The Class III phosphoinositide 3-kinase Vps34 (vacuolar protein sorting 34) plays important roles in endocytic trafficking, macroautophagy, phagocytosis, cytokinesis and nutrient sensing. Recent studies have provided exciting new insights into the structure and regulation of this lipid kinase, and new cellular functions for Vps34 have emerged. This review critically examines the wealth of new data on this important enzyme, and attempts to integrate these findings with current models of Vps34 signalling.

INTRODUCTION

Vps34 (vacuolar protein sorting 34) is a member of the phosphoinositide 3-kinase (PI3K) family of lipid kinases, and it acts by phosphorylating phosphatidylinositol at the 3′ position on the inositol ring. First identified and characterized in the early 1990s by Herman et al. [1,2], Vps34 and its functions are highly conserved from yeast to mammals, and it plays critical roles in a wide range of cellular processes. The last five years have seen the discovery of exciting new information on the structure of Vps34, the regulation of its enzyme activity, and its functions in eukaryotic cells. This review will briefly summarize the canonical functions of Vps34, and then focus primarily on more recent studies. The reader is referred to an earlier review from 2008 that describes the first 18 years of Vps34 research [3].

Vps34 acts in a complex with a probable pseudokinase, Vps15, and signals to downstream effectors through the production of phosphatidylinositol 3-phosphate (PI[3]P). This lipid supplies a binding site for proteins containing appropriate lipid-binding domains. The best understood are FYVE [Fab1, YOTB, Vac1, EEA1 (early endosomal antigen 1)] and PX (Phox homology) domains. The specificity of recruitment of a given effector to distinct PI[3]P-containing membranes is enhanced by additional protein–protein interactions. For example, recruitment of the endosomal tethering protein EEA1 to early endosomes required its binding to both PI[3]P and activated Rab5. Vps34 regulates vesicular trafficking and sorting at a number of different sites (Figure 1).

Canonical functions of Vps34

Figure 1
Canonical functions of Vps34

Vps34 acts in tetrameric complexes containing Vps15, Beclin-1 and either Atg14 (Vps34 Complex I) or UVRAG (Vps34 Complex II). Vps34 acts by producing PI[3]P in intracellular membranes. Vps34 regulates the fusion and maturation of Rab5-positive early endosomes (EE) and their maturation into Rab7-positive late endosomes (LE). PI[3]P produced by Vps34 recruits the Retromer complex, which mediates endosome to Golgi retrograde trafficking, and the ESCRT complex, which produces ILVs in multivesicular bodies (MVB). Vps34 is recruited to phagosomes after sealing to direct phagosomal maturation. It functions in the ER and in maturing autophagosomes, where it drives autophagosomal initiation and maturation.

Figure 1
Canonical functions of Vps34

Vps34 acts in tetrameric complexes containing Vps15, Beclin-1 and either Atg14 (Vps34 Complex I) or UVRAG (Vps34 Complex II). Vps34 acts by producing PI[3]P in intracellular membranes. Vps34 regulates the fusion and maturation of Rab5-positive early endosomes (EE) and their maturation into Rab7-positive late endosomes (LE). PI[3]P produced by Vps34 recruits the Retromer complex, which mediates endosome to Golgi retrograde trafficking, and the ESCRT complex, which produces ILVs in multivesicular bodies (MVB). Vps34 is recruited to phagosomes after sealing to direct phagosomal maturation. It functions in the ER and in maturing autophagosomes, where it drives autophagosomal initiation and maturation.

Early and late endosomes [4,5]

Vps34 is recruited by the binding of the WD40 domain of Vps15 to activated Rab5. Production of PI[3]P synergizes with Rab5 in the recruitment of EEA1 and Rabenosyn5 to drive endosomal tethering and fusion. Vps34–Vps15 also binds to Rab7 in late endosomes.

Multivesicular bodies [6]

ESCRT (endosomal sorting complex required for transport) is a multi-protein complex that interacts with ubiquitinated membrane proteins and drives their internalization into the intraluminal vesicles (ILVs) of the multivesicular body (MVB). ESCRT assembles in a step-wise manner involving subcomplexes (ESCRT-0, -I, -II and -III) that bind cargo, deform the late endosomal membrane to form invaginations, and finally sever the invaginated membrane to produce ILVs. Vps34 production of PI[3]P recruits the ESCRT-0 protein HRS (hepatocyte growth factor-regulated tyrosine kinase substrate)/Vps27, which contains a FYVE domain, to the endosome. HRS/Vps27 in turn recruits additional ESCRT complex members. An ESCRT-II protein, EAP45 (ELL-associated protein of 45 kDa)/Vps36, also interacts with PI[3]P through a GLUE (GRAM-like, ubiquitin binding in EAP45) domain.

Retromer [7]

Vps34 is required for the function of the Retromer complex, which drives the retrograde trafficking of endocytic cargo to the Golgi. The Retromer is composed of a dimer of sorting nexins (SNX1, SNX2, SNX5 or SNX6) that mediates membrane recruitment, and a trimer of Vps26, Vps29 and Vps35 that binds to endocytic receptors. Sorting nexins contain PX domains that bind to PI[3]P produced by Vps34.

Macroautophagy [8,9]

Macroautophagy (referred to hereinafter as autophagy) is a degradative process that drives the uptake of bulk cargo as well as specific substrates (aggregates, ubiquitinated proteins, mitochondria, peroxisomes) into a double-membrane-bound vacuole, which eventually fuses with lysosomes. Autophagy is stimulated by nutrient starvation as well as a number of cellular stresses. Vps34 is required for both early and late steps of the process. In the earliest steps of starvation-induced autophagy, production of PI[3]P in the endoplasmic reticulum (ER) recruits double-FYVE-domain-containing protein 1 (DFCP1) to the so-called omegasome, which is an autophagosomal precursor. Activation of the protein kinase ULK1/Atg1 co-operates with Vps34 in autophagosomal initiation. An additional Vps34-dependent step is the recruitment of WIPI1 and WIPI2 (WD40 repeat protein interactions with phosphoinositides 1 and 2; Atg18 in yeast), whose Phe-Arg-Arg-Gly motifs also bind to PI[3]P. At the end of the process of autophagy, Vps34 is required for the fusion of mature sealed autophagosomes with lysosomes.

Vps34 COMPLEXES AND THEIR STRUCTURAL ORGANIZATION

The core Vps34 complexes

The minimal complex is the Vps34–Vps15 dimer, which can be isolated as a stable molecule from yeast [10]. Vps15 is in excess over Vps34 in mammalian cells [11], and it is likely that most cellular Vps34 is complexed with Vps15. A distinct function for the Vps34–Vps15 dimer has been demonstrated in the yeast pheromone pathway, where both Vps34 and Vps15 are required for MAPK (mitogen-activated protein kinase) activation independently of other Vps34-associated proteins [12]. A second complex contains Vps34, Vps15 and Beclin-1 (Atg6 or Vps30 in yeast), which is a coiled-coil and BH3 (Bcl-2 homology 3) domain protein [13]. This complex appears to be the most abundant Vps34–Beclin-1 complex in mammalian cells, based on immunodepletion experiments [1416]. As will be discussed below, the stoichiometry of components within the Vps34–Vps15–Beclin-1 complex has not been defined. Finally, Vps34 is present in two tetrameric complexes, which contain Vps15, Beclin-1 and one copy of either Atg14 (initially called Barkor or Atg14L in mammalian systems) or UVRAG (UV radiation resistance-associated gene) (Vps38 in yeast) [1721]. Current nomenclature refers to the tetramer containing Vps34, Vps15, Beclin-1 (Vps30) and Atg14 as Complex I, and the tetramer containing Vps34, Vps15, Beclin-1 (Vps30) and UVRAG (Vps38) as Complex II. Vps34 signalling to the nascent autophagosome requires Vps34 Complex I, whereas autophagosome–lysosomal fusion requires Vps34 Complex II [8,9]. The functions of Vps34 in endosomes and the MVB are generally thought to involve Vps34 Complex II [17,22,23].

Structure of the core complexes

The domain organization of mammalian Vps34, Vps15, Beclin-1, Atg14 and UVRAG is shown in Figure 2; yeast Vps30, Atg14 and Vps38 are also shown, as they differ somewhat from the mammalian enzymes. The major domains of Vps34 are found in all PI3Ks, and include a C2 domain, a helical domain and a kinase domain. Vps15 contains an N-terminally myristoylated kinase-like domain, a helical domain containing a series of internal HEAT [huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and TOR (target of rapamycin)] repeats and a C-terminal WD40 domain that forms a β-propeller structure [10,24]. The kinase domain has an atypical ATP-binding site and its activity has not been unambiguously demonstrated [3]. The other core components are all coiled-coil proteins. Beclin-1 and Vps30 contain an N-terminal unstructured region, followed by a BH3 domain and two coiled-coil domains. Their C-termini contain a so-called BARA (β–α repeated, autophagy) domain [25]. Human Atg14 has an extended coiled-coil domain near its N-terminus. Its C-terminus contains a BATS [Barkor/Atg14(L) autophagosome-targeting sequence] domain, which has been implicated in the targeting of Atg14 to highly curved membranes [26]; yeast Atg14 is 100 amino acids shorter, and an equivalent domain has not been identified. Human UVRAG and yeast Vps38 both have an N-terminal C2 domain, followed by two coiled-coil domains and a C-terminal BARA-related domain, referred to as a BARA2 domain.

Domain maps for the core components of Vps34 Complex I and II

Figure 2
Domain maps for the core components of Vps34 Complex I and II

The maps show Vps34 (human); Vps15 (human); Vps30 (yeast) and Beclin-1 (human); Atg14 (yeast) and Atg14 (human); and Vps38 (yeast) and UVRAG (human). The domain organization of Vps34 and Vps15 are highly conserved between yeast and humans, so only the human proteins are shown.

Figure 2
Domain maps for the core components of Vps34 Complex I and II

The maps show Vps34 (human); Vps15 (human); Vps30 (yeast) and Beclin-1 (human); Atg14 (yeast) and Atg14 (human); and Vps38 (yeast) and UVRAG (human). The domain organization of Vps34 and Vps15 are highly conserved between yeast and humans, so only the human proteins are shown.

The organization of the two tetrameric complexes has been described by a crystal structure of yeast Complex II [10], and a cryo-EM structure of mammalian Complex I [27]. These structures are not consistent with previously published domain interactions maps derived from co-immunoprecipitation and pull-down experiments. For example, a direct contact between Vps34 and the evolutionarily conserved domain (ECD) of Beclin-1 was proposed [28], when in fact the ECD has no direct contacts with Vps34 in the structures. A common problem with binding studies in the older literature is the use of pull-down or co-immunoprecipitation experiments in which only some of the components of the Vps34 complexes are expressed. In such studies, apparently direct contacts may in fact be dependent on endogenous proteins present in the cell lysates. Binding studies that are not validated by experiments with purified or in vitro translated proteins should be viewed with caution.

The structures of Complex I and II (Figure 3) share an overall architecture governed by interactions between the Vps15–Vps34 dimer and the parallel arrangement of coiled-coil domains of Vps30 and Vps38, in yeast Complex II, and Beclin-1 and Atg14, in human Complex I. The tetramer forms a V-shape. In the structure of yeast Complex II, the tips of the V are formed in one arm by the Vps34 kinase domain, and in the other arm by the C-terminal BARA and BARA2 domains of Vps30 and Vps38 respectively (Figure 3A). In mammalian Complex I, the tips are formed by the Vps34 kinase domain in one arm, and the C-terminal BARA domain of Beclin-1; the C-terminal BATS domain of Atg14 is not seen in the structure (Figure 3B). The base of the V is formed by the N-terminal domains of Vps30 and Vps38 in the yeast Complex II, or Beclin-1 and Atg14 in the mammalian Complex I, and the HEAT repeat region of Vps15. Unlike the C2 domains of Class I PI3Ks, which interact extensively with the helical and kinase domains [29,30], the C2 domain of Vps34 is distant from the rest of the protein, where it sits at the hub of the two arms of the tetramer.

Structures of Vps34 Complex I and II

Figure 3
Structures of Vps34 Complex I and II

(A) The crystal structure of yeast Vps34 Complex II. (B) The cryo-EM structure of human Vps34 Complex I. The colour codes for members of Vps34 Complex I and II are preserved from Figures 1 and 2. 

Figure 3
Structures of Vps34 Complex I and II

(A) The crystal structure of yeast Vps34 Complex II. (B) The cryo-EM structure of human Vps34 Complex I. The colour codes for members of Vps34 Complex I and II are preserved from Figures 1 and 2. 

Vps34-mediated phosphorylation of phosphatidylinositol is governed by the tips of the two arms. At one tip, the Vps34 kinase domain interacts with the N-terminal pseudokinase domain of Vps15. The crystal structure of the helical/kinase domain fragment of Vps34 (HelCat) suggests that its C-terminal helix kα12 can exist in closed or open conformations, with the open conformation required for catalytic activity [31]. The cryo-EM analysis of Complex I suggests substantial mobility of the HelCat region of Vps34, which moves as a single module [27]; this motion of the Vps34 HelCat fragment may be important for interactions with membranes. The N-terminal myristoylation of Vps15 also contributes to membrane binding [3].

At the other tip, the C-termini of Vps30 and Vps38 in yeast Complex II, or Beclin-1 and Atg14 in mammalian Complex I, contain lipid-binding domains that are likely to regulate the association of Vps34 complexes with membranes. In yeast Complex II, the C-terminal BARA domain of Vps30 interacts with membranes, as demonstrated by DXMS (hydrogen–deuterium exchange MS) studies [10,32]. In Complex I, the Beclin-1 BARA domain and the C-terminal Atg14 BATS domain presumably act in a similar manner to enhance membrane binding. Interestingly, both purified yeast Complexes 1 and 2 show high lipid kinase activity on small unilamellar vesicles. Only Complex II is able to utilize giant unilamellar vesicles, which have low membrane curvature [10]. It seems likely that the different lipid-binding characteristics of the complexes help to define the distinct roles of Complex I and II in autophagy and vesicular trafficking respectively. However, since both the enlarging autophagosome and the tubule networks of the endocytic and recycling systems have regions of high curvature, these data do not explain why Complex I would not be active in both contexts.

The specific activity of Vps34 Complex I or II is greater than that of the Vps34–Vps15 dimer [10,33]. Overexpression of UVRAG or Atg14 in mammalian cells increases Vps34 activity [19,34], presumably by shifting Vps34–Vps15 dimers into their tetrameric states. Furthermore, there is co-dependence among the Vps34-binding proteins for protein stability: in both yeast and mammalian cells, deletion or knockdown of the members of Complex I and II lead to decreases in the expression of other complex members, presumably due to destabilization and degradation [17,18].

