVps34 have been hampered by the lack of specific in-hibitors. Interestingly, both yeast and human Vps34 share a lysineresidue that, in human Class I PI3Ks, is the site of covalent modi-fication by the pan-PI3K inhibitor wortmannin (Lys833 in PI3Kγ )[34,35]. However, whereas Class I enzymes and mammalianhVps34 are extremely sensitive to wortmannin (IC50 = 5–15 nM),yeast Vps34p is relatively resistant (IC50 = 3 µM) [28]. Thereason for this disparity in inhibitor sensitivity is not understood.Yeast Vps34p is also inhibited by LY294002 (IC50 = 50 µm) ina range similar to that for other PI3Ks and PI3K-related proteinkinases such as mTOR [28]. At the present time, specific small-molecule inhibitors of hVps34 have not been identified, althougha recent upsurge in pharmaceutical company interest in hVps34as a potential drug target should alleviate this problem. Although


The regulation and function of Class III PI3Ks 3

3-methyladenine has been suggested to be a specific inhibitor ofhVps34 [9], it in fact inhibits Class I and II PI3Ks as well [36].

Courtneidge and co-workers demonstrated that antibodies tothe C-terminus of Class IA PI3Ks are specific inhibitors of theseenzymes [37,38]. This approach has been successfully applied tomammalian hVps34 [25], and microinjection of inhibitory anti-hVps34 antibodies has been a useful tool for the identificationof hVps34-dependent functions in mammalian cells (see below)[5,39–46].

Methods for measuring Vps34 activity in cells and in vitroare similar to methods used for other PI3K isoforms, and sharesome of the same limitations; this area has been comprehensivelyreviewed recently [47]. PtdIns3P, the product of Vps34, can beextracted from [32P]orthophosphate- or [3H]myo-inositol-labelledcells, and the deacylated lipids can be separated by ion-exchangeHPLC [48]. This method does not, of course, provide informationas to the spatial localization of the lipids. MS-based methods arealso being developed for analysis of phosphoinositide abundancein cells and tissues [47]. A blotting method, in which lipid extractsfrom cells are probed with recombinant FYVE domains fromSARA (Smad anchor for receptor activation), has been used tomeasure PtdIns3P in intact cells [49].

A widely used alternative method, analogous to the useof GFP (green fluorescent protein)–PH (pleckstrin homology)domains for detection of PtdIns(3,4,5)P3 and PtdIns(3,4)P2, isthe expression of GFP-linked FYVE or PX (phox homology)domains, which are specific for PtdIns3P; tandem domains aresometimes used to increase binding affinity [50,51]. Althoughthese constructs do provide information about spatial localizationof PtdIns3P, they cannot be used to accurately quantify the netproduction of PtdIns3P in cells. The threshold for detection ofcytosolic eGFP (enhanced GFP) by fluorescence microscopyis approx. 200 nM [52], meaning that small vesicles labelledwith an eGFP reporter may not be visible above backgroundautofluorescence. Thus a change in the size of eGFP–FYVE-labelled membranous structures (e.g. through the fusion oraggregation of pre-existing eGFP-labelled vesicles) will causean increase in the fluorescence signal independently of anychange in PtdIns3P production. This is a particular problemin 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 plasmamembrane can be accurately quantified using TIRF (total internalreflection fluorescence) microscopy (reviewed in [53]).

Even as a probe of the sites of PtdIns3P production, thereare a number of caveats involved in the use of FYVE and PXdomain constructs. First, as is the case with PH-domain probes[54], FYVE-domain probes tend to identify sites of PtdIns3Pabundance, such as endosomes, but may fail to detect PtdIns3P inmembranes with lower, but still physiologically important, con-centrations of the lipid. Secondly, lipid-binding domains with sim-ilar lipid specificities may localize differently in cells [54]. For ex-ample, TAFF1 (tandem FYVE fingers 1) contains two PtdIns3P-specific FYVE domains yet localizes to the Golgi, rather than toendosomes [56]. A third problem is that FYVE-domain probescan perturb PtdIns3P-dependent processes. Thus overexpressionof a 2X-FYVE construct blocks endosomal fusion and EGF (epi-dermal growth factor) receptor sorting [57]. As an alternative toFYVE-domain-based probes, antibodies specific for PtdIns3P arecommercially available, but they have not been as well character-ized 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 forevaluation of intracellular PtdIns3P levels are not necessarilyindicative of Vps34 activity. For example, previous studies haveshown that Class II PI3Ks also produce PtdIns3P in vivo [21,62],

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 anothersource of endosomal PtdIns3P [63]. Alternatively, the Type Iαinositol polyphosphate 4-phosphatase localizes to endosomes,where it produces PtdIns3P through the dephosphorylation ofPtdIns(3,4)P2 [64]. The existence of Vps34-independent sourcesof PtdIns3P in vivo was directly demonstrated in C. elegans,where the phenotype of a Vps34 null mutant (let-512) isrescued by siRNA (small interfering RNA) knockdown of MTM(myotubularin) 1 and MTM6 [65]. Myotubularins are PtdIns3P-specific lipid phosphatases (see below), and their ability to rescuea null allele of Vps34 means that additional sources of PtdIns3Pmust exist in C. elegans. Consistent with this, siRNA knockdownof the C. elegans Class II PI3K, F39B1.1, reduces the rescue oflet-512 mutants by the myotubularin knockdown.

The enzymatic activity of recombinant Vps34 is readilyassayed in vitro [19], and the activity of endogenous Vps34can be measured in immunoprecipitates of Vps34 or its bindingpartners [5,66]. As antibodies to Vps34 can be inhibitory,care must be taken to test antibodies for inhibition usingrecombinant Vps34, and to elute Vps34 from the antibodyprior to assay if necessary [5]. Although lipid kinase activitycan be measured in immunoprecipitates from cells expressingepitope-tagged Vps34, we have found that the specific activityof overexpressed mammalian hVps34 is in fact approx. 5 % ofthat of endogenous hVps34 (Y. Yan and J.M. Backer, unpublishedwork). This presumably reflects the requirement for hVps15 andother hVps34-associated proteins for full activity (see below).Thus studies on the regulation of hVps34 that depend on itsoverexpression in the absence of hVps15, and perhaps otherbinding partners, may not accurately reflect the physiologicalregulation of the enzyme.

The hVps34 gene has not yet been disrupted in mice. Geneticapproaches to studying Vps34 in metazoans has been usefulin C. elegans, where mutations of the let-512 gene lead toL3/L4 embryonic lethality, disruption of endocytic uptake and anexpansion of the outer nuclear membrane [17]. In Drosophila,mutations in ird1 (the Drosophila homologue of the Vps34-related kinase Vps15; see below), which would presumably alsoinhibit Drosophila dVps34, lead to a loss of starvation-inducedinduction of antimicrobial peptides, suggesting a link betweenVps34 and innate immunity [67]. However, ird1 mutants alsoshow constitutive activation of the Toll pathway, in contrastwith results in RAW264.7 macrophages, where expression ofkinase-dead hVps34 blocks TLR9 (Toll-like receptor 9)-mediatedresponses [68]. These could reflect differences in flies comparedwith mammals, or differences between the phenotype of inhibitionof Vps34 compared with its presumed upstream regulator Vps15(see below).