Scaffolds/chaperones for Vps34 complex assembly

A number of proteins have been shown to regulate the formation or stability of Vps34 complexes. NRBF2 (nuclear receptor binding factor 2) (Atg38 in yeast) was described by four groups as specifically interacting with Vps34 Complex I [3538]. Originally defined as a regulator of nuclear hormone receptors [39,40], NRBF2 forms a homodimer, and has an N-terminal MIT (microtubule and trafficking) domain [35]. MIT domains are found in proteins involved in the regulation of vesicular trafficking, including the AAA (ATPase associated with various cellular activities) proteins involved in disassembly of the ESCRT complex [41]. Several of the studies show that NRBF2 enhances the binding of Vps15–Vps34 to Beclin–Atg14, suggesting a chaperone or scaffolding function [35,36,38]. However, other aspects of NRBF2 biology remain controversial: depending on the study, NRBF2 binds directly to Atg14, or to Vps15, or to both; it increases Atg14-associated Vps34 activity or has no effect; it enhances starvation-induced autophagy or suppresses it. Hopefully, subsequent studies will address these inconsistencies.

A recent study described a second scaffold specific for Vps34 Complex I, PAQR3 (progestin and adipoQ receptor family member III) [42]. PAQR3 is a heptahelical membrane protein that is localized to the Golgi and is constitutively associated with Vps34 Complex I. Binding studies suggest that it interacts directly with Beclin-1, Atg14 and Vps15, but not Vps34. PARQ3 knockdown in cells or knockout in mice leads to reductions in Atg14-associated Beclin-1, Vps34 and Vps15. PARQ3 co-localizes with members of Vps34 Complex I in the Golgi under basal conditions, and in punctate structures in glucose-starved cells. PAQR3 is also phosphorylated by AMPK (AMP-dependent protein kinase) in glucose-starved cells; its role in the regulation of Vps34 Complex I by AMPK is discussed below. Interestingly, knockdown of PAQR3 and NRBF2 have additive effects on Complex I assembly and autophagy, suggesting that their activities are mechanistically distinct [42].

Dapper1 was originally described as an inhibitor of the Wnt signalling pathway [43]. Yeast two-hybrid studies show direct interactions between Dapper1 and Atg14 or Beclin-1 [44]. Dapper1 overexpression increases co-immunoprecipitation of Beclin-1 with Atg14 and Vps34, as well as Atg14 with Vps34, and increases Vps34 activity; its knockout reduces these interactions. Although Dapper1 appears to have no effect on the assembly of Vps34 Complex II, it decreases the binding of the inhibitory protein Rubicon (RUN domain protein Beclin-1-interacting and cysteine-rich containing) (described below) to UVRAG. Thus, although its chaperone/scaffold activity may be limited to Complex I, Dapper1 may regulate the activity of Complex II by displacing the inhibitory Rubicon molecule.

Liver-specific knockout of the WD-40-only protein RACK1 (receptor for activated C kinase 1) causes significant decreases in Beclin-1 and Atg14 binding to Vps34 and Vps15, but only minor changes in Beclin-1–Atg14 binding [45]. This suggests that, under basal conditions, RACK1 might serve as a chaperone for the assembly of Beclin-1–Atg14 dimers with Vps34–Vps15 dimers. Interestingly, RACK1 is phosphorylated in starved cells by AMPK; AMPK-mediated phosphorylation increases RACK1 binding to in vitro translated Beclin-1, Vps15 and Atg14. This would suggest that its chaperone activity is enhanced during nutrient stress. Atg14-associated Vps34 activity in the livers of starved mice is blunted by RACK1 knockout, as is starvation-induced increases in PI[3]P in RACK1 knockdown HepG2 cells. Whether this is due to an allosteric regulation of the lipid kinase activity of Vps34 Complex I, as opposed to increased assembly of Complex I, remains an interesting question for future studies.

The death-effector domain-containing DNA-binding protein DEDD, whose expression in tumour cells leads to the epithelial-to-mesenchymal transition, binds to Beclin-1 and Vps34 and increases the half-time for Vps34 turnover [46], presumably by stabilizing the complex. Finally, both the canonical chaperone heat-shock protein 70 (Hsp70) [47] and the G-protein-coupled receptor (GPCR)-specific scaffold β-Arrestin 2 [48] enhance complex formation between Vps34 and Beclin-1; specificity for Vps34 Complex I or II has not been examined. Interestingly, the requirement for β-Arrestin 2 in Vps34–Beclin-1 binding is seen in neurons during oxygen/glucose starvation but not under basal conditions, which might suggest activity towards the autophagy-specific Vps34 Complex I.

Vps34-independent functions for the components of Complex I and II

Although not strictly within the sphere of this review, both UVRAG and Atg14 have Vps34-independent functions in the endocytic system. The maturation of early endosomes involves a co-ordinated transition from Rab5 activation to Rab7 activation [49]. UVRAG plays an important role in the maturation of early endosomes via its interactions with the HOPS (homotypic fusion and vacuole protein sorting) complex, which acts as a Rab7 effector in late endosomal membrane fusion events [50]. Independently of its interactions with Beclin-1, UVRAG binds to the Vps16 subunit of the HOPS complex, which enhances the GTP loading of Rab7, late endosome–lysosome fusion, and cargo degradation [51]. Rubicon, which binds to and inhibits Vps34 Complex II (discussed below), inhibits the binding of UVRAG to the HOPS complex [52]. Interestingly, Rubicon also binds to activated GTP-bound Rab7, which displaces Rubicon from UVRAG [34,52]. The authors propose a feed-forward cycle in which activation of Rab7 releases UVRAG from Rubicon-mediated inhibition, allowing UVRAG to engage with HOPS and further activate Rab7.

UVRAG also acts as a PI[3]P effector in the regulation of Golgi-to-ER retrograde trafficking [53]. UVRAG binds to PI[3]P in the ER via its C2 domain, allowing it to associate with the RINT1 (Rad50 interactor 1)–ZW10–NAG (neuroblastoma amplified gene) tethering complex. The RINT1–ZW10–NAG complex is required for Golgi-derived coatomer protein I (COPI)-mediated vesicular traffic to the ER [54]. UVRAG knockdown blocks the retrograde trafficking of KDEL-tagged cargo and disrupts Golgi structure.

Atg14 functions in a Vps34-independent manner in the regulation of endosomal fusion through interactions with SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) and associated proteins. Atg14 binds to Snapin, a SNARE-binding protein that drives the fusion of target (t-)SNARE–vesicle (v-)SNARE complexes between donor and target membranes in the endocytic system [55,56]. Knockdown/rescue experiments with Atg14 mutants show that its role in endocytic trafficking requires its binding to Snapin-1 but not its binding to Beclin-1. More recently, it has been shown that purified Atg14 enhances the tethering and fusion of liposomes reconstituted with autophagosomal SNAREs [57]. Atg14 binds to the autophagic SNARE through its coiled-coil domain, and in addition requires homo-oligomerization through cysteine residues in its N-terminus. Mutation of these cysteine residues blocks autophagosome–lysosome fusion but has no effect on Atg14 localization to sites of autophagosomal initiation in the ER (discussed below) or to autophagosomes, suggesting functions for Atg14 that are independent of its Vps34-related role in autophagy.

MECHANISMS OF Vps34 REGULATION

The amount of cellular PI[3]P increases during the induction of autophagy. Similarly, a loss of PI[3]P production, and presumably Vps34 activity, is seen when cells are treated with growth factors and cytokines that inhibit autophagy [58]. How does this regulation of Vps34 occur? There is evidence for a number of overlapping mechanisms, including (a) phosphorylation, ubiquitination, SUMOylation and acetylation of Vps34 and members of the Vps34 complexes; (b) changes in the assembly of Vps34 Complex I and II; (c) the binding of auxiliary proteins and inhibitors; (d) intracellular targeting; and (e) selective degradation of members of the Vps34 complexes. Longer-term regulation of Vps34 activity by changes in transcriptional levels have been described (for example [59,60]), but will not be reviewed here.

Regulation by phosphorylation during autophagy

Until recently, the question of whether the specific activity of Vps34 increases during induction of autophagy was controversial. Many studies claiming to show Vps34 activation in fact show an increase in intracellular PI[3]P that could also be caused by mechanisms such as intracellular targeting. Some studies that directly examined Vps34 activity rely on expression of Vps34 in the absence of the associated Vps15 pseudokinase. However, Vps34 is not fully active as a monomer, and its regulation by amino acids and binding to Beclin-1–UVRAG requires the presence of Vps15 [33]. Finally, there are direct contradictions in the literature between studies that show increased activity compared with decreased activity of Beclin-associated and total Vps34 during nutrient starvation (see, for example, [61,62]).

A better understanding of Vps34 regulation during the induction of autophagy was provided by a series of studies that used an immunodepletion strategy to define four distinct Vps34 pools: complexes containing Vps34 and Vps15 but not Beclin-1, complexes containing Vps34, Vps15 and Beclin-1, and complexes containing Vps34, Vps15, Beclin-1 and either Atg14 or UVRAG [1416]. Quantitative immunodepletion of Beclin-1 in mouse embryonic fibroblasts (MEFs) reduces the amount of Vps34 and Vps15 remaining in the supernatant, although there is a significant residual suggesting a large pool of Vps34–Vps15 dimers. In contrast, quantitative immunodepletion of Atg14 or UVRAG has modest effects on the amount of Vps34 and Beclin-1 remaining, and no effect on the amount of Vps15 remaining. These data suggest that the bulk of Vps34 is in complexes with Vps15 and Beclin, but not Atg14 or UVRAG.

Intriguingly, amino acid or glucose starvation causes a decrease in the specific activity of Vps34–Vps15 and Vps34–Vps15–Beclin-1 complexes [1416]. However, 2–3-fold increases in activity are seen in complexes containing Atg14 (Vps34 Complex I) or UVRAG (Vps34 Complex II). These increases are presumably not reflected in the activity of the total Beclin-associated Vps34 pool, because Complex I and Complex II represent a small fraction of total Vps34–Vps15–Beclin-1 complexes. These studies explain why a decrease in the overall specific activity of Vps34 in starved cells is compatible with increases in autophagosomal PI[3]P during the induction of autophagy; one result reflects the bulk pool of Vps34, whereas the other reflects a specifically targeted and regulated fraction of the total pool.

The induction of autophagy is intimately related to regulation of the mammalian target of rapamycin (mTOR) complex I (mTORC1) pathway, and involves three key protein kinases: mTOR itself, which as part of mTORC1 is activated by nutrients, and AMPK and ULK1, which are activated during nutrient stress [8]. A combination of phosphosite mapping and mutagenesis was used to show that all three of these kinases regulate Vps34 [1416]. In glucose-starved cells, activation of Vps34 Complex I is mediated though AMPK, which phosphorylates Beclin-1 at Ser91 and Ser94 [16] (Figure 4). Purified AMPK is unable to phosphorylate Beclin-1 in vitro in the context of a Vps34–Vps15–Beclin-1 complex, but the addition of recombinant Atg14 restores the in vitro phosphorylation of Beclin-1. This suggests that Atg14 alters the conformation of the Beclin-1 N-terminus so as to allow its recognition by AMPK. Beclin-1 within the context of Vps34 Complex II is also phosphorylated by AMPK, leading to increased Complex II lipid kinase activity. Notably, induction of autophagy by glucose starvation is blocked in cells expressing S91A/S94A Beclin-1. A recent study has also implicated the Vps34 Complex I scaffold PAQR3 in AMPK regulation. PAQR3 is constitutively bound to Vps34 Complex I, and undergoes AMPK-mediated phosphorylation at Thr32 in glucose- but not amino acid-starved cells [42] (Figure 4). Reconstitution of PAQR3-knockdown cells with T32A PAQR3 inhibits autophagy and activation Vps34 Complex I in glucose-starved cells, whereas the mutant rescues autophagy in response to amino acids. The effect of PAQR3 on AMPK-mediated phosphorylation of Beclin-1 was not examined, but it seems possible that the two events are mechanistically linked.

Regulation of Vps34 complexes by phosphorylation during starvation

Figure 4
Regulation of Vps34 complexes by phosphorylation during starvation

Activating phosphorylation sites are in blue. Inhibitory sites are in red. Pathways that are known to regulate a specific Vps34 complex are shown with unbroken lines. Pathways that regulate Vps34 Complex I, but might also regulate Complex II, are shown with dashed lines.

Figure 4
Regulation of Vps34 complexes by phosphorylation during starvation

Activating phosphorylation sites are in blue. Inhibitory sites are in red. Pathways that are known to regulate a specific Vps34 complex are shown with unbroken lines. Pathways that regulate Vps34 Complex I, but might also regulate Complex II, are shown with dashed lines.

In amino acid-starved cells, Beclin-1 is phosphorylated by ULK1 at Ser14 when it is present in either Vps34 Complex I or Complex II (Figure 4); this modification activates the lipid kinase activity of these complexes [15]. Expression of a S14A mutant of Beclin-1 blocks the induction of autophagy in response to amino acid starvation. ULK1 is be co-immunoprecipitated along with both Atg14 and UVRAG, suggesting that direct binding to the complexes enhances their regulation by ULK1. Interestingly, the Atg14-dependent phosphorylation of Beclin-1 at the AMPK sites (Ser91/Ser94) is also observed in cells HCT116 colon cancer cells incubated in amino acid-free medium [63]. As in the case of glucose starvation [16], expression of S91A/S94A Beclin-1 could not rescue autophagic responses in amino acid-starved Beclin-1-knockout cells.

Under nutrient-replete conditions, the activating signals from ULK1 and AMPK are inhibited, and mTORC1 is activated. Activation of mTORC1 directly inhibits Vps34 Complex I by phosphorylation of Atg14 at Ser3, Ser223, Ser233, Ser383 and Ser440 [14] (Figure 5). Similar to the phosphorylation of Complex I by AMPK, incubation of mTORC1 with Vps34–Vps15–Beclin-1 complexes in vitro has no effect on lipid kinase activity, but addition of recombinant Atg14 renders the complex susceptible to inhibition by mTORC1-mediated phosphorylation. Cells expressing mutant Atg14 containing serine-to-alanine mutations at the five phosphorylation sites show constitutive increases in autophagy, consistent with a physiological role for mTORC1-mediated Atg14 phosphorylation in the suppression of autophagy under nutrient-rich conditions.

Regulation of Vps34 complexes by phosphorylation under nutrient-replete conditions or during growth factor stimulation

Figure 5
Regulation of Vps34 complexes by phosphorylation under nutrient-replete conditions or during growth factor stimulation

Inhibitory phosphorylation sites are in red. Pathways that are known to regulate a specific Vps34 complex are shown with unbroken lines. Pathways that regulate Vps34 Complex I, but might also regulate Complex II, are shown with dashed lines.

Figure 5
Regulation of Vps34 complexes by phosphorylation under nutrient-replete conditions or during growth factor stimulation

Inhibitory phosphorylation sites are in red. Pathways that are known to regulate a specific Vps34 complex are shown with unbroken lines. Pathways that regulate Vps34 Complex I, but might also regulate Complex II, are shown with dashed lines.