REGULATION OF Vps34 BY Vps15

Like Vps34p, Vps15p was cloned from S. cerevisiae by theEmr laboratory [69]. Although Vps15 has been described asa Vps34 regulatory protein, the sequence of Vps15 suggeststhat it functions as a protein kinase. Homologues have beendescribed in mammals and Drosophila [29,67], and a share similardomain organization (Figure 1). Vps15 contains an N-terminalmyristoylation consensus sequence, and the yeast and mammalianenzymes are myristoylated [29,70]. Mutation of the Glu2 acceptorsite has no effect on partitioning of Vps15p into particulatefractions, but does partially reduce Vps15p phosphorylation andis additive with other Vps15p mutations for temperature-sensitive


4 J. M. Backer

growth [70]. Vps15 consists of a predicted protein kinase domainfollowed by a central region containing multiple HEAT repeatsand a series of C-terminal WD40 domains. It was suggested byMurray et al. [71] that these WD40 repeats might form a β-propeller-like structure, similar to the C-terminus of Gβ subunits[71]; in fact recent studies from Dohlman and co-workers suggestthat Vps15p binds to yeast Gα subunits (see below) [7].

The relationship between the Vps34 and Vps15 proteins wasfirst suggested by the similar phenotypes caused by their deletionin S. cerevisiae, which do not cleanly fall into any of the fiveclasses (A–E) of vps mutants defined by Stevens, Emr andtheir co-workers [72–74]. �vps34 and �vps15 stains resembleClass D vps mutants [72], with defects in the sorting of newlysynthesized hydrolases to the yeast vacuole and in vacuolarsegregation during mitosis, but with a relatively normal vacuolemorphology. However, �vps15 and �vps34 strains also showsensitivity to growth at 37 ◦C and to osmotic stress, and haveabnormal cytoplasmic membranous structures, which are morecharacteristic of Class C mutants [10,73,74]. Thus Vps15p andVps34p appear to form a distinct subset of vps gene products [75].

The similarities between the Vps34p and Vps15p phenotypesled to experiments showing that overexpression of Vps34pcomplements for growth at elevated temperatures in Vps15p-mutant strains containing a single mutation in the Vps15pkinase domain, but not Vps15p-null strains or strains containingtriple mutants in the kinase domain [76]. Production of theVps34p product, PtdIns3P, is abolished in �vps15 strainsor in strains expressing predicted kinase-dead mutants ofVps15p, and strains expressing a temperature-sensitive alleleof Vps15p are temperature-sensitive for PtdIns3P production[77]. These results clearly show that a functional Vps15pwith an intact kinase domain is required for Vps34p activ-ity in vivo. Vps15p and Vps34p are both membrane-associatedin yeast [76]. They also co-immunoprecipitate, and the deletionof Vps15p leads to a loss of Vps34p membrane association[76]. Interestingly, a native kinase domain of Vps15p is alsorequired for its binding to Vps34p; point mutations at residuespredicted to abolish kinase activity, based on homology with otherserine/threonine kinases, led to a marked inhibition of Vps15p–Vps34p co-immunoprecipitation [77]. More recent analysis of theVps34p–Vps15p interaction identified residues 837–864 in the C-terminus of Vps34p as sufficient to bind to the kinase and HEATdomains of Vps15p [24].

Although Vps15p is a protein kinase that binds Vps34p andwhose putative kinase activity is required for Vps34p acti-vity, Vps15p does not appear to be a Vps34p kinase. Vps34pis phosphorylated in yeast, but the preponderance of thisphosphorylation reflects autophosphorylation rather than phos-phorylation by Vps15p [28]. Thus Vps34p phosphorylation issimilar in �vps15 compared with wild-type strains, and disablingmutations in the kinase domain of Vps34p eliminate most of thein vivo phosphate incorporation into Vps34p. Additional resultsquestion whether Vps34p is regulated by phosphorylation, asits activity is minimally affected by partial dephosphorylationwith potato acid phosphatase [28]. However, this experimentwas performed with Vps34p overexpressed in �vps15 yeast, andit is not clear how dephosphorylation would affect the activityof Vps34p from wild-type strains. Overall, the contribution ofphosphorylation to the regulation of Vps34p activity is unclear.

If Vps15 does not activate Vps34p by phosphorylation, whyis an intact Vps15p kinase domain required for Vps34p activity?Vps15p-mediated autophosphorylation could be required for anallosteric activation of Vps34p. However, deletion of 30 aminoacids from the Vps15p C-terminus (residues 1426–1455) but not anearby internal deletion (residues 1412–1427) abolishes Vps15p

phosphorylation at 30 ◦C, but only inhibits vacuolar protein sortingat elevated temperatures [70]. It is not clear whether the effectof the C-terminal deletion on Vps15p phosphorylation is due toan inhibition of Vps15p kinase activity, or to the removal of itsmajor sites of autophosphorylation. Nonetheless, the C-terminaltruncation mutant supports vacuolar sorting, and therefore Vps34pactivity, at permissive temperatures in the absence of Vps15pphosphorylation.

An alternative hypothesis is that Vps15p acts on Vps34pprimarily by recruiting it to cellular membranes. Consistentwith this, overexpression of Vps34p in both �vps15 strains andstrains expressing putative kinase-dead Vps15p (E220R) causesa significant increase in production of PtdIns3P. However, thislipid production correlates with a partial restoration of vacuolarsorting only in the mutant vps15 strain and not in the �vps15 strain[77]. Rescue of sorting in the mutant Vps15p yeast presumablyreflects the fact that the E200R mutation weakens but does notabolish Vps15p–Vps34p binding [76,77]. The authors note thatPtdIns3P levels are higher in the kinase-dead vps15 strain thanin the �vps15 strain, and suggest that the different phenotypesmay reflect a threshold level of PtdIns(3)P required for sorting.Alternatively, these results may suggest that the critical role ofVps15p involves targeting of Vps34p as opposed to its activation.Indeed, the in vitro lipid kinase activity of Vps34p extractedfrom wild-type and �vps15 yeast has not been compared, andthe activity of mammalian hVps34 was increased only 2-fold bycomplex formation with hVps15 [29]. On the other hand, siRNAknockdown of hVps15 in mammalian cells leads to a decreasein hVps34 protein levels (Y. Yan and J.M. Backer, unpublishedwork), suggesting that hVps15 may be required for hVps34stability. The precise role of Vps15 in the regulation of Vps34is still uncertain.

It is not clear why the same mutations that disrupt theprotein kinase activity of Vps15p (as measured by its autophos-phorylation) also weaken or abolish its binding to Vps34p[77]. It is possible that binding of Vps15p and Vps34p inyeast requires Vps15p-mediated phosphorylation of an as yetunidentified accessory protein. Vps34p and Vps15p do existin complexes with other proteins (discussed below), but thedeletion of these accessory proteins does not disrupt the Vps34p–Vps15p association in vivo [78]. Furthermore, directVps34–Vps15 binding was detected in a two-hybrid assay [24].Similarly, recombinant mammalian hVps34 and hVps15 do dir-ectly associate in vitro [29], although the requirement for an intacthVps15 kinase domain for this interaction has not been tested.

Interestingly, Vps15 has not been formally proven to possessprotein kinase activity. A comparison of the hVps15 sequencewith that of other protein kinases shows significant differences;hVps15 lacks, for example, the canonical GXGXXG motifinvolved in ATP binding [79] (the sequence is GSTRFFin hVps15). The major evidence that Vps15p is a proteinkinase comes from experiments in which point mutationspredicted to disrupt kinase function also abolish Vps15pautophosphorylation [28,69]. However, since the Vps15p mutantswere immunoprecipitated from yeast, and given that the samepoint mutations are known to abolish interactions betweenVps15p and other proteins (e.g. Vps34p [77]), it remains possiblethat in vitro phosphorylation of Vps15p is in fact due to a co-purifying kinase. This kinase would presumably be present insub-stoichiometric amounts, and might not have been detectedin parallel immunoprecipitations from [35S]methionine-labelledyeast [69]. Similarly, the kinase activity of yeast Vps15p towardsexogenous substrates has never been demonstrated, and thekinase activity of mammalian hVps15 has only been assayedin a complex with hVps34 [29]. Like yeast Vps34p, mammalian



hVps34 has protein as well as lipid kinase activity [19], so theidentity 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 membranerecruitment of proteins containing modular domains that bind toPtdIns3P. These domains included FYVE domains, zinc-fingerdomains named for the first four proteins known to containthe domain [Fab1p, YOTB, Vac1p, EEA1 (early endosomalantigen 1)] [80] and PX domains, named for the Phox homologydomain of the p47phox and p40phox subunits of the phagocyteNADPH oxidase [81]. FYVE and PX domain proteins have beenextensively reviewed [82–86] and will not be discussed at lengthhere. FYVE-domain containing proteins that regulate scaffoldingand/or sorting steps in the endosomal system include EEA1 [87–89] and Hrs (hepatocyte-growth-factor-regulated tyrosine kinasesubstrate)/Vps27 [90–92]. The PtdIns3P 5-kinase Fab1/PIKfyve,which contains a FYVE domain, is involved in endosome/Golgisorting and late-endosomal trafficking [93–98]. Whereas most ofthese 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 endocyticvesicles [99]. This may be related to the observation thatdisruption of the Vps34 gene in C. elegans (let-512) altersthe morphology of the nuclear membrane [17]. PX-domain-containing proteins regulated by PtdIns3P include the phagocyteNADPH 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-sensitivefusion protein-attachment protein receptor) Vam7 [107], the CISK(cytokine-independent survival kinase) protein kinase, whichis related to the Akt kinase [108,109], and the PX-domain-containing RGS (regulator of G-protein signalling) protein, RGS-PX1, a Gαs-specific GAP (GTPase-activating protein) that inhibitsEGF 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 traffickingin mammalian cells by PI3K inhibitors such as wortmannin(reviewed in [111]). However, it was not clear from these studieswhich PI3K isoform was responsible. The late 1990s saw theidentification of EEA1 as a wortmannin-sensitive component ofendocytic vesicles [87], and the identification of FYVE domainsas PtdIns3P-specific lipid-binding domains that direct EEA1 toearly endosomes [80,112,113]. Given that GFP–FYVE domaintargeting to endosomes requires Vps34p in yeast [112], it seemedlikely that hVps34 would be responsible for many of the PI3K-dependent steps in the endocytic system.

In fact, different experimental approaches to perturbing hVps34signalling have yielded somewhat different assignments ofhVps34 function in the endosomal system of mammalian cells.The first studies specifically addressing the role of hVps34in mammalian cells used inhibitory antibodies, and showedthat specific inhibition of hVps34 activity delays passage ofinternalized PDGF (platelet-derived growth factor) receptorsthrough the early endosome, slows the recycling of transferrinreceptors, inhibits EEA1 recruitment to early endosomes [25],inhibits homotypic early endosomal fusion [39] and inhibitsthe formation of internal vesicles in multivesicular bodies [43].

hVps34 is also implicated in the movement of endosomes onmicrotubule tracks [40], through the recruitment of KIF16B, aPX domain-containing kinesin that is targeted to endosomes in aPI3K-dependent manner [114]. Anti-hVps34 antibodies inhibitboth apical and basolateral endocytic trafficking in polarizedhepatocytes [42]. Expression of a kinase-dead hVps34 inhibitsmaturation of procathepsin D in lysosomes, consistent with a rolefor hVps34 in endosomal sorting [115]. In contrast, inhibitionof PtdIns3P signalling by overexpression of FYVE domainsspecifically inhibits EGF receptor degradation but does not affectfluid-phase transport or formation of endosomal carrier vesiclesfrom donor early endosomes [57]. Similarly, stable knockdownof hVps34 blocks multivesicular body formation and slows thedegradation of the EGF receptor, but does not affect fluid-phaseendocytosis or EEA1 binding to early endosomes [116]. Finally,inhibition of PtdIns3P production in the early endosome viaspecific targeting of the PtdIns3P phosphatase MTM1 causespronounced tubulation of the early endosome, with impairedtransferrin receptor egress but only slight effects on EEA1targeting or EGF-receptor degradation [117].

The differential methodologies used in these studies havedistinct experimental drawbacks. Methods aimed at globallyblocking PtdIns3P signalling through expression of FYVEdomains [57] will also inhibit signalling by Class II PI3Ks, whichproduce PtdIns3P in the plasma membrane [21,62]. Specifictargeting of MTM1 to the early endosome [117] may be lessaffected by this problem. Inhibition of PtdIns3P productionusing hVps34 inhibitors (antibodies, kinase-dead mutants ordrugs, when available) and hVps34 knockdown will block bothPtdIns3P signalling as well as any signalling by the proteinkinase activity of hVps34. The knockdown strategy also altersthe composition of multiprotein complexes containing beclin-1and hVps15 (see below), which could have additional effects onhVps34-independent functions of these proteins. An additionalcomplication is that PI3K inhibitors increase the activation levelof Rab5 in phagosomes and only block a subset of Rab5-dependent events [118]. Secondary effects of hVps34 inhibitiondue to enhanced Rab5 signalling in the early endosome might beexpected to spare early endosomal trafficking events relative toevents in the late endosome or multivesicular body.

Finally, it should be noted that some effects of hVps34inhibition might be due to a decrease in the availability ofPtdIns3P as a substrate for PIKfyve/Fab1, a PtdIns3P-5-kinase[94,119,120]. PIKfyve/Fab1 has been independently implicated inthe 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/vacuolelumen [98,119,123,124].

Mechanism of Vps34 action in the endosomal system

A model for the regulation of Vps34 in the early endosomecame from the identification of mammalian hVps34 and hVps15in the eluate from a RAB5–GTP affinity column [39]. In vitroexperiments showed direct GTP-dependent binding of Rab5 tohuman hVps15 and hVps15–hVps34 dimers, but not to hVps34itself. Rab5 binding requires the HEAT and WD40 domains ofhuman hVps15 [71]. These experiments suggest that hVps34is 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 ofendogenous or overexpressed hVps34 and hVps15 to enlargedEEA1-positive endosomes in HeLa cells [71]. The finding thatoverexpressed hVps34 is recruited to endosomes without co-expression of hVps15 was explained by the fact that hVps15


6 J. M. Backer

Figure 2 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 oftargeting. C/C, coiled-coil; PI[3]P, PtdIns3P; RBD, Rab5-binding domain.

is in excess compared with hVps34 in HeLa cells. Interestingly, anet movement of hVps34 or hVps15 from cytosolic to membranecompartments was not observed in cell fractionation studies [71],suggesting that the Rab5 induces a redistribution of hVps34–hVps15 between membrane compartments, rather than a directrecruitment from cytosol to membrane.