In contrast with Vps34 Complexes I and II, the lipid kinase activity of Vps34–Vps15 and Vps34–Vps15–Beclin-1 complexes is inhibited during starvation [61]. In glucose-starved cells, AMPK phosphorylates Vps34 at Thr163 and Ser165 in the C2 domain, leading to a decrease in lipid kinase activity [16] (Figure 5). During incubations with AMPK in vitro, Vps34 in anti-Vps34 immunoprecipitates but not in Atg14 immunoprecipitates is phosphorylated, suggesting that the presence of Atg14, or the AMPK-mediated phosphorylation of Atg14-bound Beclin-1, blocks the inhibitory phosphorylation of Vps34 by AMPK. Mutation of the Thr163/Ser165 phosphorylation sites in Vps34 does not affect the induction of autophagy but reduces the survival of cells during prolonged (24 h) glucose starvation. During amino acid starvation, inhibition of Vps34–Vps15 and Vps34–Vps15–Beclin-1 complexes requires mTORC1 but not ULK1; inhibition of VPs34 activity by mTORC1 could not be reconstituted in vitro, suggesting an indirect mechanism [14].

Several other kinases have been implicated in the regulation of Vps34 during the induction of autophagy. MAPK-activated protein kinase 2 and 3 (MAPKAPK2 and MAPKAPK3), which function downstream from the stress-activated kinase p38, phosphorylate Beclin-1 at Ser90 [64] (Figure 4). Consistent with a regulatory role for Ser90 phosphorylation, induction of autophagy by amino acid starvation is reduced in cells expressing S90A Beclin-1. However, the mechanism by which Ser90 phosphorylation regulates Vps34 activity is not clear. Basal Beclin-1-associated Vps34 activity is decreased in cells expressing S90A Beclin-1, and increased in cells expressing S90E Beclin-1. However, Vps34 activity associated with S90E Beclin-1 is increased by amino acid starvation similarly to cells expressing wild-type Beclin-1. These data suggest that the effect on Ser90 phosphorylation on the activity of the Vps34 complex is indirect, since regulation still occurs in the non-phosphorylatable S90E mutant. A plausible explanation would involve the recruitment of a regulatory protein or activator, which would lead to enhanced activity under basal and starvation conditions.

Death-associated protein kinase (DAPK) is a positive regulator of autophagy. This is due in part to the phosphorylation of Thr119 in the BH3 domain of Beclin-1 [65], which leads to dissociation of Bcl-2–Beclin-1 complexes (discussed below). However, induction of autophagy by DAPK is blocked by knockdown of PKD (protein kinase D), and overexpression of PKD activates autophagy [66]. Overexpressed PKD and Vps34 can be co-immunoprecipitated, and PKD phosphorylates Vps34 in vitro. Mass spectrometry studies identified Thr677 as one of several phosphorylation sites (Figure 4), but mutation of the site does not diminish the in vitro phosphorylation of Vps34 by PKD. The effect of PKD-mediated phosphorylation on Vps34 activity has not been determined.

Regulation by phosphorylation during growth factor stimulation, mitosis and transformation

Autophagy is inhibited by nutrients but also by growth factor stimulation. Two studies have shown that phosphorylation of Beclin-1 downstream from receptor tyrosine kinases (RTKs) inhibits autophagy. Phosphorylation of Beclin-1 by Akt at Ser234 and Ser295 leads to a decrease in autophagy [67] (Figure 5). Akt can be co-immunoprecipitated with Beclin-1 under fed but not starved conditions, leading to Beclin-1 phosphorylation and increased Beclin-1 binding to 14-3-3 proteins and vimentin; these changes correlate with a reduction in starvation-induced autophagy. With regard to Vps34, overexpression of constitutively active myristoylated Akt (Myr-Akt) causes a loss of Vps34 binding to wild-type Beclin-1, whereas binding to a S234A/S295A Beclin-1 mutant is unaffected. The effect of these mutations on Beclin-1 binding to Vps34 during starvation, or the regulation of Vps34–Beclin-1 binding by physiological activation of the PI3K/Akt pathway, were not tested. In addition, activation of epidermal growth factor receptors (EGFRs) with ligand or by mutation leads to the tyrosine phosphorylation of Beclin-1 on Tyr229, Tyr233 and Tyr352, in the coiled-coil domain [68] (Figure 5). Tyrosine phosphorylation of Beclin-1, or mutation of the tyrosine residues to glutamate, leads to a loss of Vps34–Beclin-1 binding and a decrease in autophagy. The authors propose a mechanism in which tyrosine phosphorylation enhances Beclin-1 homodimerization and reduces its participation in Vps34 complexes; this model is discussed in detail below.

It has been known for some time that autophagy is inhibited during mitosis [69]. A mechanism for this inhibition is provided by the finding that Vps34 is directly phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5/p25, at Thr159 in the C2 domain of Vps34 and at Thr668 in the kinase domain [70] (Figure 5). Phosphorylation of Vps34 leads to a loss of binding to Beclin-1 as well as a loss of Vps34 lipid kinase activity. Starvation-induced autophagy is inhibited by overexpression of Cdk5, and this inhibition is lost in cells expressing T159A Vps34. Similarly, mitotic arrest in paclitaxel-treated cells leads to an increase in Thr159 phosphorylation and a decrease in autophagy [71] (Figure 5).

In contrast, Vps34 is activated in Src-transformed cells by phosphorylation at Tyr231 and Tyr310, and kinase-dead or Y231F Vps34 blocks Src-mediated transformation [72] (Figure 4). Src-mediated phosphorylation of Vps34 at Tyr231 and Tyr310 is also observed in insulin-stimulated NIH3T3 cells, and expression of Y231F Vps34 causes a reduction in insulin-stimulated activation of S6K1 [73]. These studies suggest that Src-mediated phosphorylation of Vps34 plays a positive role in proliferative responses.

The mechanism by which phosphorylation regulates the activity of Vps34 Complex I and II has not yet been determined. In general, the activating phosphorylation sites in Beclin-1 (Ser14, Ser90/91 and Thr119) are within its N-terminal domain [15,16,65], whereas inhibitory sites are in the coiled-coil (Ser234/235, Tyr229/233/352) or BARA (Ser295) domains [67,68]. An exception is the inhibitory Mst1 site (Thr108) [74]. For Vps34, phosphorylation sites within the C2 domain are inhibitory (Thr159, Thr163 and Ser165) as is phosphorylation of Thr668 in the kinase domain [14,70]. Activating phosphorylation sites are in the C2-helical linker (Tyr231) or in the kinase domain (Tyr310 and Thr667) [66,72,73]. Inhibitory phosphorylation sites in Atg14 (Ser3, Ser223, Ser233, Ser383 and Ser440) are spread throughout the proteins [14].

The Beclin-limited model of Vps34 activation

The most commonly cited model for the activation of Vps34 during autophagy involves the regulated assembly of Vps34 Complex I and Complex II, driven by the release of sequestered Beclin-1 to form complexes with Vps34, Vps15 and either Atg14 or UVRAG (Figure 6). The crux of this model is that the availability of Beclin-1 is rate-limiting for the formation of productive Vps34 complexes. Despite the widespread acceptance of this model, a substantial body of published work is inconsistent with its major facets.

The Beclin-limited model of autophagy initiation

Figure 6
The Beclin-limited model of autophagy initiation

Beclin-1 homodimers are stabilized by EGFR-mediated phosphorylation and by binding to Bcl-2. Phosphorylation of Bcl-2 by JNK1 leads to dissociation of Bcl-2, which destabilizes the Beclin-1 dimer. This frees Beclin-1 to form complexes with Vps34, Vps15 and Atg14 or UVRAG. Despite its widespread acceptance, the proposed mechanism is inconsistent with data on the relative abundance of Beclin-1 as compared with other members of Complex I and II, and studies in which changes in the abundance of Beclin-1/Bcl-2 heterodimers and Beclin-1 homodimers do not correlate with effects on Vps34 complex formation.

Figure 6
The Beclin-limited model of autophagy initiation

Beclin-1 homodimers are stabilized by EGFR-mediated phosphorylation and by binding to Bcl-2. Phosphorylation of Bcl-2 by JNK1 leads to dissociation of Bcl-2, which destabilizes the Beclin-1 dimer. This frees Beclin-1 to form complexes with Vps34, Vps15 and Atg14 or UVRAG. Despite its widespread acceptance, the proposed mechanism is inconsistent with data on the relative abundance of Beclin-1 as compared with other members of Complex I and II, and studies in which changes in the abundance of Beclin-1/Bcl-2 heterodimers and Beclin-1 homodimers do not correlate with effects on Vps34 complex formation.

The Beclin-limited model was initially built on the observations that Beclin-1 binding to the anti-apoptotic protein Bcl-2 decreases upon nutrient starvation, and that Beclin-1 mutants that are deficient for Bcl-2 binding cause an increase in autophagy [75]. The ability of Bcl-2 to inhibit autophagy is localization-dependent, as ER-targeted Bcl-2, but not mitochondria-targeted Bcl-2, blocks starvation-induced autophagy. The Beclin-limited model suggests that Bcl-2-bound Beclin-1 is unable to participate in its autophagic functions. A reduction in Beclin-1–Bcl-2 binding in response to starvation would release Beclin-1 and promote formation of complexes with UVRAG, Atg14 and Vps34–Vps15.

An important element of the Beclin-1 sequestration model is the finding that the Beclin-1 coiled-coil domain forms both homodimers and heterodimers with other coiled-coil proteins. For example, the structure of Vps34 Complex II from yeast [10] shows the coiled-coil domains of Vps30 pairing in a parallel fashion with the coiled-coil domains of Vps38. A similar arrangement is likely for Complex I [27]. In contrast, Beclin-1 homodimers form in an antiparallel fashion [76,77]. Studies evaluating the stability of the Beclin-1 homodimer have been inconsistent. An isothermal calorimetry study found that the isolated Beclin-1 coiled-coil domain forms a relatively weak homodimer (Kd=89 μM) due to unfavourable residues at the a’ and d’ positions of the heptad coiled-coil repeat [76]. In contrast, an analytical ultracentrifugation study found that the formation of dimers of the Beclin-1 coiled-coil domain is not concentration–dependent, suggesting a stable structure that is not in rapid equilibrium [78]. The reason for the disparity between these studies is not clear.

Beclin-1 dimers are further stabilized by binding to Bcl-2-family proteins [78]. This is mediated by contacts between the BH3 domain of Beclin-1 and the hydrophobic groove of Bcl-2 [79]. Given the involvement of the Beclin-1 coiled-coil domain in both homodimerization and heterodimerization with Atg14 and UVRAG, the enhancement of Beclin-1 homodimerization by Bcl-2 family members is predicted to inhibit the formation of Beclin-1 complexes containing Vps34. Consistent with this idea, the UVRAG and Atg14 coiled-coil domains bind more tightly to the Beclin-1 coiled-coil domain than does Beclin-1 itself (Kd=0.24 μM and 3.22 μM respectively) [76]; in the absence of Bcl-2-mediated stabilization, Beclin-1 would be preferentially in the form of heterodimers with Atg14 or UVRAG. The formation of higher-order Beclin-1–Bcl-2 oligomers, which might further enhance stability of the homodimeric state, has also been proposed [80]. However, this study relied on gel filtration, which is an unreliable measure of molecular mass for rod-like coiled-coil proteins; the possibility of Beclin-1–Bcl-2 oligomers in vitro and in cells remains an interesting question.

In support of the Beclin-limited model, there are numerous papers demonstrating that Beclin-1 binding to Bcl-2 inhibits autophagy and is regulated by autophagic stimuli. Beclin-1 binding to Bcl-2 is inhibited during starvation by c-Jun N-terminal kinase 1 (JNK1)-mediated phosphorylation of Bcl-2 [81]. Consistent with the Beclin-limited model, other proteins that modify Beclin-1 binding to Bcl-2 regulate autophagy. For example, parkin-mediated mono-ubiquitination of Beclin-1 inhibits binding to Bcl-2 and activates autophagy [82]; knockdown of the resident ER protein nutrient-deprivation autophagy factor 1 (Naf1) decreases Beclin-1–Bcl-2 binding and increases autophagy [83]; knockdown of high-mobility group box 1 (HMGB1) inhibits Bcl-2–Beclin-1 dissociation and blocks autophagy [84]; and overexpression of DAPK phosphorylates the Beclin-1 BH3 domain at Thr119, blocking its binding to Bcl-2 and Bcl-xL and activating autophagy [65].

However, these studies do not address a key aspect of the model: does the regulated assembly of autophagy-related Beclin-1 complexes actually occur? That is to say, does Beclin-1 binding to Bcl-2 prevent it from forming complexes with Vps34, Vps15 and UVRAG or Atg14, and does the dissolution of Beclin-1–Bcl-2 complexes or Beclin-1 homodimers increase the formation of Vps34 Complex I or II? In the first description of the Beclin-limited model, it is shown that overexpression of Bcl-2 inhibits Beclin-1 binding to Vps34, but changes in the level of Beclin-1–Vps34 binding in response to an autophagic stimulus are not examined [75]. Similarly, the study examining JNK1 regulation of Beclin-1–Bcl-2 binding does not examine Beclin-1–Vps34 binding [81].

Numerous studies have reported increases in Beclin-1 binding to Vps34 under conditions of increased autophagy [44,53,8591]. In addition, several studies show changes in Beclin-1–Vps34 binding in the context of altered Vps34 expression [47,92], although this may be secondary to changes in Vps34 abundance rather than changes in Vps34–Beclin-1 binding. However, these studies do not specifically address the formation of Vps34 Complex I and II. Given that UVRAG- and Atg14-containing complexes are small fractions of the total Vps34–Vps15–Beclin-1 pool [16], it is not clear that these data reflect regulation of the complexes that are active in autophagy. Studies directly showing an increase in UVRAG or Atg14-associated Vps34 during the induction of autophagy are less abundant. An increase in Beclin-1 and UVRAG binding to Vps34 is seen in rapamycin treated or starved human embryonic kidney (HEK)-293 cells [53]. An increase in Atg14-associated Vps34 activity is seen after 48 h of starvation, but unfortunately no blots are presented to measure the amount of bound protein [45]. Finally, in response to myocardial infarction, the MST1 kinase phosphorylates Beclin-1 at Thr108 [74]. This leads to an increase in Beclin-1 binding to Bcl-2 and Bcl-xL, increased Beclin-1 dimerization, and a reduction in Beclin-1 binding to Atg14 and Vps34. However, no change in Vps34 binding to Atg14 is seen, which is surprising given that Vps34–Vps15 binds Atg14 only in the heterotetramer with Beclin-1. Furthermore, there is no effect on Beclin-1 binding to UVRAG; increased Beclin-1 binding to Bcl-2 would be expected to affect both Vps34 Complex I and II.

In contrast, numerous papers have been unable to detect starvation-induced changes in the abundance of Vps34 Complex I and II. No changes in Beclin-1 or Vps34 binding to UVRAG [93] or Beclin-1 binding to UVRAG [94] are seen in starved HEK-293 cells or HCT116 cells respectively. No changes in Beclin-associated Vps34 or Atg14 are observed in starved U2OS cells [64], and no changes in Vps34-associated proteins in anti-Vps34 or anti-UVRAG immunoprecipitates are seen in starved HeLa and U2OS cells and MEFs [95]. No changes in Vps34, Vps15 and Beclin-1 binding to Atg14 or UVRAG are seen in amino acid- or glucose-starved MEFs [42]. Notably, a study that separately examines Beclin-1–Vps34 binding in complexes containing Atg14 or UVRAG, as well as complexes containing Beclin-1, Vps15 and Vps34 without Atg14 or UVRAG, did not detect changes in complex formation in response to amino acid or glucose starvation [1416].