Overall, the model provides an attractive explanation for thespecificity of Rab5 signalling in the early endosome, since Rab5effectors such as EEA1 are recruited both directly, by bindingto GTP–Rab5, and indirectly, by recruitment of hVps34–hVps15and subsequent production of PtdIns3P [39,71,89] (Figure 2).Two other early endosomal regulatory proteins, Rabenosyn-5and Rabankyrin-5, share the ability to bivalently interact withboth Rab5 and, via FYVE domains, with PtdIns3P [125,126].The assembly of these Rab- and PtdIns3P-binding proteins isthought to regulate early endosomal docking and fusion. ThusEEA1 interacts with syntaxin 6 and syntaxin 13, endosomalSNARE proteins that regulate vesicle docking/priming [127,128].Rabenosyn-5 binds to hVps45 [126], which is related to Sec1proteins that are negative regulators of SNARE pairing [129].The endosomal role of Rabankyrin-5 is not clear, and it may playa 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 withwild-type or nucleotide-free (N125I) forms, as opposed toactivated (Q76L) or inhibitory (T22N) forms. Accumulation ofPtdIns3P in late endosomes, based on GFP–FYVE staining, isdecreased in cells expressing dominant-negative Rab7 mutants,suggesting a role for Rab7 in maintaining hVps34 activity in thelate endosome.

The last few years have seen rapid progress in dissectingthe role of PtdIns3P signalling in ubiquitin-dependent sortingof 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 ofubiquitination during endocytic sorting was first discovered inyeast [132,133], and the ubiquitin-dependent endocytic sortingof the EGF, CSF-1 (colony-stimulating factor 1) and otherreceptors have been reported in mammalian cells (reviewed in[134–136]). The degradative sorting of internalized ubiquitinatedcargo requires the sequential engagement of the ESCRT(endosomal sorting complex required for transport) complexesI, II and III [4,137,138], and the eventual invagination of theubiquitinated membrane proteins into an intraluminal vesicle. Akey regulator of this process is Vps27/Hrs, which contains both

a FYVE domain and an UIM (ubiquitin-interacting motif) andis itself ubiquitinated [139]. Hrs/Vps27 interacts with endosomalPtdIns3P, with ubiquitinated cargo, and with ESCRT I, therebyinitiating the assembly of the ESCRT complex on to endosomalmembranes [92,140,141]. In addition, one of the components ofESCRT III, Vps24, binds to PtdIns(3,5)P2 [142]. Thus Vps34plays a key role in the initiation of the ESCRT-mediatedprocessing of ubiquitinated cargo.

Finally, Vps34p is required for retrograde endosome-to-Golgitransport, a process that requires the assembly of Vps34p, Vps29p,Vps26p, Vps17p and Vps5p into what is termed the retromercomplex [143]. This pathway is required for the retrieval fromendosomes to the Golgi of Vps10p, the yeast homologue ofthe mammalian mannose 6-phosphate receptor [144]. Anotherprotein required for Vps10p retrieval is Vps30p [145], whichwas 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 retromerassembly, presumably by the PtdIns3P-mediated recruitment ofthe PX-domain-containing proteins Vps5p and Vps17p [146].Results from mammalian cells suggests that hVps34 might alsoinfluence the endosome-to-Golgi sorting by providing substratefor PIKfyve/Fab1, a PtdIns3P 5-kinase [94,119,120], which hasbeen shown to regulate this pathway [95].

Vps34 AND AUTOPHAGY

Vps34 is essential for macroautophagy, a process by whichcells degrade cytosolic content by the formation of a double-walled vesicular structure that eventually fuses with lysosomes[147,148]. Macroautophagy is a physiological response to nutrientdeprivation, and has also been implicated in innate immunity,development, tumour suppression and clearance of neuronalaggregates [149–151]. Whereas autophagy is activated by nutrientdeficiency, it is inhibited by nutrient sufficiency. In yeast, this ismediated by the nutrient-stimulated activation of the TOR proteinkinase, which leads to the phosphorylation and inactivation ofcomponents of the autophagy pathway [147,148]. TOR is also animportant regulator of cell growth, via its effects on transcription,protein synthesis and ribosome biogenesis (discussed below).

Autophagy has been most intensively studied in yeast, wheremutants of autophagy-related Atg genes have provided importantexperimental tools [152]. A number of yeast vps genes are alsorequired for autophagy, including VPS34 and VPS15 [8]. In



Figure 3 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 VPS34or VPS15 causes additional trafficking phenotypes not seen with deletion of VPS30, ATG14 or VPS38, suggesting the existence of additional complexes. The Vps34p–Vps15p–Gpa1 complexinvolved in pheromone signalling may also include Atg14p, which would function as a Gγ protein.

mammalian cells, hVps34 is also involved in nutrient signallingto mTOR [5,6]; this is discussed below.

Tetrameric Vps34 complexes in yeast

A major advance in Vps34 signalling was the identificationby Ohsumi and co-workers of two distinct Vps34p–Vps15p-containing complexes in yeast [8]. Both contain Vps34p, Vps15pand a third protein, Vps30p/Atg6p, which had previously beenidentified as being required for autophagy [153]. The complexeswere in fact identified by MS analysis of proteins isolated froman anti-Vps30p antibody column. Additional analysis revealedthe presence of two other proteins, Vps38p and Atg14p; theseproteins are present in mutually exclusive complexes, based ontheir ability to co-immunoprecipitate with Vps34p, Vps15p andVps30p but not with each other. Interestingly, the complexes arefunctionally distinct. Deletion of VPS38 inhibits sorting of thelysosomal hydrolase CPY (carboxypeptidase Y), but has no affecton starvation-induced autophagy, whereas deletion of ATG14inhibits autophagy, but has no effect on CPY sorting. Deletionof VPS30 causes a loss of both CPY sorting and autophagy, butdoes not affect the processing of newly synthesized proteaseA or protease B (which occurs upon lysosomal delivery). Incontrast, deletion of VPS34 or VPS15 blocks autophagy, CPY andprotease A/B processing, and also leads to decreased growth at37 ◦C. These results were the first indication of multiple Vps34p-containing complexes that have distinct intracellular functions,presumably by the differential regulation and/or targeting ofVps34p (Figure 3).

The organization of the complex was deduced by co-immuno-precipitation experiments in deletion strains lacking each com-ponent [8]. Co-immunoprecipitation of Vps30p with Vps34p–Vps15p is markedly decreased in a �vps38 strain, whereas co-immunoprecipitation of Vps30p with Vps38p is unaffected bydeletion of VPS34 or VPS15. These results suggest that Vps38pserves as a bridge between Vps34p–Vps15p and Vps30p. The re-ciprocal experiment with Atg14p was less informative, as Atg14pis expressed at low levels relative to Vps38p, making it difficultto detect the effects of its deletion on co-immunoprecipitationof Vps30p with Vps34p–Vps15p. However, it was proposed thatAtg14p interacts with both Vps34p–Vps34p and Vps30p, sincedeletion of any of these proteins resulted in decreased expressionof Atg14p, presumably due to decreased stability.

In yeast, deletion of either ATG6/VPS30 or ATG14 blocks therecruitment of Atg5p and Atg8p to a PAS (pre-autophagosomalstructure) that also contains Atg1p, Atg2p and Atg16p [154].The presumed mechanism of this inhibition is either a defect invesicular trafficking leading to the missorting of PAS componentsor a loss of PtdIns3P production in the nascent PAS, withsubsequent defects in the recruitment of later autophagic effectorproteins. These may include Etf1, which is involved in the Cvt(cytosol-to-vacuole) degradative pathway in yeast [155]; Etf1lacks FYVE or PX domains yet interacts with PtdIns3P via a basicmotif. Two additional components of the Cvt pathway, Cvt13 andCvt20, contain PtdIns3P-binding PX domains that are required fortheir association with the pre-autophagosome [156]. However, themechanisms by which Atg6/Vps30–Vps34p complexes andthe production of PtdIns3P regulate autophagy are stillincompletely understood.

Regulation of hVps34 in mammalian autophagy: beclin, Bcl2 andUVRAG

Of the Vps34p–Vps15p-associated proteins discovered in yeast,mammalian homologues have not been identified for eitherAtg14p or Vps38p. The two proteins contain coiled-coil regionsbut do not contain other structurally defined domains, and it is notclear whether these proteins are needed for hVps34-dependentfunctions in higher eukaryotes.