The question of whether the abundance of Vps34 Complex I and II changes acutely in response to an autophagic stimulus thus remains controversial. However, in cases where a change is detected, what is the evidence supporting the mechanism proposed by the Beclin-limited model? A key aspect of the Beclin-limited model is that the availability of ‘free’ Beclin-1 limits the formation of Vps34 Complex I and II. However, a recent proteomic analysis in HeLa cells shows that Beclin-1 is nearly 6-fold in excess over UVRAG, and 25-fold over Atg14 [96]. In contrast, the levels of Beclin-1 and Vps34 and Vps15 are more closely matched (ratios of 3:1 and 1.4:1 respectively) [96]. In addition, as discussed above, quantitative immunodepletion studies in MEFs show that the number of Vps34–Vps15–Beclin-1 complexes significantly exceeds the number of complexes containing UVRAG or Atg14 [16]. These data are not consistent with the overall availability of Beclin-1 as a limiting factor for assembly of Vps34 complexes.

Additional studies are inconsistent with the proposed mechanism by which dissociation of Beclin-1–Bcl-2 complexes or Beclin-1 homodimers might regulate the formation of Vps34 complexes. Rapamycin, which is a commonly used autophagic stimulus, does not lead to dissociation of Beclin–Bcl-xL complexes [97]. Beclin-1 dimerization, as measured by cross-linking and co-immunoprecipitation of tagged constructs, is unaffected by overexpression of Bcl-xL or UVRAG, or by starvation or treatment with rapamycin [98]. In addition, treatment of cells with the synthetic BH3 ligand ABT737 disrupts Beclin-1–Bcl-2 binding but has no effect on Beclin-1–Vps34 complexes [99], and inhibitors of Bcl-2/Bcl-xL induce autophagy independently of changes in Beclin-1–Vps34 binding [100]. However, these studies do not specifically look at Complex I and II, and might not have detected changes in these relatively small pools.

Finally, two studies on the regulation of Beclin-1 by phosphorylation are inconsistent with the disruption of Beclin-1–Bcl-2 complexes and Beclin-1 homodimers as rate-limiting for the formation of Vps34 complexes. During starvation, Beclin-1 is phosphorylated by MAPKAPK2, which activates autophagy [64]. A previous study showed that the activation of autophagy during starvation requires the JNK1-mediated phosphorylation of Bcl-2, which leads to the dissociation of Bcl-2 from Beclin-1 [81]. The MAPKAPK2-mediated Ser90 phosphorylation of Beclin-1 apparently requires this dissociation of Bcl-2–Beclin-1 complexes, as it is blocked by a Bcl-2 mutant lacking the JNK1 phosphorylation sites. However, Beclin-1 Ser90 phosphorylation during starvation is not associated with a change in Beclin-1-associated Vps34 or Atg14 [64]. Thus, a regulatory phosphorylation of Beclin-1 that requires the dissociation of Bcl-2–Beclin-1 complexes is not correlated with any change in the formation of Beclin-1 complexes with Vps34 or Atg14.

Even more striking is the regulation of Beclin-1 by EGFR-mediated tyrosine phosphorylation at Tyr229, Tyr233 and Tyr352 in the coiled-coil domain, which causes an enhancement of Beclin-1 homodimerization [68]. The Beclin-limited model would predict that an enhancement of Beclin-1 homodimerization would lead to a reduction in its formation of complexes with Atg14 and UVRAG, since the same coiled-coil domain is involved in both the homo- and the hetero-dimeric interactions. However, EGFR phosphorylation of Beclin-1 has no effect on Beclin-1 binding to Atg14 or UVRAG [68]. This means that changes in the level of Beclin-1 homodimerization are not correlated with changes in its ability to participate in Vps34 Complex I and II.

Although it has no effect on Beclin-1 binding to Atg14 and UVRAG, EGFR-mediated Beclin-1 phosphorylation does inhibit Beclin-1 binding to Vps34 [68]. These data could be explained by the dissociation of Beclin-1–Atg14 and Beclin-1–UVRAG dimers from Vps34–Vps15 dimers. Dimeric Vps30–Vps38 complexes are stable, and can bind to Vps34–Vps15 complexes [10]. It is also possible that the major effect of EGFR-mediated Beclin-1 phosphorylation is to disrupt the Beclin-1–Vps34–Vps15 complex; as discussed above, these are the most abundant Beclin-1–Vps34–Vps15 complexes [16]. Of note, we do not know what such a complex would look like. Based on the structures of Complex I and Complex II [10,27], Beclin-1 appears to interact with the Vps34–Vps15 dimer as a part of a pair of coiled coils, with the coiled-coil domain of Beclin-1–Vps30 interacting in a parallel manner with those of Atg14 or Vps38. Given these structures, it seems unlikely that Beclin-1 would interact with Vps34–Vps15 as an unpaired coiled coil. However, it is possible that another as yet unidentified coiled-coil protein might pair with Beclin-1 so as to stabilize a complex with Vps34–Vps15. Alternatively, although there is no structural basis for this conjecture, it is possible that antiparallel Beclin-1 homodimers could bind to Vps34–Vps15 dimers; this could be tested in vitro.

Regulation by accessory proteins for Vps34 Complex I and II

The core Vps34 complexes bind to a host of additional cytosolic proteins, some of which regulate lipid kinase activity or intracellular targeting. The co-ordination of these various factors, and their role in specific Vps34-dependent cellular processes, is still an area of active investigation.

Ambra1 is a WD40-containing protein that is critical for neural development [85]. Ambra1 interacts directly with Beclin-1 in a yeast two-hybrid analysis, and its knockdown inhibits starvation-induced autophagy and decreases the amount of Vps34 in Beclin-1 immunoprecipitates in both fed and starved cells. This is due to the binding of Ambra1 to the TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) E3 ubiquitin ligase, with ubiquitinates Beclin-1 at Lys437 [101,102] (discussed below). Beclin-1 binding to Ambra1 is inhibited by Ambra1 binding to mitochondrial Bcl-2; this binding is reduced in starved cells, presumably increasing the pool of available Ambra1 [103]. In addition to its contribution to Vps34 complex assembly, Ambra1 regulates the targeting of Vps34–Beclin-1 complexes to microtubules, which inhibits autophagy [104] (discussed below). Unfortunately, it has not been unambiguously demonstrated whether Ambra1 interacts preferentially with Vps34 Complex I or II, or whether it interacts with Beclin-1–Vps34–Vps15 complexes that lack UVRAG and Atg14. Ambra1 is found in the basal body of cilia, along with Vps15, Vps34 and Atg14 [105]. However, since Beclin-1 is not present in this structure, it is not clear that this is related to Ambra1 binding to Vps34 Complex I.

Bif-1 is a positive regulator of autophagy, based on overexpression and knockdown studies [106]. Co-immunoprecipitation studies with overexpressed protein suggest that Bif-1 binds to UVRAG but not Beclin-1, suggesting that it regulates Vps34 Complex II, and endogenous Bif-1 associates with Beclin-1 and UVRAG in response to starvation. In MEFs transfected with FLAG–Vps34 without Vps15 other members of Complex I or II, a 2-fold increase in lipid kinase activity is seen upon starvation; this response is attenuated in Bif-1-knockout MEFs.

Bif-1 is a member of the endophilin family of cytosolic proteins, and contains an N-terminal BAR (bin-ampiphysin-Rvs) domain and a C-terminal SH3 (Src homology 3) domain. BAR domains preferentially interact with lipid membranes that show a high degree of curvature, and incubation of endophilins with lipid vesicles in vitro leads to the production of narrow tubules [107]. It has been proposed that the Bif-1 BAR domain contributes to Vps34 interactions with highly curved membranes during autophagosome formation [108]. Thus, the BAR domain of Bif-1 in VPs34 Complex II may play an analogous role to that of the BATS domain of Atg14 in Vps34 Complex I [26]. Bif-1 also regulates the Vps34 Complex II-dependent trafficking of the transmembrane autophagy protein Atg9 during starvation [53].

Rubicon contains FYVE and coiled-coil domains and an N-terminal RUN domain (RPIP8, UNC-14 and NESCA); RUN domains have been implicated in signalling by Rab GTPases [109,110]. Rubicon was identified by several laboratories as a specific inhibitor of autophagy that binds to Vps34 Complex II [19,20]. In vitro binding studies with purified proteins suggest that the RUN domain binds directly to Vps34; a larger region of Rubicon that includes its coiled-coil and FYVE domains binds directly to UVRAG [34]. Binding of Rubicon to Vps34 Complex II reduces its lipid kinase activity [19,34].

TAB2 and TAB3. Inhibition of nuclear factor κB (NFκB) signalling blocks the induction of autophagy by starvation and pharmacological stimuli [111]. Two binding partners of transforming growth factor β-activated kinase (TAK1), an upstream activator of the NFκB signalling cascade, are the coiled-coil proteins TAB2 and TAB3 (TAK1-binding proteins 2 and 3). In addition to their roles in NFκB signalling, TAB2 and TAB3 constitutively bind to the coiled-coil domain of Beclin-1; this binding is disrupted upon starvation, when TAB2 and TAB3 instead bind to TAK1 [112]. Knockdown of TAB2 or TAB3 leads to increased autophagy, suggesting that they inhibit the pro-autophagic activity of Beclin-1. Although TAB2 and TAB3 knockdown increases the level of intracellular PI[3]P, effects on the kinase activity of Vps34 complexes have not been measured. Similarly, if TAB2 and TAB3 bind to the coiled-coil domain of Beclin-1, this could disrupt Beclin-1 binding to Atg14 or UVRAG and might disrupt VPS34 complexes. The role of TAB2 and TAB3 in Vps34 complex assembly deserves further study.

WDR91/WDR81 (SORF-1/SORF-2). In a screen for suppressors of endocytic defects in Caenorhabditis elegans expressing a mutant HOPS complex member, two genes, SORF-1 and SORF-2, were identified as regulators of Vps34 in the early endosome [113]. The mammalian homologues, WDR91 and WDR81, bind to Beclin-1 in immunoprecipitation assays, and indirectly interact with Vps34. Their knockdown increases the specific activity of anti-Vps34 immunoprecipitates and leads to enlarged Rab5 endosomes, which is characteristic of disruptions in endosomal maturation [49]. Interestingly, SORF-1 and SORF-2 bind to PI[3]P, and to a lesser extent PI[4]P, in membrane lipid-binding assays. It is suggested that PI[3]P sensing by these proteins could provide feedback inhibition of PI[3]P production in the early endosome.

Regulation by ubiquitination, acetylation and SUMOylation

Several studies show that Lys63-linked ubiquitination of Beclin-1 enhances its pro-autophagic activity (Figure 7). In macrophages, induction of autophagy in response to interleukin (IL)-1, interferon γ (IFNγ) or activation of Toll-like receptors (TLRs) is linked to Lys63-linked ubiquitination of Beclin-1 at Lys117, mediated by the TRAF6 E3 ligase and reversed by the deubiquitinating enzyme A20 [114]. Cells expressing K117R Beclin-1 show a reduction in PI[3]P levels, suggesting an effect on the activity or targeting of Vps34. In response to starvation, Beclin-1 undergoes Lys63-linked ubiquitination at Lys437 [53]. This modification is mediated by Ambra1, which under the name CDAF3 has been identified as part of a family of substrate receptors that interact with the DDB1–Cullin 4 ubiquitin ligase complex [101]. Ambra1 regulates Beclin-1 ubiquitination by acting as a substrate adapter for the TRAF6 E3 ligase activity [102]. Ambra1 is also required for the ubiquitination of ULK1, which enhances ULK1 dimerization during the induction of autophagy [102]. Ambra1-mediated ubiquitination of Beclin-1 is antagonized by the Wiskott–Aldrich syndrome protein (WASP) family member WASH [53]. WASH binds to Beclin-1 in fed but not starved cells, inhibiting Beclin-1 binding to Ambra1 and Vps34. The authors propose a model in which starvation causes the release of WASH, leading to Ambra1-mediated Lys437 ubiquitination of Beclin-1 and enhanced Beclin-1 binding to Vps34. Ambra1-mediated ubiquitination of ULK1, and presumably Beclin-1, is inhibited under nutrient replete conditions by mTORC1-mediated phosphorylation of Ambra1 at Ser52 [102]. Taken together, these data suggest that ubiquitination of Beclin-1 at Lys117 and Lys437 enhances its functions in autophagy, but it is not clear whether this is due to an effect on Vps34 complex assembly, targeting, or the specific activity of Vps34 complexes.

Regulation of Vps34 Complex I by ubiquitination (U), ISGylation (I), acetylation (A) and SUMOylation (S)

Figure 7
Regulation of Vps34 Complex I by ubiquitination (U), ISGylation (I), acetylation (A) and SUMOylation (S)

Sites that stimulate Vps34 activity are in blue. Sites that inhibit Vps34 activity are in red.

Figure 7
Regulation of Vps34 Complex I by ubiquitination (U), ISGylation (I), acetylation (A) and SUMOylation (S)

Sites that stimulate Vps34 activity are in blue. Sites that inhibit Vps34 activity are in red.

In contrast, Beclin-1–Vps34 complexes are negatively regulated when modified by interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, in interferon-treated H4 cells [87] (Figure 7). ISGylation at Lys117, Lys263, Lys265 and Lys266 inhibits Lys63-linked ubiquitination of Beclin-1 at these sites. ISGylation is inhibited by ubiquitin-specific peptidase (USP)18/USP43, which are members of the ubiquitin protease family. Enhancement of Beclin-1 ISGylation by treatment of cells with interferon or knockdown of USP18 inhibits autophagy and EGFR degradation, apparently by reducing Beclin-1-associated Vps34 activity. Finally, Beclin-1 has recently been shown to undergo acetylation at Lys430 and Lys437 by the p300 acetyltransferase. The modification requires a priming phosphorylation at Thr406 by an unknown kinase, and subsequent casein kinase 1-mediated phosphorylation of Beclin-1 at Ser409; acetylation is reversed by Sirtuin 1 [115]. Beclin-1 acetylation enhances the recruitment of Rubicon, presumably inhibiting the activity of Vps34 Complex II. Consistent with this view, mutation of Lys430/Lys437 to arginine has no effect on autophagosomal formation, but enhances autophagosome–lysosome fusion and EGFR degradation.

Induction of autophagy by long-term (48 h) treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat leads an increase in acetylated Hsp70, whose binding to Vps34 and Beclin-1 recruits the SUMO E3 ligase KAP1 [47] (Figure 7). KAP1 SUMOylates Vps34 at Lys840, which increases Vps34 lipid kinase activity in vitro. Unfortunately, the connection between KAP1-mediated SUMOylation of Vps34 and the induction of autophagy was not directly tested, as the K840R mutant of Vps34 is catalytically inactive, and the effect of KAP1 knockdown on panobinostat-stimulated autophagy was not measured. The physiological role of Vps34 SUMOylation during stress-induced autophagy deserves further study.

As with phosphorylation, discussed above, the mechanism by which these post-translational modifications regulate the activity of Vps34 Complex I and II is not yet known. Ubiquitination of Beclin-1 at Lys117, in the BH3 domain, or at Lys437, in the BARA domain, enhance autophagic signalling [53,114]. ISGylation in the BH3 (Lys117) or coiled-coil domains (Lys263, Lys265 and Lys266) inhibits autophagy [87], as does acetylation at Lys430/437 in the BARA domain. The stimulatory SUMOylation of Vps34 at Lys840 is in the C-terminus of the kinase domain [47].

Regulation by site-specific targeting

In yeast, targeting of Vps34 to the pre-autophagosomal structure (PAS) requires Atg14 (although, surprisingly, targeting of Vps15 to the PAS is Atg14-independent) [116]. Similarly, in mammalian cells, the targeting of Vps34 to sites of autophagic initiation in the ER requires Atg14 [117]. Recruitment of Vps34 and Beclin-1 to Atg14 does not require an autophagy-specific locale, as ectopic targeting of Atg14 to the plasma membrane causes plasma membrane targeting of Vps34 and Beclin-1. Similar amounts of Atg14 are found in the ER in both fed and starved cells. However, starvation causes a change in Atg14 localization from a diffuse ER localization to punctae that partially co-localize with the PI[3]P effector DFCP1 and the autophagy proteins Atg16 and light chain 3 (LC3). The authors conclude that targeting of Vps34 Complex I to the ER occurs under basal conditions, and is not the switch that initiates the autophagic response. The events that mediate the condensation of Atg14 into ER-associated punctae, and the mechanism by which this increases production of PI[3]P, are not yet known. However, recent work suggests that, in yeast, Atg14 recruitment to the PAS requires its binding to the HORMA (HOP1, REV7 and MAD2) domain of the Atg1 scaffold Atg13 [118]. Mammalian Atg13 forms a complex with FIP200 [200 kDa FAK (focal adhesion kinase) family-interacting protein] and the ULK1 kinase, which are activated early in the formation of nascent autophagosomes [8,9]; it is not known whether Atg13–Atg14 interactions regulate Atg14 localization within the ER.

Several alternative models for the regulated production of PI[3]P at sites of autophagosomal initiation involve the sequestration of Vps34 complexes. In fed cells, Ambra1 binds to the dynein microtubule motor complex via dynein light chain 1 (DLC1/LC8) [104]. This binding is inhibited by an ULK1-dependent phosphorylation of Ambra1, which correlates with a loss of Ambra1–DLC1 co-immunoprecipitation and translocation of both Ambra1 and Beclin-1 to the ER. Consistent with a model in which DLC1 binding restrains the localization of Ambra1, an Ambra1 mutant that is defective for binding to DLC1 is constitutively localized to the ER. Furthermore, DLC1 knockdown enhances autophagy. A subsequent study suggested a different mechanism by which Beclin-1 in fed cells is sequestered to a microtubule compartment. In this study, sequestration is mediated by the simultaneous binding of the BH3-only pro-apoptotic protein Bim to Beclin-1 and DLC1/LC8 [119]. Starvation leads to the phosphorylation of Bim on a predicted JNK1 site, leading to its displacement from both DLC1/LC8 and Beclin-1. Starvation or Bim knockdown causes a decrease in the co-localization of Beclin-1 with microtubules, which correlates with enhanced autophagy. Unfortunately, neither of these studies examined other members of the Vps34 complexes, so it is not clear whether the sequestration of Beclin-1 to the microtubule cytoskeleton actually regulates the localization of Vps34 Complex I or II. The sequestration of Vps34 complexes by microtubule-binding proteins remains an intriguing hypothesis.

Recruitment of Vps34 to sites other than the ER may regulate its activity. The plasma membrane has been proposed as a site of autophagy induction [120]. Knockdown of connexins, which are subunits of gap junctions, increases autophagy, suggesting an inhibitory role [121]. In fed cells, plasma membrane connexins bind to Atg16 as well as Vps15, Vps34 and Beclin-1. The authors propose that connexins maintain the Vps34 complex in an inactive state in fed cells, but facilitate activation upon starvation. In contrast with the connexins, a positive effect on Vps34 activity in neurons is caused by the cellular prion protein (PRNP), which binds to the BH3 domain of Beclin-1 and recruits Beclin-1 to lipid rafts in response to amyloid β1–42 [122]. Finally, recruitment of Vps34 to mitochondria has been implicated in the induction of mitophagy. In cells treated with the mitochondrial poison carbonyl cyanide m-chlorophenylhydrazone (CCCP), the AMPKβ subunit becomes N-terminally myristoylated and localizes to mitochondria, along with the Atg12–Atg5–Atg16 complex and Vps34–Vps15 [123]. Recruitment of these proteins, as well as induction of mitophagy, is blocked in AMPKβ-knockout cells.

Recent studies show that the small GTPase RalB stimulates autophagy by regulating the binding of Vps34 Complex I to the exocyst complex [124]. The exocyst is a complex of eight distinct polypeptides that regulates the tethering of secretory vesicles to the plasma membrane prior to their fusion [125]. Several of the exocyst subunits, including Exo84 and Sec5, bind directly to RalB in its GTP-bound state. In addition to the fully assembled exocyst octamer, subcomplexes containing either Sec5 or Exo84 have been described in yeast [126]. In starved cells, RalB is activated, and Vps34 Complex I exchanges from Sec5-containing complexes to Exo84-containing complexes. The mechanism for the transfer is not clear, since (a) inhibition of RalB action by expression of a RalB effector domain-binding protein inhibits Vps34 Complex I binding to both Sec5 and Exo84 [124], and (b) activation of RalB has been shown to stimulate the condensation of Sec5- and Exo84-containing subcomplexes [126]. An additional level of regulation is provided by the serine/threonine kinase 38 (STK38)/nuclear Dbf2-related kinase 1 (NDR1) protein kinase, which promotes binding of RalB to Beclin-1 and Exo84 [127]. STK38 colocalizes with Vps34 Complex I in starved cells and co-immunoprecipitates with Beclin-1, and its kinase activity (as judged by autophosphorylation) is increased by starvation. The substrates of STK38 that regulate targeting of Vps34 Complex I are not known.

Regulation by degradation

Members of Vps34 Complex I and II are regulated by a number of selective degradation systems. Several adapters and E3 subunits of ubiquitin ligases have been linked to proteasomal down-regulation of components of the complexes. Under basal conditions, Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4)-mediated Lys11-linked ubiquitination of Beclin-1 regulates its expression levels [128]. Nedd4 competes with Vps34 for Beclin-1 binding, such that Vps34 knockdown leads to increased Beclin-1 ubiquitination and degradation. Similarly, basal levels of Beclin-1 are regulated by the deubiquitinases USP10 and USP13, whose inhibition by the drug Spautin-1 causes the proteosomal degradation of Beclin-1 [129]. As noted above, Ambra1 acts as an adapter for E3 ubiquitin ligases to promote the Lys63-linked ubiquitination of Beclin-1 in starved cells [53]. Ambra1 itself undergoes Lys48-linked ubiquitination at Lys45, which is mediated by the E3 ligase RNF2 and leads to Ambra1 degradation [130].

Members of Vps34 Complex I undergo enhanced degradation in response to a number of stimuli. During prolonged starvation, the Cul3-KLHL20 ubiquitin ligase mediates the degradation of Beclin-1, Vps34 and ULK1 by direct binding and ubiquitination [131]. Atg14 also undergoes Cul3-KLH20-dependent degradation, although Atg14 is not directly ubiquitinated by Cul3-KLH20. Interestingly, Atg14, but not other members of the Vps34 Complex I, is degraded by zinc finger- and BTB domain-containing 16 (ZBTB16)-Cullin3–Roc1-mediated ubiquitination in cells stimulated with serum or a variety of GPCR ligands. This degradation is inhibited upon serum withdrawal, when activation of GSK3 leads to the phosphorylation, auto-ubiquitination and degradation of ZBTB16 [132]. In response to DNA damage, the p53-mediated transcriptional induction of FBX120 [a component of the Skp1-Cul1-F-box (SCF) complex] leads to the ubiquitination and degradation of Vps34 [133]. Recognition of Vps34 by FBX120 requires the phosphorylation of Vps34 at Thr159 by Cdk1, which is activated during mitotic arrest caused by DNA damage. Given that Thr159 phosphorylation also disrupts Vps34 binding to Beclin-1 [70], dissociation of the Vps34 complex may also facilitate Vps34 degradation. During apoptosis induced by expression of the pro-apoptotic protein BAX, the caspase-mediated cleavage of Beclin-1 at Asp149 disrupts its binding to Vps34–Vps15 [134].

NEW FUNCTIONS FOR Vps34

The critical roles of Vps34 in both endocytic trafficking and macroautophagy mean that it is involved in a wide range of cellular and organismic functions. For example, Vps34 knockdown or conditional knockouts have suggested important roles in heart, skeletal muscle, kidney, the immune system and the brain (Table 1). In most cases, the phenotypes are a consequence of altered endocytic trafficking, macroautophagy or both. Vps34 signalling in the endocytic and autophagosomal systems has been discussed extensively in other reviews, as have the roles of these systems in maintaining cellular and organismic health (see, for example, [135140]). An exciting new development has been the design of specific inhibitors of the Vps34 lipid kinase [141144], which hopefully will supplant the use of the modestly selective inhibitor 3-methyladenine and identify new functions for Vps34 (Table 2). This section will focus on emerging roles for Vps34 that are not a direct consequence of its roles in trafficking and autophagy.

Table 1
Knockout (KO) and knockdown (KDn) studies in mice and flies reveal organismic roles for Vps34
Organ/cell type Experiment Phenotype References 
Heart Vps34 KO Cardiomegaly, decreased contractility [146
 Rubicon KO Resistance to lipopolysaccharide (LPS)-induced reductions in cardiac output [205
 Vps34 inhibition Reduced progression to maladaptive cardiac hypertrophy [87
Skeletal muscle Vps15 KO Autophagic vacuolar myopathy [206
 Vps34 KDn Impaired myoblast differentiation [156
 Vps34 KO Murine muscular dystrophy [207
Kidney Vps34 KO (podocytes) Proteinuria, glomerular scarring [208
Nervous system Vps34 KO (hippocampus, pyramidal neurons) Loss of dendritic spines, neurodegeneration [209
 Vps34 KO (sensory neurons) Vacuolization in large diameter sensory neurons, increased lysosomes in small diameter neurons, neurodegeneration [210
 Vps34 KDn (cortical neurons) Increased amyloidogenic processing of amyloid precursor protein (APP), reduced sorting of APP to MVBs [211
 UVRAG or Vps15 mutation Loss of axon pruning (Drosophila[212
Immune system Vps34 KO (T-cells) Reduced T-cell number, increased cell death and reduced IL-7 receptor expression [213
 Vps34 KO (T-cells) Normal development but reduced T-cell survival, increased mitochondrial mass and ROS [214
 Vps34 KO (T-cells) Normal development but reduced natural killer (NK) cells, inflammatory wasting syndrome with reduced CD4+Foxp3+ regulatory T-cells (Tregs) [215
Salivary gland Vps15 KO Decreased salivary gland protein secretion (Drosophila[216
Organ/cell type Experiment Phenotype References 
Heart Vps34 KO Cardiomegaly, decreased contractility [146
 Rubicon KO Resistance to lipopolysaccharide (LPS)-induced reductions in cardiac output [205
 Vps34 inhibition Reduced progression to maladaptive cardiac hypertrophy [87
Skeletal muscle Vps15 KO Autophagic vacuolar myopathy [206
 Vps34 KDn Impaired myoblast differentiation [156
 Vps34 KO Murine muscular dystrophy [207
Kidney Vps34 KO (podocytes) Proteinuria, glomerular scarring [208
Nervous system Vps34 KO (hippocampus, pyramidal neurons) Loss of dendritic spines, neurodegeneration [209
 Vps34 KO (sensory neurons) Vacuolization in large diameter sensory neurons, increased lysosomes in small diameter neurons, neurodegeneration [210
 Vps34 KDn (cortical neurons) Increased amyloidogenic processing of amyloid precursor protein (APP), reduced sorting of APP to MVBs [211
 UVRAG or Vps15 mutation Loss of axon pruning (Drosophila[212
Immune system Vps34 KO (T-cells) Reduced T-cell number, increased cell death and reduced IL-7 receptor expression [213
 Vps34 KO (T-cells) Normal development but reduced T-cell survival, increased mitochondrial mass and ROS [214
 Vps34 KO (T-cells) Normal development but reduced natural killer (NK) cells, inflammatory wasting syndrome with reduced CD4+Foxp3+ regulatory T-cells (Tregs) [215
Salivary gland Vps15 KO Decreased salivary gland protein secretion (Drosophila[216
Table 2
Comparison of recently described Vps34 inhibitors to 3-methyladenine and pan-PI 3K inhibitors

ND, not determined.

 IC50in vitro (μM) 
Drug Vps34 p110α p110β p110δ p110γ C2α C2β C2γ Reference 
Vps34-IN-1 0.025 >10 >10 >10 >10 >10 >10 >10 [141
PIK-III 0.018 3.96 >9 1.2 3.04 ND ND ND [143
SAR405 0.0012 >10 >10 >10 >10 >10 >10 >10 [144
Compound 31 0.002 2.7 4.5 2.5 >10 >10 >10 >10 [142
3-Methyladenine 0.036 0.039 0.590 0.120 0.004 ND ND ND [31
PI-103 0.488 0.026 0.045 0.048 0.560 >10 0.490 0.250 [144
GDC-0941 >10 0.003 0.033 0.003 0.075 ND 0.670 ND [217
 IC50in vitro (μM) 
Drug Vps34 p110α p110β p110δ p110γ C2α C2β C2γ Reference 
Vps34-IN-1 0.025 >10 >10 >10 >10 >10 >10 >10 [141
PIK-III 0.018 3.96 >9 1.2 3.04 ND ND ND [143
SAR405 0.0012 >10 >10 >10 >10 >10 >10 >10 [144
Compound 31 0.002 2.7 4.5 2.5 >10 >10 >10 >10 [142
3-Methyladenine 0.036 0.039 0.590 0.120 0.004 ND ND ND [31
PI-103 0.488 0.026 0.045 0.048 0.560 >10 0.490 0.250 [144
GDC-0941 >10 0.003 0.033 0.003 0.075 ND 0.670 ND [217

mTORC1 signalling and phospholipase D1

Insulin stimulation of S6 kinase 1 (S6K1) is blunted by siRNA knockdown of Vps34, microinjection of inhibitory anti-Vps34 antibodies, or overexpression of FYVE domains to sequester the Vps34 product PI[3]P [61]. These data led to the hypothesis that Vps34 is a positive regulator of mTORC1 signalling. An initial attempt to test the link between Vps34 and TORC1 in vivo found that in the fat body of Drosophila embryos, knockout or expression of kinase-dead Vps34 has no effect on basal levels of pS6K1, whereas autophagy and endocytic uptake of transferrin are impaired [145]. This negative result may be explained by the finding that amino acid-stimulated but not basal S6K1 phosphorylation is inhibited by Vps34 knockout in liver [146]. Similarly, in skeletal muscle, high-resistance contractions lead to parallel increases in intramuscular leucine, Vps34 activity and S6K1 phosphorylation [147]. Genetic disruption of the PI[3]P phosphatase MTM1 in skeletal muscle leads to increased mTORC1 signalling [148]. Formation of phosphatidylinositol 3,5-bisphosphate (PI[3,5]P2) from PI[3]P by the PI(3)P 5-kinase PIKFYVE has been implicated in mTORC1 activation in adipocytes, although this requires the Class II PI 3K PIK3C2α, and not Vps34 [149].