The mammalian homologue of Atg6p/Vps30p, beclin-1,was first isolated as a Bcl2-interacting tumour suppressor inmammalian cells [157,158]. The 450-amino-acid mammalianprotein contains a nuclear export signal [159], distinct Bcl2-binding and coiled-coil domains, and a structurally unchar-acterized region [the ECD (evolutionarily conserved domain)]that is highly conserved among beclin zoologues (Figure 1)[66]. In cells overexpressing both epitope-tagged beclin-1 andmammalian hVps34, co-immunoprecipitation of the two speciesis readily observed [66]. The overexpressed proteins greatlyexceed the amount of endogenous hVps15 or any potentialVps38p homologue; these results suggest that, unlike yeastVps30p/Atg6p, beclin-1 can bind directly to mammalian hVps34.This binding is mediated by the coiled-coil domain and ECD ofbeclin, binding to the C2 domain of hVps34 [66,161]. Cross-linking studies have suggested that 50% of mammalian hVps34


8 J. M. Backer

is bound to beclin-1, as compared with 20% of Vps34p bound toVps30/Atg6 in yeast [162].

As mentioned above, Atg6p/Vps30p plays distinct roles invesicular trafficking compared with autophagy in yeast [8].Beclin-1 is also required for normal vesicular trafficking inC. elegans, where its knockout leads to a loss of PtdIns3P-rich vesicular structures and inhibition of GFP–vitellogeninuptake by oocytes [17]. Although beclin-1 and hVps34 wereshown to co-localize in the trans-Golgi network in HeLa cells[162], the role of beclin-1 in vesicular trafficking in mammaliancells may be limited. Recent studies suggest that processingof newly synthesized cathepsin D in the mammalian lysosomeis normal in cell lines that express little beclin, or in beclin-knockdown cells [66,164]. Fluid-phase endocytosis, EGF receptorendocytosis and degradation, and maintenance of normal Golgiand endosome/lysosome morphology are also normal in beclin-1-knockdown cells [164]. In contrast, stable knockdown of hVps34inhibits EGF receptor trafficking and cathepsin D processing[116]. Thus the trafficking functions of Vps30p/Atg6p/Beclin-1in 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 autophagyis inhibited by overexpression of Bcl2, which also causes adecrease in beclin-1–hVps34 binding [169]. Thus the inhibitionof autophagy by Bcl2 may be in part via a disruption ofhVps34–beclin-1 interactions. The authors propose that therelative balance of beclin–Bcl2 compared with beclin–hVps34complexes defines a continuum between apoptotic cell death,survival with normal levels of autophagy, and cell death dueto hyperactive autophagy [169]. Bcl2 may also be involvedin the regulation of autophagy by nutrients, as Bcl2–beclin-1binding is inhibited by starvation [169]. A decrease in Bcl2–beclin-1 binding in starved cells would presumably lead toan increase in beclin-1–hVps34 binding, thereby promotingautophagy. Liang et al. [161] identified the UVRAG (UV radiationresistance-associated gene) tumour suppressor as another beclin-1-binding partner that regulates hVps34 [161]. UVRAG is a 699-amino-acid protein that is frequently mutated in human coloncancer and contains an N-terminal proline-rich domain as wellas C2 and coiled-coil domains [170–173] (Figure 1). Cross-immunoprecipitation experiments demonstrated the presenceof beclin-1–Bcl2–hVps34–UVRAG complexes; expression ofisolated domains were used to show that the coiled-coil domainsof beclin-1 and UVRAG mediates their binding, independently ofbeclin-1 binding to Bcl2. Expression of the isolated coiled-coildomain of UVRAG inhibits autophagy, presumably by disruptingbeclin-1–UVRAG complexes. Similarly, autophagy is reduced byUVRAG knockdown.

Whereas Bcl2 binding to beclin is decreased in nutrient-starved cells [169], UVRAG binding to beclin is unaffected bystarvation [161]. Moreover, increased UVRAG expression leads toan increase in the amount of beclin-1-associated hVps34, and alsoleads to an increase in the lipid kinase activity of overexpressedhVps34 in cells co-transfected with beclin-1. This activationrequires UVRAG–beclin binding, as it is not observed with abinding-defected UVRAG mutant. These findings led to a modelin which beclin-1-associated hVps34 is negatively regulated byBcl2, and positively regulated by UVRAG. Under nutrient-repleteconditions, Bcl2 binding would balance the effects of UVRAGand reduce the amount of beclin-associated hVps34 activity.Under conditions of nutrient starvation, dissociation of Bcl2would cause a loss of its inhibitory input on beclin-1-associatedhVps34 activity. In this case, the continued tonic positive inputfrom UVRAG would result in an increase in beclin-1-associatedhVps34 activity. It is not yet clear whether Bcl2 acts primarily to

inhibit hVps34 binding to beclin-1 [169] or to inhibit the activityof the hVps34–beclin-1–UVRAG complex [161].

Recently, several other UVRAG-binding proteins have beenimplicated as regulators of autophagy that modulate the hVps34–beclin-1–UVRAG complex (Figure 1). The 1300-amino-acidprotein Ambra1, originally identified as a gene involved inneural development, interacts with beclin-1 in a two-hybridscreen, and overexpressed Ambra1 binds to overexpressed beclin-1 via a structurally undefined central domain [174]. Moreover,Ambra1–beclin-1 complexes can be co-immunoprecipitated withendogenous hVps34. Embryos with homozygous defects inAmbra1 expression show decreased autophagy, and Ambra1knockdown in cultured cells inhibits autophagy and decreasesthe amount of endogenous beclin-1-associated hVps34. ThusAmbra1 may regulate autophagy at least in part through its effectson hVps34–beclin-1 binding. In contrast with Ambra1, whichinteracts directly with Beclin-1, the endophilin family memberBif-1 binds directly to UVRAG [175]. The interaction wasdemonstrated with endogenous proteins, and subsequent analysisshowed that the Bif-1 SH3 (Src homology 3) domain binds tothe N-terminal proline-rich domain of UVRAG. Autophagy isinhibited in Bif-1−/− mouse embryonic fibroblasts, and isolatedSH3 domains from Bif-1 inhibit autophagy, presumably bycompetitively inhibiting the formation of endogenous Bif-1–UVRAG complexes. Bif-1 knockout also reduces the activityof overexpressed hVps34 in both fed and starved cells, andoverexpression of the BIF-1 SH3 domain inhibits the activityof hVps34 in starved cells. These results suggest that the role ofBif-1 in autophagy is related in part to an enhancement of hVps34activity in beclin–UVRAG complexes. Bif-1 may also act on theformation of the PAS by inducing membrane curvature throughits BAR (Bin/amphiphysin/Rvs) domain [176].