A potential mechanism for Vps34 as an upstream regulator of mTORC1 regulation involves the production of phosphatidic acid (PA) by PLD (phospholipase D) (Figure 8A). It has previously been shown that phosphatidic acid activates mTORC1 by binding to the mTOR FRB [FKBP12 (FK506-binding protein 12) rapamycin-binding] domain, which is the binding site for the mTORC1 inhibitor rapamycin [150,151]. More recently, it was found that PLD1, but not PLD2, is activated by amino acids and glucose in a Vps34-dependent manner [152,153]. PLD1 contains a PX domain that mediates the Vps34-dependent translocation of PLD1 to the lysosome in response to amino acids, presumably by binding to PI[3]P. Importantly, this translocation is independent of the Rag GTPases, which mediate the amino acid-stimulated translocation of mTORC1 to the lysosome [152]. The translocation of PLD1 to the lysosome is critical for its function in the regulation of mTORC1, as exogenous PI[3]P activates PLD1 but does not cause its translocation to lysosomes and does not activate mTORC1 signalling. Thus, the Vps34/PLD1/PA pathway operates in parallel with the targeting of mTORC1 to the lysosomal membrane by the Rag GTPases [154], and provides a second, Vps34-dependent, mechanism for nutrient regulation of mTORC1. Interestingly, PA can also activate mTORC2, which is not thought to be nutrient-sensitive [155]. Although knockdown of Vps34 does not affect the phosphorylation of Akt at the mTORC2 phosphorylation site, Ser473 [61], the potential role of Vps34 in mTORC2 signalling warrants further study.

Novel functions for Vps34

Figure 8
Novel functions for Vps34

(A) Vps34-dependent production of PI[3]P recruits PLD1 to the lysosome, where it produces PA to activate mTORC1. mTORC1 is targeted to the lysosome by its binding to Rag GTPases. It is not known whether this function of Vps34 requires Vps34 Complex I or II. (B) Vps34 Complex II is targeted to the midbody by binding to the GEF Ric-8A and to activated Gαi. Vps34 produces PI[3]P in endosomes that cluster at the midbody during abscission. The centrosomal protein FYVE-CENT moves to the midbody along microtubules by binding to TTC19 and the associated microtubule motor KIF13A. The localization of FYVE-CENT to the midbody is stabilized by binding to PI[3]P. TTC19 in turn recruits members of the ESCRT complex, which drives abscission. (C) During phagocytosis mediated by receptors for opsonized particles, Vps34 Complex II is recruited to the newly internalized phagosome by binding to Rab5. Production of PI[3]P displaces the PI[3,4,5]P3 phosphatase Inpp5B, which allows the accumulation of PI[3,4,5]P3 and phagosomal rocketing on actin comet tails (in red). Vps34 Complex II also causes the recruitment of the phagosomal NADPH oxidase complex, through the binding of NADPH oxidase components to both PI[3]P and to Rubicon. During phagocytosis of bacteria by the SLAM receptor, Vps34 Complex II is directly recruited to the phagosome by binding to SLAM.

Figure 8
Novel functions for Vps34

(A) Vps34-dependent production of PI[3]P recruits PLD1 to the lysosome, where it produces PA to activate mTORC1. mTORC1 is targeted to the lysosome by its binding to Rag GTPases. It is not known whether this function of Vps34 requires Vps34 Complex I or II. (B) Vps34 Complex II is targeted to the midbody by binding to the GEF Ric-8A and to activated Gαi. Vps34 produces PI[3]P in endosomes that cluster at the midbody during abscission. The centrosomal protein FYVE-CENT moves to the midbody along microtubules by binding to TTC19 and the associated microtubule motor KIF13A. The localization of FYVE-CENT to the midbody is stabilized by binding to PI[3]P. TTC19 in turn recruits members of the ESCRT complex, which drives abscission. (C) During phagocytosis mediated by receptors for opsonized particles, Vps34 Complex II is recruited to the newly internalized phagosome by binding to Rab5. Production of PI[3]P displaces the PI[3,4,5]P3 phosphatase Inpp5B, which allows the accumulation of PI[3,4,5]P3 and phagosomal rocketing on actin comet tails (in red). Vps34 Complex II also causes the recruitment of the phagosomal NADPH oxidase complex, through the binding of NADPH oxidase components to both PI[3]P and to Rubicon. During phagocytosis of bacteria by the SLAM receptor, Vps34 Complex II is directly recruited to the phagosome by binding to SLAM.

The amino acid- and Vps34-dependent activation of PLD1 also plays a role in myogenic differentiation, through up-regulation of insulin-like growth factor (IGF)-II production [156]. In this case, PLD1 acts in the opposite way from mTORC1 activation, which inhibits differentiation in C2C12 cells. In addition, several groups have identified PLD1 as a positive regulator of starvation-induced macroautophagy [157]. Given that PLD1 is activated by PI[3]P, it is possible that its regulation during autophagy is dependent on Vps34 Complex I or II, which are activated by starvation, as opposed to the larger population of Vps34–Vps15, which is inhibited by nutrient deprivation. This activation might be specific for growing autophagosomes, as PLD1 does partially co-localize with LC3 in starved cells [157].

Cytokinesis

The components of Vps34 Complex II, including Vps34, Vps15, Beclin-1, UVRAG and BIF-1 but not Atg14, were identified as novel regulators of cytokinesis [22] (Figure 8B). An siRNA screen of FYVE domain-containing proteins identified FYVE-CENT (FYVE domain-containing centrosomal protein) as a positive regulator of cytokinesis. FYVE-CENT in turn interacts with tetratricopeptide repeat protein 19 (TTC19) and the plus-ended microtubule motor protein KIF13A, a member of the kinesin family [158]. Knockdown of Vps34 or KIF13A reduces the midbody localization of FYVE-CENT and TTC19, whereas Vps34 knockdown does not affect midbody localization of KIF13A. These data suggest that the microtubule-dependent recruitment of FYVE-CENT and TTC19 to the midbody is stabilized by the production of PI[3]P by Vps34 Complex II. The association of Vps34 with microtubule motors during cytokinesis has some parallels to the role of Vps34 in the microtubule-dependent movement of Rab5-positive endosomes [159].

TTC19 binds to the ESCRT III component charged multivesicular body protein 4B (CHMP4B); the ESCRT system has previously been implicated in midbody abscission [160]. ESCRT-1 proteins such as Tgs101 and Vps28 are also implicated in abscission [160]. Although a role for the PI[3]P-binding ESCRT-0 proteins in cytokinesis has not been described, it is possible that Vps34-dependent production of PI[3]P might influence recruitment of the ESCRT machinery by interacting with the ESCRT-II component EAP45/Vps36, which binds to PI[3]P through its GLUE domain [161]. Finally, Vps34 is likely to play a role in the disposal of the midbody after abscission, which occurs via selective macroautophagy mediated by the autophagic receptor NRB1 [162]. Vps34 itself is recruited to the midbody via interactions with Gαi and the guanine-nucleotide-exchange factor (GEF) Ric-8A, which localize to the midbody and bind to Vps34 and Vps15 [163]. Binding of the Gα protein Gpa to Vps15 has been previously described in yeast [12].

The role of Vps34 in cytokinesis may be a very ancient phenomenon. Vps15 and Vps34 have both been implicated in pollen development in Arabidopsis [164,165]. The latter study suggests a defect in mitosis, which may be related to the functions of Vps34 at the midbody of dividing mammalian cells [22].

Phagocytosis in macrophages and lymphocytes

The clearance of bacteria and other foreign bodies from the body is mediated by the phagocytic cells of the immune system. Receptors on the surface of phagocytic cells can either bind directly to the foreign body (via pattern-recognition receptors and receptors for apoptotic corpses), or to foreign bodies coated with antibody (via Fcγ receptors) and complement (via CR3, CR4 and integrins) [166]. This stimulates the actin-mediated protrusion of the phagocytic cup, which eventually engulfs the phagocytic cargo and seals to form a phagosome. Studies with fluorescent probes for distinct phosphoinositides have demonstrated that PI[3]P, produced by Vps34, accumulates shortly after sealing [167,168]. Vps34 is recruited by Rab5, which also begins to accumulate in the phagosome just prior to sealing [169,170] (although a genetic study in C. elegans suggests instead that Vps34 is recruited to the phagosome by dynamin and functions upstream of Rab5 recruitment [171]).

Although some of the functions of Vps34 in the autophagosome are analogous to its functions in Rab5-positive early endosomes, it plays several additional roles. During CR3-mediated phagocytosis, an initial phosphatidylinositol 3,4,5-trisphosphate (PI[3,4,5]P3)-dependent accumulation of filamentous actin (F-actin) drives phagocytic closure. PI[3,4,5]P3 levels fall after sealing, due in part to the PI[3,4,5]P3 5-phosphatase Inpp5B. Vps34-mediated PI[3]P production in the sealed phagosome leads to the displacement of Inpp5B, allowing Class I PI3K to produce a second wave of PI[3,4,5]P3 that enhances phagosome maturation by driving the formation of actin comet tails [172] (Figure 8C). In macrophages and neutrophils, phagocytosis mediated by Fc and TLR receptors lead to an activation of the NADPH oxidase system and reactive oxygen species (ROS) production. Activation of the NADPH oxidase by bacterial chemotaxis in neutrophils and macrophages requires Vps34 [173], which acts by recruiting the p40phox subunit to PI[3]P [174]. Rubicon also contributes to activation of the oxidase complex, by binding to the p22phox subunit, and by recruiting the Vps34 Complex II to phagosomes induced by TLR ligands [175,176]. This example of Rubicon as a positive effector of a Vps34-dependent process is unusual, given the inhibitory effect of Rubicon on Vps34 in other contexts. Finally, Vps34 and Rubicon are required for the recruitment of some components of the macroautophagic pathway, including LC3, to phagosomes formed during engulfment of dead [177] or live [178] cells and bacteria [176].

Of note, the production of PI[3]P in the autophagosome may depend on other lipid kinases. Studies in C. elegans suggest that Class II PI3K also produces phagosomal PI[3]P; the Class II PI3K PIKI-1 and the Class III PI3K act sequentially to produce to waves of PI[3]P [179]. Recently the mammalian Class II PI3K PI3KC2γ was demonstrated to be a Rab5 effector that is recruited to early endosomes [180]; the potential role of the mammalian Class II PI3Ks in phagocytosis has not been addressed. The sequential dephosphorylation of PI[3,4,5]P3 to PI[3]P has been described in endosomes [181] and may also contribute to PI[3]P production during phagocytosis.

In addition to its interactions with Rab5, Vps34–Vps15 complexes can be recruited to phagosomes by binding to the signalling lymphocyte-activation molecule (SLAM) family of myeloid cell receptors [182] (Figure 8C). SLAM binds to bacterial surface proteins and enters the phagosome along with the internalized bacterium. SLAM immunoprecipitates contain the components of Vps34 Complex II, but not Rubicon or Atg14 [93]. Interestingly, antibody-mediated ligation of SLAMF1 activates JNK1/2, leading to phosphorylation of Bcl-2, dissociation of Bcl-2 from Beclin-1, and increased autophagy [91]. These data suggest that SLAM might both serve as a scaffold for recruitment of Vps34 Complex II during phagocytosis as well as a positive regulator of autophagy. In addition to its binding to bacteria, SLAM is a receptor for measles virus [183]. A second receptor for measles (as well as other pathogens), CD46, also binds to Vps34–Beclin-1 complexes via its associated scaffold protein Golgi-associated PDZ- and coiled-coil motif-containing (GOPC) [184]. Like SLAM, CD46 engagement activates autophagy.

The secretory pathway

Despite its discovery in the context of lysosomal enzyme sorting [1,2], Vps34 has not been thought to be a core component of the canonical ER/Golgi secretory pathway. However, several recent studies have identified roles for Vps34 in biosynthetic and secretory trafficking. In yeast, Vps34 is required for the association of two proteins involved in Golgi function: Rsp5, a ubiquitin ligase, and Sec7, the Arf GEF [185]. In addition, Vps34 is required for the compartment for unconventional proteins secretion (CUPS), a specialized compartment that forms in response to glucose starvation and mediates the secretion of proteins that do not contain signal sequences [186]. Formation of CUPS also requires Sec7, suggesting a possible relationship between these two Vps34-dependent pathways.

Lysosomes

During prolonged starvation, activation of the autophagy pathway leads to be formation of multiple autophagolysosomes and depletion of lysosomes. At a certain point, lysosomes are reformed through a process of mTORC1-dependent tubulation called autophagosome–lysosome reformation (ALR) [187]. mTORC1 is initially inhibited during starvation but then becomes reactivated under the influence of intracellular amino acids provided by the autophagy pathway. Recent studies have implicated Vps34 as an important regulator of this process [95]. When mTORC1 is activated by the rebound in amino acids, it phosphorylates UVRAG at Ser550 and Ser571 (Figure 4), leading to activation of Vps34 Complex II. Expression of UVRAG mutant containing alanine at these sites, or treatment of cells with the Vps34-specific inhibitor Vps34-IN1, causes an increase in the number and length of lysosomal tubules. Although trafficking of both endocytic and autophagic cargo to the lysosome is not affected by expression of the serine-to-alanine mutant, survival after long-term starvation or treatment with L-leucyl-L-leucine methyl ester, a lysosomotropic agent, is markedly reduced. These studies suggest a novel role for Vps34 Complex II in the regulation lysosomal morphology and responses to prolonged starvation.

In neurons, transport of lysosomes, as well as synaptic vesicles and other organelles, occurs along microtubules and is mediated by microtubule motors and their adapters. The dynactin complex, an adapter for dynein-mediated retrograde trafficking, is recruited to vesicles through the Ankyrin B (AnkB) adapter [188]. Ank B, in turn, binds to PI[3]P produced by Vps34. Thus, axonal transport is disrupted in hippocampal neurons from AnkB−/− mice or Vps34fl/fl mice transfected with Cre.