The identification of UVRAG–Beclin-1–hVps34 complexes issignificant, and the results clearly show that these complexesplay important roles in autophagy (Figure 4). Taken together[161,169,174,175], the studies suggest a general model in whichrates of autophagy can be controlled by the regulated assemblyof a UVRAG–Beclin-1–hVps34 complex, whose production ofPtdIns3P is critical for formation of the PAS in starved cells.However, with regard to the regulation of hVps34, several aspectsof the model have not been tested. First, the model predictsa net increase in beclin-associated hVps34 in nutrient-starvedcells. This has been examined in MCF-7 cells overexpressingwild-type beclin, where starvation-induced changes in beclin–hVps34 binding were not observed [5]; the effect of starvation onendogenous hVps34–beclin-1 binding has not been examined.Similarly, this model predicts an increase in the activity ofbeclin-associated hVps34 activity in nutrient-starved cells. Thishas been addressed in two studies. Byfield et al. [5] showeda decrease in the specific activity of hVps34 in FLAG–beclinimmunoprecipitates from starved MCF-7 cells, consistent witha decrease in total hVps34 activity; the decrease was apparentwithin 15 min, and reached approx. 50% after 4 h of starvation.In contrast, Tassa et al. [177] concluded that beclin-associatedhVps34 activity increases transiently in starved C2C12 cells.However, the specificity of the lipid kinase assay used in thisstudy is unclear, as the beclin-associated activity was insensitive to50 nM wortmannin, a concentration of inhibitor that fully inhibitshVps34 [19] and blocks starvation-induced proteolysis [177].

To fully evaluate the UVRAG model, it will be necessary todetermine what proportion of cellular beclin-1 and hVps34–beclin-1 complexes are bound to UVRAG. The differentialregulation of a subset of beclin–hVps34 complexes through theirassociation with UVRAG could be critical for autophagy, and theregulation of hVps34 activity in UVRAG–beclin-1 complexes



Figure 4 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-1is 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 whethersignalling 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 totalbeclin-associated hVps34. Consistent with this possibility, siRNAknockdown of UVRAG markedly inhibits beclin-dependentautophagy in MCF-7 cells, suggesting that beclin-associatedhVps34 is not sufficient to effectively drive autophagy in theabsence of UVRAG [161]. Similarly, if the enhancement ofautophagy by UVRAG overexpression [161] is due to an increasein hVps34 binding to beclin-1, this suggests that only a fractionof beclin–Vps34 complexes are bound to endogenous UVRAG incontrol fibroblasts. Alternatively, UVRAG could have effects onautophagy independently of effects on hVps34, for instance byrecruiting 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 studiedin the presence of overexpressed beclin and hVps34, but withendogenous, and hence sub-stoichiometric, levels of hVps15[161,174,175]. As mentioned above, we find that overexpressedhVps34 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 benon-physiological. For example, Takahashi et al. [175] foundthat starvation caused an increase in the activity of epitope-tagged hVps34, overexpressed in the absence of hVps15; thisis in disagreement with experiments measuring the activity ofendogenous hVps34, which decreases in starved cells [5,6]. Inyeast, Vps34p activity clearly requires Vps15p. Although it isnot yet known whether this will be the case with the mammalianenzyme, we have found that hVps34 protein levels are markedlyinhibited by siRNA knockdown of hVps15 (M. Byfield and J.M.Backer, unpublished work). Thus the effect of UVRAG, Bif-1and Ambra1 on the activity of overexpressed hVps34 might beto stabilize the enzyme via an enhancement of hVps34–beclin-1 binding. It remains to be seen whether these proteins actuallyregulate the activity of endogenous hVps34 complexes.

REGULATION OF Vps34 BY NUTRIENTS AND ITS ROLE IN mTORSIGNALLING

The TOR protein kinase is a central regulator of protein synthesisand cell growth in eukaryotes [178,179]. In both yeast andmammalian cells, TOR is present in two functionally distinctmultiprotein complexes [180–182]. In mammalian cells, TORC1

(TOR complex 1) is sensitive to cellular nutritional state andregulates protein synthesis via the activation of a downstreamkinase, S6K1 (S6 kinase 1), and the inhibition of an inhibitor ofcap-dependent translation, 4E-BP1 (eukaryotic initiation factor4E-binding protein 1) [183–187]. TORC2 (TOR complex 2) doesnot respond to changes in nutritional conditions, and has beenimplicated in cytoskeletal regulation as well as the regulatoryphosphorylation of Akt at the Ser473 site [188,189]. The TORC1 isregulated by insulin and by nutrients, including glucose and aminoacids, as well as a variety of cellular stresses [179,185,190,191].The insulin-responsive inputs to mTOR include Akt-mediatedinhibition of the TSC1–TSC2 dimer (where TSC is tuberoussclerosis complex), which is a GAP for the Rheb GTPase[190,192–195]. Rheb is required for activation of mTOR byinsulin and directly binds to mTOR [185]. Nutrient inputs tomTOR include AMPK (AMP-activated kinase), whose activationby decreasing ATP/AMP ratios in glucose-starved cells leads toinhibition of mTOR [195–197]. The mechanism of amino acidregulation of mTOR is not known.

Recent studies have shown that hVps34 contributes to theregulation of mTOR by nutrients (Figure 5). Inhibition of hVps34by microinjection of inhibitory antibodies, overexpression ofFYVE domains to sequester PtdIns3P or siRNA-mediatedknockdown of hVps34 expression blocks insulin-stimulatedphosphorylation of both S6K1 and 4E-BP1 [5,6]. hVps34knockdown also blocks amino acid stimulation of S6K1 [6].Conversely, overexpression of hVps34 activates S6K1 in theabsence of insulin stimulation. Given that hVps34 is not inhibitedby the TORC1 inhibitor rapamycin [5], these results suggest thathVps34 is upstream of mTOR.

If hVps34 is upstream of mTOR, which of the inputs to mTORdoes it regulate? hVps34 activity is not regulated by insulin, andknockdown of hVps34 does not inhibit Akt or block insulin-stimulated phosphorylation of TSC2 [5,6]. Consistent with a rolefor hVps34 in the nutrient input to mTOR, Byfield et al. [5] showedthat mammalian hVps34 is inhibited by amino-acid deprivation.Conversely, addition of amino acids to starved cells leads to anincrease in hVps34 activity in an immune complex assay, and anincrease in intracellular PtdIns3P as detected by anti-PtdIns3Pstaining [6]. These studies are in disagreement with an earlierstudy that measured total hVps34 activity in starved C2C12 cells[177]. However, the methods used to measure hVps34 in this studywere indirect, and included (i) assays of total lipid kinase activity


10 J. M. Backer

Figure 5 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 thathVps34–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 ofautophagy, and as a positive effector of mTOR, which inhibits autophagy.

remaining in p85/p110-depleted lysates, despite the fact that suchlysates still contain Class II PI3Ks and highly abundant PI4K, and(ii) in vitro lipid kinase activity of hVps34 after purification withan inhibitory antibody, but without elution of the enzyme prior toassay.

Studies in GRC LR+73 cells {a CHO (Chinese-hamster ovary)cell derivative [198]} also showed a decrease in hVps34 activityafter incubation in glucose-free medium, even in the presenceof amino acids [5]. Glucose starvation is known to activateAMPK [196,197], and treatment of cells with pharmacologicalactivators of AMPK [the energy poison oligomycin and the AMPanalogue AICAR (5-amino-4-imidazolecarboxamide riboside)]inhibit hVps34 activity [5]. This latter finding has been questionedby Meley et al. [199] in a study showing that the previouslydescribed inhibition of autophagy by AICAR [200] was probablydue to off-target effects of the drug; the authors suggest asimilar explanation for the effects of AICAR on hVps34.However, preliminary results suggest that the inhibition of hVps34activity in glucose-starved cells is blocked by infection witha dominant-negative AMPK adenovirus (M. Byfield and J.M.Backer, unpublished work), suggesting that AMPK is in fact anegative regulator of hVps34.

Interestingly, the phenotypes of hVps34 siRNA knockdownas opposed to mTOR inhibition by rapamycin are not identicalwith regard to S6K1. Whereas Thr389 phosphorylation isblocked in hVps34 siRNA-treated cells, other insulin-stimulatedphosphorylation sites are unaffected, as measured by a gel-shift (slower electrophoretic migration) or by immunoblottingwith anti-(phospho-Thr421/Ser424) antibodies [5]. In contrast, thisselectivity for the Thr389 site is seen after brief treatment of cellswith rapamycin, but longer treatment leads to a complete loss ofthe insulin-stimulated gel shift and loss of other phosphorylationsites [5,201]. Thus inhibition of mTOR has more pleiotropiceffects on S6K1 phosphorylation than does inhibition of hVps34.Finally, it should be noted that to date the published results use

the phosphorylation of mTOR substrates as a readout for mTORactivity [5,6]; a direct effect of hVps34 on mTOR kinase activityhas not been demonstrated.

The finding that hVps34 is a positive regulator of mTORsignalling that is inhibited by starvation presents a paradox(Figure 5). Vps34p and hVps34 are required for starvation-induced 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 shouldbe activated by starvation, and that its activity would be inverselycorrelated with that of mTOR. In fact, hVps34 is inhibited bystarvation, and its activity is positively correlated with mTORsignalling [5,6]. However, it is important to note that studies inDrosophila show that dS6K (Drosophila S6K) plays a similarlyparadoxical role with regard to autophagy and mTOR signalling:dS6K is positively regulated by mTOR, yet autophagy is blockedin dS6K−/− larvae [205]. Thus two distinct proteins in the mTORpathway are required for autophagy, yet are inhibited by starvationand activated under conditions that activate TOR and inhibitautophagy. Neufeld and co-workers suggest that inhibition ofdS6K by starvation might serve to limit cellular damage due toexcessive autophagy [205]. Additional studies will be requiredto 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 ofopsonized particles by macrophages, which in fact requires ClassIA PI3Ks [206], but is required for phagosomal maturationand fusion with late endosomes/lysosomes. The accumulationof PtdIns3P in nascent phagosomes is transient, beginning afterclosure of the phagocytic cup and declining within 5–10 min;additional waves of PtdIns3P accumulation are seen at later



times [41,207]. Nascent phagosomes also contain activated Rab5,which may mediate the recruitment of hVps34 to these structuresimmediately after closure [118]. In contrast, the later waves ofPtdIns3P accumulation are blocked by inhibitors of calmodulinand calmodulin kinase II (see below) [207]. It is not yet clearwhether these increases in PtdIns3P are caused by a translocationof hVps34 as opposed to an increase in hVps34 specific activity.

Mycobacterium tuberculosis evades destruction by macro-phages by inhibiting recruitment of EEA1 to phagosomes, therebyinhibiting phagosome maturation and fusion with lysosomes.[45]. Infection of macrophages with M. tuberculosis alsoblocks increases in cytosolic calcium that normally accompanyphagocytosis of opsonized particles [208]; this inhibition hasbeen attributed to either the production of a toxin, LAM(lipoarabinomannan) [209], or inhibition of sphingosine kinase[210]. Interestingly, Deretic and co-workers suggested thatincreases in cytosolic calcium might also regulate hVps34, sincehVps34–hVps15 complexes bind to Ca2+/calmodulin beads, andtreatment of macrophages with the calmodulin inhibitor W7blocks phagosomal accumulation of EEA1 and PtdIns3P [209].However, later studies showed that M. tuberculosis secrete a lipidphosphatase, SapM, that hydrolyses PtdIns3P in the phagosomeand may account for the loss of EEA1 binding in infectedmacrophages [211].

The effects of Ca2+/calmodulin inhibitors on PtdIns3P produc-tion in macrophages may to be due to modulation of hVps34targeting rather than hVps34 activity, since treatment ofmacrophages with W7 has no effect on hVps34 activity measuredin immunoprecipitates (M. Byfield and J.M. Backer, unpublishedwork). These effects are also apparently cell-type-specific [207],since calmodulin inhibitors have no effect on endosomal PtdIns3Plevels or hVps34 activity in COS-7 cells, but do inhibit EEA1association with endosomes in vivo and EEA1 binding toPtdIns3P-containing membranes in vitro [49]. This latter studyproposed that Ca2+/calmodulin helps to stabilize the structure ofthe EEA1 C-terminal FYVE domain, presumably via interactionswith the IQ domain of EEA1 [212]. hVps34 does in fact containtwo predicted non-IQ calmodulin-binding sites, at residues 320–334 and 800–816 [as predicted using the calmodulin target data-base (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html)],consistent with its binding to calmodulin–agarose [209].However, recombinant hVps34 and endogenous hVps34–hVps15complexes are active in the absence of added calcium and inthe presence of 1 mM BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N ′,N ′-tetra-acetic acid] (Y. Yan and J.M. Backer, unpublishedwork). The physiological significance of calmodulin–hVps34interactions in cells other than macrophages is not currently clear.

Vps34 AND MAPK SIGNALLING: REGULATION BY TRIMERICG-PROTEINS

A number of studies suggest that mammalian hVps34 may beregulated by interactions with trimeric G-proteins. In HT-29colon cancer cells, autophagy is inhibited by overexpression ofan activated mutated form of Gαi3 but is restored by treatmentof cells with synthetic PtdIns3P [9]. In RBL-2H3 basophilicleukaemia cells, antigen-stimulated degranulation is mediatedby Fcε-receptor signalling to Class I PI3Ks. However, in cellsoverexpressing the M1 muscarinic receptor, carbachol-stimulateddegranulation 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 linkbetween trimeric G-proteins and Vps34p–Vps15p. Dohlman andco-workers identified both VPS34 and VPS15 in a screen forsuppressors of GPA1-mediated transcriptional responses involved

in pheromone signalling [7]. Deletion of either VPS34 or VPS15inhibits α-factor stimulation of transcriptional responses andMAPK 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 Vps15phave signalling functions independent of their role in traffickingand autophagy. Both proteins were shown to bind to Gpa1p.Vps34p bound in the manner of a Gpa1p effector: it exhibitedpreferential binding to GTPase-deficient mutants (Gpa1pQ323L),and expression of active Gpa1p increased endogenous productionof PtdIns3P and caused the recruitment of a PX domain–GFP fusion protein to endosomes. In contrast, Vps15p boundpreferentially to GDP-loaded Gpa1p. The authors propose thatVps15p functions as a Gβ protein, binding to the Gα Gpa1through its C-terminal WD40 domains, which are predicted toform a β-propeller [7,71]. The known Vps34p-associated proteinAtg14p is proposed to serve as a Gγ protein, based on sequencehomology with known Gγ proteins, and Atg14p does in fact bindto Gpa1p in a GDP-dependent manner (H. Dohlman, personalcommunication). Of note, a Gβγ pair composed of Vps15p–Atg14p would be somewhat atypical, in that membrane targetingis achieved via a myristoylated Vps15p (instead of a prenylatedGγ ). Furthermore, Atg14p is not required for Vps15p stability(unlike traditional Gβγ pairs), although Vps15p does stabilizeAtg14p [8].

ANTAGONISTS OF Vps34 SIGNALLING: LIPID PHOSPHATASES

hVps34 signalling is terminated by degradation of PtdIns3P. Twomajor pathways for PtdIns3P turnover have been elucidated.The first involves the sequestration of PtdIns3P in the internalvesicles of multivesicular bodies, where the lipids are presumablydegraded by subsequent fusion of the multivesicular bodies withlysosomes [214]. However, hVps34 signalling is also antagonizedby the myotubularin family of lipid phosphatases, whose mutationresults in severe genetic diseases affecting skeletal muscle andthe nervous system [215,216]. Myotubularin family membersresemble tyrosine phosphatases but show activity toward PtdIns3Pand PtdIns(3,5)P2 [217–220]. Interestingly, six out of 14 familymembers are catalytically inactive; the finding that mutations inthe inactive MTM5 and MTM13 cause human disease [221,222]presumably involves the heterodimerization of inactive with activemyotubularin isoforms [223]. All myotubularins contain proteintyrosine phosphatase and PH-GRAM (PH glucosyltransferase,Rab-like GTPase activators and myotubularins) domains, andmost contain coiled-coil domains. Some isoforms also containSID (SET-interacting domain), PH and FYVE domains [216,224].