RTK signalling

Given its role in endocytic trafficking and MVB formation, it is not surprising that disruption of Vps34 activity or expression has effects on signalling by RTKs. However, several recent studies suggest new sites of Vps34 action during RTK signalling. APPL is an adapter protein containing BAR, PH and phosphotyrosine-binding (PTB) domains that associates with Rab5 endosomes that do not contain the early endosomal marker EEA1 [189]. The APPL compartment is thought to regulate signalling by Akt, which binds to APPL1 [190]. There has been disagreement as to whether the APPL compartment is an intermediate step in the transition to an EEA1-positive endosome, as opposed to a distinct stable Rab5 compartment [191,192]. However, knockdown of Beclin-1 decreases PI[3]P production in APPL endosomes and increases the duration of co-localization of labelled epidermal growth factor (EGF) with this compartment [193]. Interestingly, Beclin-1 knockdown also leads to sustained activation of Akt and extracellular-signal-regulated kinase (ERK) activation in response to IGF-I and EGF, and increases the invasion of breast cancer cells. The authors suggest that enhancement of growth factor signalling in the APPL compartment might provide an alternative mechanism for tumour induction seen in Beclin-1 heterozygous knockout mice [194].

Signalling by another RTK, the insulin receptor, is enhanced in the livers of Vps15 conditional knockout mice [195]. Vps34 Complex II can be co-immunoprecipitated with insulin receptors in hepatocytes, and Rubicon binding to Vps34 Complex II is decreased upon insulin stimulation. This suggests the presence of a negative feedback loop, in which activation of Complex II-dependent insulin receptor trafficking down-regulates receptor signalling. In contrast, Vps34 promotes proliferation in a different EGFR signalling pathway: Vps34 knockdown blocks the nuclear translocation of EGFR in non-small-lung-cancer cells expressing the activated L858R mutation of the EGFR [196]. Nuclear EGFR acts as a transcriptional repressor of the p14ARF tumour suppressor, and Vps34 knockdown increases p14ARF expression.

Downstream from RTKs and GPCRs, signalling by the Class I PI3Ks works in part through the activation of Akt. Interestingly, breast cancer cells expressing constitutively active alleles of PIK3CA show low levels of activated Akt. Instead, Akt signalling is supplanted by SGK3 (serum- and glucocorticoid-stimulated kinase 3) [197]. Also known as CISK (cytokine-independent survival kinase), SGK3 has a PX domain that binds to the Vps34 product PI[3]P [198]. Interestingly, treatment of cells with the Vps34-specific inhibitor Vps34-IN1 leads to a 50–60% decrease in SGK3 activation; treatment with a combination of both Class I PI3K and Vps34-specific inhibitors reduces SGK3 activity by 80–90% [141]. Thus, activation of SGK3 seems to involve both the direct production of PI[3]P by Vps34, as well as the sequential dephosphorylation of PI[3,4,5]P3 by phosphoinositide 4- and 5-phosphatases. These data suggest a novel and interesting intersection in signalling by Vps34 and Class I PI3Ks.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN)

An immunofluorescence analysis of the PTEN tumour suppressor, which antagonizes Class I PI3K signalling through its PI[3,4,5]P3 3-phosphatase activity, shows localization to cytosolic vesicles bound to microtubules [199]. This localization is governed by the PTEN C2 domain, which binds to PI[3]P. Knockdown of Vps34, or mutation of the C2 domain, abolishes targeting of PTEN and causes an increase in Akt activation, consistent with an increase in PI[3,4,5]P3 levels in the cells. Strikingly, fusion of a FYVE domain to the C2 domain mutant of PTEN restores targeting and reduced Akt activation.

These data suggest a role for Vps34 as a positive regulator of PTEN function, and hence a negative regulator of Class I PI3K signalling. However, a role for Vps34 in Class I PI3K signalling has not been previously observed. Vps34 knockdown [61] or inhibition [141,143,200] has no effect on insulin-stimulated Akt activity. The authors in the PTEN localization study do not test whether Vps34 knockdown causes an up-regulation of Akt activity; future work will be needed to better define potential cross-talk between Vps34 and PTEN in the regulation of Class I PI3K signalling.

Vps34 in lower organisms

Many of the components of the Vps34 signalling system are conserved from yeast through mammals, and Vps34 is important for virulence in some pathogens. In some cases, for example in Cryptococcus neoformans, the loss of virulence in Vps34-null strains is mimicked by deletion of Atg8, suggesting that it is secondary to a disruption in autophagy [201]. However, in Candida albicans, Vps34 may play a more complex role, as the loss of virulence in Vps34-null strains is linked to its binding to Ade5,7p, an enzyme involved in purine nucleotide biosynthesis [202]. Strains lacking Ade5,7p are avirulent and phenocopy the loss of Vps34 in some, but not all aspects, for example sensitivity to copper and silver but not other divalent cations that inhibit the growth of Vps34-null strains. In Salmonella-infected cells, host cell Vps34 is required for bacterial invasion [203]. The virulence factor SopB, a phosphoinositide phosphatase, is required for the recruitment of Rab5 to the outer surface of internalized Salmonella-containing vacuoles. This in turn recruits Vps34–Vps15 to produce PI[3]P and drive maturation of the vacuole.

CONCLUSION

The wealth of new information about Vps34 and its binding partners over the Past few years has greatly expanded our understanding of this evolutionarily ancient lipid kinase. As our knowledge of the different Vps34 complexes and their distinct regulation and targeting has increased, new questions have emerged. First, we need an integrated description of whether the complexes and the accessory proteins overlap or lie in distinct pools. Vps34 Complex I binds to Ambra1, NRBF2, PAQR3, RINT1 and RACK1. Similarly, Complex II binds to Rubicon, Bif-1 and probably WDR91/WDR81. Are all of these proteins engaged simultaneously with the same complexes? What are the stoichiometries? Do any complete with each other, or synergize? How do the auxiliary proteins affect regulation of Complex I and II by phosphorylation and other post-translational modifications?

Secondly, the question of whether Vps34 complex assembly is regulated in response to an acute autophagic stimuli remains controversial. As discussed above, a widely cited model in which the availability of Beclin-1 limits autophagy is problematic, and the mechanism by which by Bcl-2 binding to Beclin-1 inhibits autophagy remains unclear. Studies on the assembly/disassembly of Vps34 complexes in vivo, perhaps using methods such as fluorescence fluctuation spectroscopy [204] to monitor changes in assembly dynamics in live cells, will be important. Also, although we now understand the structural organization of Complex I and II, the structure of the Beclin-1–Vps34–Vps15 complex is unknown; what, if anything, pairs with the Beclin-1 coiled coil in the absence of Atg14 or UVRAG?

Thirdly, the identification of so many sites of post-translational modification in Vps34 Complex I and II opens up new mechanistic questions. Structural and biochemical studies to determine how these modifications affect activity and membrane binding are a clear priority for future work.

Fourthly, which biological activities of Vps34 are mediated by which complexes? Although there are clear roles for Complex I and II in autophagy and trafficking respectively, some of Vps34-dependent events (PLD1/mTORC1 signalling) have not yet been assigned to either complex. In addition, although endocytic functions of Vps34 are likely to involve Complex II, the role of Complex II in Rab5-mediated Vps34 signalling during early endosomal fusion and microtubule-mediated movement has not been clearly established. Are there specific functions of Beclin-1–Vps34–Vps15 and Vps34–Vps15 complexes? And finally, are there functions for Vps34 that we do not yet know about? The new Vps34-specific inhibitors should provide important new information in this area.

With so much new information, as well as the availability at long last of selective Vps34 inhibitors, the next few years of Vps34 science should bring many new surprises.

I thank Dr Daniel Klionsky, Univ. Michigan for helpful discussions, and Dr James H. Hurley, University of California, Berkley, Berkley, CA, U.S.A., for helpful discussions and for providing data files for the structure of Vps34 Complex I. I also thank Dr Anne R. Bresnick, Albert Einstein College of Medicine, for insightful comments on the paper.

FUNDING

This work was supported by the National Institutes of Health [grant numbers AG039632 and GM112524].

Abbreviations

     
  • AMPK

    AMP-dependent protein kinase

  •  
  • AnkB

    Anykyrin B

  •  
  • BARA

    β–α repeated, autophagy

  •  
  • BATS

    Barkor/Atg14(L) autophagosome-targeting sequence

  •  
  • BH3

    Bcl-2 homology 3

  •  
  • Cdk

    cyclin-dependent kinase

  •  
  • CUPS

    compartment for unconventional proteins secretion

  •  
  • DAPK

    death-associated protein kinase

  •  
  • DFCP1

    double-FYVE-domain-containing protein 1

  •  
  • DLC1

    dynein light chain 1

  •  
  • EAP45

    ELL-associated protein of 45 kDa

  •  
  • ECD

    evolutionarily conserved domain

  •  
  • EEA1

    early endosomal antigen 1

  •  
  • EGF

    epidermal growth factor

  •  
  • EGFR

    EGF receptor

  •  
  • ER

    endoplasmic reticulum

  •  
  • ESCRT

    endosomal sorting complex required for transport

  •  
  • FYVE-CENT

    FYVE domain-containing centrosomal protein

  •  
  • GEF

    guanine-nucleotide-exchange factor

  •  
  • GLUE

    GRAM-like, ubiquitin binding in EAP45

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • HEK

    human embryonic kidney

  •  
  • HOPS

    homotypic fusion and vacuole protein sorting

  •  
  • Hsp70

    heat-shock protein 70

  •  
  • IGF

    insulin-like growth factor

  •  
  • IL

    interleukin

  •  
  • ILV

    intraluminal vesicle

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LC3

    light chain 3

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MAPKAPK

    MAPK-activated protein kinase

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • MIT

    microtubule and trafficking

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • mTORC1

    mTOR complex 1

  •  
  • MVB

    multivesicular body

  •  
  • NAG

    neuroblastoma amplified gene

  •  
  • Nedd4

    neural-precursor-cell-expressed developmentally down-regulated 4

  •  
  • NFκB

    nuclear factor κB

  •  
  • NRBF2

    nuclear receptor binding factor 2

  •  
  • PA

    phosphatidic acid

  •  
  • PAQR3

    progestin and adipoQ receptor family member III

  •  
  • PAS

    pre-autophagosomal structure

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PI[3]P

    phosphatidylinositol 3-phosphate

  •  
  • PI[35]P2

    phosphatidylinositol 3,5-bisphosphate

  •  
  • PI[3

    4,5]P3, phosphatidylinositol 3,4,5-trisphosphate

  •  
  • PKD

    protein kinase D

  •  
  • PLD

    phospholipase D

  •  
  • PTEN

    phosphatase and tensin homologue deleted on chromosome 10

  •  
  • PX

    Phox homology

  •  
  • RACK1

    receptor for activated C kinase 1

  •  
  • RINT1

    Rad50 interactor 1

  •  
  • ROS

    reactive oxygen species

  •  
  • RTK

    receptor tyrosine kinase

  •  
  • Rubicon

    RUN domain protein Beclin-1-interacting and cysteine-rich containing

  •  
  • S6K1

    S6 kinase 1

  •  
  • SGK3

    serum- and glucocorticoid-stimulated kinase 3

  •  
  • SLAM

    signalling lymphocyte-activation molecule

  •  
  • SNARE

    soluble N-ethylmaleimide-sensitive factor-attachment protein receptor

  •  
  • SNX

    sorting nexin

  •  
  • STK38

    serine/threonine kinase 38

  •  
  • TAB

    TAK1-binding protein

  •  
  • TAK1

    transforming growth factor β-activated kinase

  •  
  • TLR

    Toll-like receptor

  •  
  • TRAF6

    tumour-necrosis-factor-receptor-associated factor 6

  •  
  • TTC19

    tetratricopeptide repeat protein 19

  •  
  • USP

    ubiquitin-specific peptidase

  •  
  • UVRAG

    UV radiation resistance-associated gene

  •  
  • Vps

    vacuolar protein sorting

  •  
  • ZBTB16

    zinc finger- and BTB domain-containing 16

References

References
1
Herman
P.K.
Stack
J.H.
Emr
S.D.
An essential role for a protein and lipid kinase complex in secretory protein sorting
Trends Cell Biol.
1992
, vol. 
2
 (pg. 
363
-
368
)
[PubMed]
2
Herman
P.K.
Emr
S.D.
Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae
Mol. Cell. Biol.
1990
, vol. 
10
 (pg. 
6742
-
6754
)
[PubMed]
3
Backer
J.M.
The regulation and function of Class III PI3Ks: novel roles for Vps34
Biochem. J.
2008
, vol. 
410
 (pg. 
1
-
17
)
[PubMed]
4
Miaczynska
M.
Zerial
M.
Mosaic organization of the endocytic pathway
Exp. Cell Res.
2002
, vol. 
272
 (pg. 
8
-
14
)
[PubMed]
5
Lindmo
K.
Stenmark
H.
Regulation of membrane traffic by phosphoinositide 3-kinases
J. Cell Sci.
2006
, vol. 
119
 (pg. 
605
-
614
)
[PubMed]
6
Henne
W.M.
Buchkovich
N.J.
Emr
S.D.
The ESCRT pathway
Dev. Cell
2011
, vol. 
21
 (pg. 
77
-
91
)
[PubMed]
7
Bonifacino
J.S.
Hurley
J.H.
Retromer
Curr. Opin. Cell Biol.
2008
, vol. 
20
 (pg. 
427
-
436
)
[PubMed]
8
Lamb
C.A.
Yoshimori
T.
Tooze
S.A.
The autophagosome: origins unknown, biogenesis complex
Nat. Rev. Mol. Cell Biol.
2013
, vol. 
14
 (pg. 
759
-
774
)
[PubMed]
9
Parzych
K.R.
Klionsky
D.J.
An overview of autophagy: morphology, mechanism, and regulation
Antioxid. Redox Signal.
2014
, vol. 
20
 (pg. 
460
-
473
)
[PubMed]
10
Rostislavleva
K.
Soler
N.
Ohashi
Y.
Zhang
L.
Pardon
E.
Burke
J.E.
Masson
G.R.
Johnson
C.
Steyaert
J.
Ktistakis
N.T.
Williams
R.L.
Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes
Science
2015
, vol. 
350
 pg. 
aac7365
 
[PubMed]
11
Murray
J.T.
Panaretou
C.
Stenmark
H.
Miaczynska
M.
Backer
J.M.
Role of Rab5 in the recruitment of hVps34/p150 to the early endosome
Traffic
2002
, vol. 
3
 (pg. 
416
-
427
)
[PubMed]
12
Slessareva
J.E.
Routt
S.M.
Temple
B.
Bankaitis
V.A.
Dohlman
H.G.
Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome
Cell
2006
, vol. 
126
 (pg. 
191
-
203
)
[PubMed]
13
Sinha
S.
Levine
B.
The autophagy effector Beclin 1: a novel BH3-only protein
Oncogene
2008
, vol. 
27
 