Interestingly, hVps34–hVps15 is found in a complex withMTM1 [225]. Binding is mediated by the WD40 domainof hVps15, which also binds to Rab5 and Rab7 [71,130].Furthermore, co-immunoprecipitation experiments suggest thathVps15 binding to Rab5 or Rab7 and MTM1 is mutuallyexclusive. The binding of hVps15 to both activators (Rab5 andRab7) and inhibitors (MTM1) of hVps34 signalling suggest thatfine-tuning the magnitude or duration of endosomal PtdIns3Plevels is important for endosomal function.

hVps34 AND HUMAN DISEASE

With the exception of two studies suggesting a linkage betweenmutations in the hVps34 promoter and schizophrenia [226,227],disease-related mutations or changes in hVps34 expression havenot been identified in humans. In contrast, mutations in themyotubularin family of PtdIns3P phosphatases are involved


12 J. M. Backer

in myotubular myopathy and Charcot–Marie–Tooth neuropathy[215,217], presumably due to a deregulation of PtdIns3Psynthesis. Although it is not yet known what cellular functionsare disrupted in patients with these disorders, pharmacologicalinhibition of hVps34 might be useful in restoring normal levelsof PtdIns3P.

Nonetheless, hVps34 is strongly implicated in a number ofcellular processes that are involved in human disease, whichcould make hVps34 a candidate target for pharmacologicalmodulation. hVps34 is a key regulator of autophagy, whoserole in the immune system, in the clearance of pathologicalprotein aggregations in neurodegenerative disease and in tumoursuppression is increasingly clear [228]. For example, up-regulation of autophagy by inhibition of mTOR reduces toxicityin a Huntington’s disease model [229], whereas inhibitionof autophagy by ablation of Atg7 in Purkinje cells leads toneurodegeneration [230]. Similarly, knockdown of either beclin-1 or hVps34 blocked the IGF-1 (insulin-like growth factor 1)-stimulated clearance of mutant huntingtin aggregates in HeLacells [230a]. If pharmacological up-regulation of hVps34 leadsto enhanced autophagy, then this might provide an alternative oradjunct approach to the treatment of neurodegenerative disorders.Alternatively, hyperactivation of the mTOR pathway by nutrientexcess has been implicated in the development of insulinresistance [231] through the phosphorylation of IRS-1 (insulinreceptor substrate 1) [232,233]. hVps34 is required for signallingthrough the mTOR pathway [5,6], and suppression of hVps34activity might be useful in the management of insulin resistancein obesity. It is a significant problem that treatment of neurode-generation compared with obesity/insulin resistance wouldrequire opposite pharmacological modulation of hVps34. Thisis in some ways the same problem faced by drugs targeting theClass IA PI3K p85/p110α: inhibition of the enzyme may beuseful in the treatment of cancer [234], but could exacerbateinsulin resistance or diabetes due to the role of p110α inglucose homoeostasis [235,236]. However, in this latter case,the development of diabetes is a temporary and treatable sideeffect of what would presumably be a short-term treatment for alife-threatening malignancy. In contrast, neurodegeneration andinsulin resistance are both long-term term problems requiringchronic therapy, and it is not clear how the risks and benefits ofhVps34-targeted therapy would balance out.

UNANSWERED QUESTIONS IN Vps34 SIGNALLING

The present review has attempted to highlight aspects of Vps34biology that remain unclear or controversial. First, at thebiochemical level, we know surprisingly little about Vps34, andeven less about Vps15. Does hVps34 activity in mammalian cellsrequire hVps15 as it does in yeast? Is the Vps34–Vps15 interactionregulated, and if so by what? Is Vps15 a protein kinase, and,if so, what are its substrates? How many Vps34 complexes arethere? 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 complexesalso exist in mammalian cells, and are there mammalianhomologues of ATG14 and VPS38? Are they related to theUVRAG–beclin-1–hVps34–Vps15 (presumably)–Bif-1–Ambra1complexes [66,161,174,175]? How do distinct Vps34 com-plexes confer differential signalling: by regulation of Vps34activity or by differential targeting of Vps34? Granting that therehas been disagreement as to whether subsets of Vps34 showincreases or decreases in activity during starvation [5,6,177], whatis the mechanism of the nutrient regulation of hVps34 activity andhVps34 binding to beclin-1?

Secondly, recent research has identified functions for Vps34that extend beyond its well-established role in endocytictrafficking. These include nutrient sensing in the mTOR pathway,and GPCR (G-protein-coupled receptor)-mediated regulation ofthe MAPK pathways [5–7]. Thus a lingering question is whetherthese new functions are related to the old ones. That is to say,are the novel functions secondary to Vps34-mediated vesiculartrafficking, or do they represent independent Vps34 activities? Theresults are not yet clear. There are multiple systems in whichthe endocytosis of signalling receptors plays a role in signalling[237], either by down-regulation of a signalling input (forexample, in the CSF-1 receptor system [238]), or in some cases bymoving receptors to a compartment that contains a signalling co-factor {for example, the role of the endosomal SARA proteinin TGFβ (transforming growth factor β) signalling [239]}.In this regard, inhibition of hVps34 does not block receptorinternalization or sorting to early endosomes [25], although it doesdelay receptor degradation and might therefore enhance someaspects of receptor signalling.

However, signalling in the mTOR system suggests a subtlerrequirement for normal endomembrane trafficking. Both mTORand Rheb have been observed in Golgi and endoplasmic reticulummembranes [240], and mTOR is activated by overexpression ofa Golgi-targeted Rheb, but not by Rheb targeted to other cellularmembranes [241]. Moreover, the Rheb-GAP TSC2 also has GAPactivity towards the early endosomal GTPase Rab5 [242], andoverexpression of Rheb leads to enlarged early endosomal vesicles[243], a phenotype reminiscent of Rab5 hyperactivation. TORitself appears to regulate trafficking; Neufeld and co-workers haveshown that mTOR signalling inhibits the endocytic degradationof amino acid transporters, but stimulates fluid phase uptakeof bulk nutrients [244]. Thus it will be important to determinewhether site-specific disruption of endosomal compartments, orsite-specific amplification or inhibition of hVps34, modulatesmTOR signalling.

Finally, there remains the apparently contradictory require-ments for hVps34 during autophagy and mTOR signalling inmammalian cells [5,6,9]. To date, the involvement of hVps34in 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 dVps34is not required for mTOR signalling in Drosophila (T. Neufeld,personal communication). In this case, the convergence of thehVps34 and mTOR signalling pathways may have occurred onlyin more complex organisms. The physiological role of hVps34 innutrient sensing in mammals, and in the control of bothautophagy and mTOR-regulated protein synthesis, will be animportant and exciting area for future study.

This work was supported by RO1-DK070679 and RO1-GM55692 (J. M. B.) and by a grantfrom the Janey Fund. I thank Dr Erik Snapp (Albert Einstein College of Medicine, NewYork, 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 2007Published on the Internet 29 January 2008, doi:10.1042/BJ20071427


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

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