Suppl. 1
(pg. 
S137
-
S148
)
[PubMed]
14
Yuan
H.X.
Russell
R.C.
Guan
K.L.
Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy
Autophagy
2013
, vol. 
9
 (pg. 
1983
-
1995
)
[PubMed]
15
Russell
R.C.
Tian
Y.
Yuan
H.
Park
H.W.
Chang
Y.Y.
Kim
J.
Kim
H.
Neufeld
T.P.
Dillin
A.
Guan
K.L.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase
Nat. Cell Biol.
2013
, vol. 
15
 (pg. 
741
-
750
)
[PubMed]
16
Kim
J.
Kim
Y.C.
Fang
C.
Russell
R.C.
Kim
J.H.
Fan
W.
Liu
R.
Zhong
Q.
Guan
K.L.
Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy
Cell
2013
, vol. 
152
 (pg. 
290
-
303
)
[PubMed]
17
Kihara
A.
Noda
T.
Ishihara
N.
Ohsumi
Y.
Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae
J. Cell Biol.
2001
, vol. 
152
 (pg. 
519
-
530
)
[PubMed]
18
Itakura
E.
Kishi
C.
Inoue
K.
Mizushima
N.
Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG
Mol. Biol. Cell
2008
, vol. 
19
 (pg. 
5360
-
5372
)
[PubMed]
19
Zhong
Y.
Wang
Q.J.
Li
X.
Yan
Y.
Backer
J.M.
Chait
B.T.
Heintz
N.
Yue
Z.
Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex
Nat. Cell Biol.
2009
, vol. 
11
 (pg. 
468
-
476
)
[PubMed]
20
Matsunaga
K.
Saitoh
T.
Tabata
K.
Omori
H.
Satoh
T.
Kurotori
N.
Maejima
I.
Shirahama-Noda
K.
Ichimura
T.
Isobe
T.
, et al. 
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages
Nat. Cell Biol.
2009
, vol. 
11
 (pg. 
385
-
396
)
[PubMed]
21
Sun
Q.
Fan
W.
Chen
K.
Ding
X.
Chen
S.
Zhong
Q.
Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase
Proc. Natl. Acad. Sci. U.S.A.
2008
, vol. 
105
 (pg. 
19211
-
19216
)
[PubMed]
22
Thoresen
S.B.
Pedersen
N.M.
Liestol
K.
Stenmark
H.
A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic
Exp. Cell Res.
2010
, vol. 
316
 (pg. 
3368
-
3378
)
[PubMed]
23
McKnight
N.C.
Zhong
Y.
Wold
M.S.
Gong
S.
Phillips
G.R.
Dou
Z.
Zhao
Y.
Heintz
N.
Zong
W.X.
Yue
Z.
Beclin 1 is required for neuron viability and regulates endosome pathways via the UVRAG-VPS34 complex
PLoS Genet.
2014
, vol. 
10
 pg. 
e1004626
 
[PubMed]
24
Heenan
E.J.
Vanhooke
J.L.
Temple
B.R.
Betts
L.
Sondek
J.E.
Dohlman
H.G.
Structure and function of Vps15 in the endosomal G protein signaling pathway
Biochemistry
2009
, vol. 
48
 (pg. 
6390
-
6401
)
[PubMed]
25
Noda
N.N.
Kobayashi
T.
Adachi
W.
Fujioka
Y.
Ohsumi
Y.
Inagaki
F.
Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy
J. Biol. Chem.
2012
, vol. 
287
 (pg. 
16256
-
16266
)
[PubMed]
26
Fan
W.
Nassiri
A.
Zhong
Q.
Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)
Proc. Natl. Acad. Sci. U.S.A.
2011
, vol. 
108
 (pg. 
7769
-
7774
)
[PubMed]
27
Baskaran
S.
Carlson
L.A.
Stjepanovic
G.
Young
L.N.
Kim do
J.
Grob
P.
Stanley
R.E.
Nogales
E.
Hurley
J.H.
Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex
eLife
2014
, vol. 
3
 pg. 
e05115
 
[PubMed]
28
Furuya
N.
Yu
J.
Byfield
M.
Pattingre
S.
Levine
B.
The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function
Autophagy
2005
, vol. 
1
 (pg. 
46
-
52
)
[PubMed]
29
Mandelker
D.
Gabelli
S.B.
Schmidt-Kittler
O.
Zhu
J.
Cheong
I.
Huang
C.H.
Kinzler
K.W.
Vogelstein
B.
Amzel
L.M.
A frequent kinase domain mutation that changes the interaction between PI3Kalpha and the membrane
Proc. Natl. Acad. Sci. U.S.A.
2009
, vol. 
106
 (pg. 
16996
-
17001
)
[PubMed]
30
Zhang
X.
Vadas
O.
Perisic
O.
Anderson
K.E.
Clark
J.
Hawkins
P.T.
Stephens
L.R.
Williams
R.L.
Structure of lipid kinase p110beta/p85beta elucidates an unusual SH2-domain-mediated inhibitory mechanism
Mol. Cell
2011
, vol. 
41
 (pg. 
567
-
578
)
[PubMed]
31
Miller
S.
Tavshanjian
B.
Oleksy
A.
Perisic
O.
Houseman
B.T.
Shokat
K.M.
Williams
R.L.
Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34
Science
2010
, vol. 
327
 (pg. 
1638
-
1642
)
[PubMed]
32
Huang
W.
Choi
W.
Hu
W.
Mi
N.
Guo
Q.
Ma
M.
Liu
M.
Tian
Y.
Lu
P.
Wang
F.L.
, et al. 
Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein
Cell Res.
2012
, vol. 
22
 (pg. 
473
-
489
)
[PubMed]
33
Yan
Y.
Flinn
R.J.
Wu
H.
Schnur
R.S.
Backer
J.M.
hVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells
Biochem. J.
2009
, vol. 
417
 (pg. 
747
-
755
)
[PubMed]
34
Sun
Q.
Zhang
J.
Fan
W.
Wong
K.N.
Ding
X.
Chen
S.
Zhong
Q.
The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression
J. Biol. Chem.
2011
, vol. 
286
 (pg. 
185
-
191
)
[PubMed]
35
Araki
Y.
Ku
W.C.
Akioka
M.
May
A.I.
Hayashi
Y.
Arisaka
F.
Ishihama
Y.
Ohsumi
Y.
Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity
J. Cell Biol.
2013
, vol. 
203
 (pg. 
299
-
313
)
[PubMed]
36
Lu
J.
He
L.
Behrends
C.
Araki
M.
Araki
K.
Jun Wang
Q.
Catanzaro
J.M.
Friedman
S.L.
Zong
W.X.
Fiel
M.I.
, et al. 
NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity
Nat. Commun.
2014
, vol. 
5
 pg. 
3920
 
[PubMed]
37
Cao
Y.
Wang
Y.
Abi Saab
W.F.
Yang
F.
Pessin
J.E.
Backer
J.M.
NRBF2 regulates macroautophagy as a component of Vps34 Complex I
Biochem. J.
2014
, vol. 
461
 (pg. 
315
-
322
)
[PubMed]
38
Zhong
Y.
Morris
D.H.
Jin
L.
Patel
M.S.
Karunakaran
S.K.
Fu
Y.J.
Matuszak
E.A.
Weiss
H.L.
Chait
B.T.
Wang
Q.J.
Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels
J. Biol. Chem.
2014
, vol. 
289
 (pg. 
26021
-
26037
)
[PubMed]
39
Yasumo
H.
Masuda
N.
Furusawa
T.
Tsukamoto
T.
Sadano
H.
Osumi
T.
Nuclear receptor binding factor-2 (NRBF-2), a possible gene activator protein interacting with nuclear hormone receptors
Biochim. Biophys. Acta
2000
, vol. 
1490
 (pg. 
189
-
197
)
[PubMed]
40
Flores
A.M.
Li
L.
Aneskievich
B.J.
Isolation and functional analysis of a keratinocyte-derived, ligand-regulated nuclear receptor comodulator
J. Invest. Dermatol.
2004
, vol. 
123
 (pg. 
1092
-
1101
)
[PubMed]
41
Monroe
N.
Hill
C.P.
Meiotic clade AAA ATPases: protein polymer disassembly machines
J. Mol. Biol.
2015
 
doi:10.1016/j.jmb.2015.11.004
[PubMed]
42
Xu
D.Q.
Wang
Z.
Wang
C.Y.
Zhang
D.Y.
Wan
H.D.
Zhao
Z.L.
Gu
J.
Zhang
Y.X.
Li
Z.G.
Man
K.Y.
, et al. 
PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity
EMBO J.
2016
, vol. 
35
 (pg. 
496
-
514
)
[PubMed]
43
Zhang
L.
Gao
X.
Wen
J.
Ning
Y.
Chen
Y.G.
Dapper 1 antagonizes Wnt signaling by promoting dishevelled degradation
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
8607
-
8612
)
[PubMed]
44
Ma
B.
Cao
W.
Li
W.
Gao
C.
Qi
Z.
Zhao
Y.
Du
J.
Xue
H.
Peng
J.
Wen
J.
, et al. 
Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation
Cell Res.
2014
, vol. 
24
 (pg. 
912
-
924
)
[PubMed]
45
Zhao
Y.
Wang
Q.
Qiu
G.
Zhou
S.
Jing
Z.
Wang
J.
Wang
W.
Cao
J.
Han
K.
Cheng
Q.
, et al. 
RACK1 promotes autophagy by enhancing the Atg14L-Beclin 1-Vps34-Vps15 complex formation upon phosphorylation by AMPK
Cell Rep.
2015
, vol. 
13
 (pg. 
1407
-
1417
)
[PubMed]
46
Lv
Q.
Wang
W.
Xue
J.
Hua
F.
Mu
R.
Lin
H.
Yan
J.
Lv
X.
Chen
X.
Hu
Z.W.
DEDD interacts with PI3KC3 to activate autophagy and attenuate epithelial-mesenchymal transition in human breast cancer
Cancer Res.
2012
, vol. 
72
 (pg. 
3238
-
3250
)
[PubMed]
47
Yang
Y.
Fiskus
W.
Yong
B.
Atadja
P.
Takahashi
Y.
Pandita
T.K.
Wang
H.G.
Bhalla
K.N.
Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy
Proc. Natl. Acad. Sci. U.S.A.
2013
, vol. 
110
 (pg. 
6841
-
6846
)
[PubMed]
48
Wang
P.
Xu
T.Y.
Wei
K.
Guan
Y.F.
Wang
X.
Xu
H.
Su
D.F.
Pei
G.
Miao
C.Y.
ARRB1/beta-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia
Autophagy
2014
, vol. 
10
 (pg. 
1535
-
1548
)
[PubMed]
49
Rink
J.
Ghigo
E.
Kalaidzidis
Y.
Zerial
M.
Rab conversion as a mechanism of progression from early to late endosomes
Cell
2005
, vol. 
122
 (pg. 
735
-
749
)
[PubMed]
50
Balderhaar
H.J.
Ungermann
C.
CORVET and HOPS tethering complexes–coordinators of endosome and lysosome fusion
J. Cell Sci.
2013
, vol. 
126
 (pg. 
1307
-
1316
)
[PubMed]
51
Liang
C.
Lee
J.S.
Inn
K.S.
Gack
M.U.
Li
Q.
Roberts
E.A.
Vergne
I.
Deretic
V.
Feng
P.
Akazawa
C.
Jung
J.U.
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking
Nat. Cell Biol.
2008
, vol. 
10
 (pg. 
776
-
787
)
[PubMed]
52
Sun
Q.
Westphal
W.
Wong
K.N.
Tan
I.
Zhong
Q.
Rubicon controls endosome maturation as a Rab7 effector
Proc. Natl. Acad. Sci. U.S.A.
2010
, vol. 
107
 (pg. 
19338
-
19343
)
[PubMed]
53
Xia
P.
Wang
S.
Du
Y.
Zhao
Z.
Shi
L.
Sun
L.
Huang
G.
Ye
B.
Li
C.
Dai
Z.
, et al. 
WASH inhibits autophagy through suppression of Beclin 1 ubiquitination
EMBO J.
2013
, vol. 
32
 (pg. 
2685
-
2696
)
[PubMed]
54
Schmitt
H.D.
Dsl1p/Zw10: common mechanisms behind tethering vesicles and microtubules
Trends Cell Biol.
2010
, vol. 
20
 (pg. 
257
-
268
)
[PubMed]
55
Buxton
P.
Zhang
X.M.
Walsh
B.
Sriratana
A.
Schenberg
I.
Manickam
E.
Rowe
T.
Identification and characterization of Snapin as a ubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells
Biochem. J.
2003
, vol. 
375
 (pg. 
433
-
440
)
[PubMed]
56
Kim
H.J.
Zhong
Q.
Sheng
Z.H.
Yoshimori
T.
Liang
C.
Jung
J.U.
Beclin-1-interacting autophagy protein Atg14L targets the SNARE-associated protein Snapin to coordinate endocytic trafficking
J. Cell Sci.
2012
, vol. 
125
 (pg. 
4740
-
4750
)
[PubMed]
57
Diao
J.
Liu
R.
Rong
Y.
Zhao
M.
Zhang
J.
Lai
Y.
Zhou
Q.
Wilz
L.M.
Li
J.
Vivona
S.
, et al. 
ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes
Nature
2015
, vol. 
520
 (pg. 
563
-
566
)
[PubMed]
58
Lipinski
M.M.
Hoffman
G.
Ng
A.
Zhou
W.
Py
B.F.
Hsu
E.
Liu
X.
Eisenberg
J.
Liu
J.
Blenis
J.
, et al. 
A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions
Dev. Cell
2010
, vol. 
18
 (pg. 
1041
-
1052
)
[PubMed]
59
Sandri
M.
Sandri
C.
Gilbert
A.
Skurk
C.
Calabria
E.
Picard
A.
Walsh
K.
Schiaffino
S.
Lecker
S.H.
Goldberg
A.L.
Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy
Cell
2004
, vol. 
117
 (pg. 
399
-
412
)
[PubMed]
60
Yamada
E.
Bastie
C.C.
Koga
H.
Wang
Y.
Cuervo
A.M.
Pessin
J.E.
Mouse skeletal muscle fiber-type-specific macroautophagy and muscle wasting are regulated by a Fyn/STAT3/Vps34 signaling pathway
Cell Rep.
2012
, vol. 
1
 (pg. 
557
-
569
)
[PubMed]
61
Byfield
M.P.
Murray
J.T.
Backer
J.M.
hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
33076
-
33082
)
[PubMed]
62
Tassa
A.
Roux
M.P.
Attaix
D.
Bechet
D.M.
Class III phosphoinositide 3-kinase–Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes
Biochem. J.
2003
, vol. 
376
 (pg. 
577
-
586
)
[PubMed]
63
Fogel
A.I.
Dlouhy
B.J.
Wang
C.
Ryu
S.W.
Neutzner
A.
Hasson
S.A.
Sideris
D.P.
Abeliovich
H.
Youle
R.J.
Role of membrane association and Atg14-dependent phosphorylation in beclin-1-mediated autophagy
Mol. Cell. Biol.
2013
, vol. 
33
 (pg. 
3675
-
3688
)
[PubMed]
64
Wei
Y.
An
Z.
Zou
Z.
Sumpter
R.
Su
M.
Zang
X.
Sinha
S.
Gaestel
M.
Levine
B.
The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation
eLife
2015
, vol. 
4
 pg. 
e05289
 
[PubMed]
65
Zalckvar
E.
Berissi
H.
Mizrachy
L.
Idelchuk
Y.
Koren
I.
Eisenstein
M.
Sabanay
H.
Pinkas-Kramarski
R.
Kimchi
A.
DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy
EMBO Rep.
2009
, vol. 
10
 (pg. 
285
-
292
)
[PubMed]
66
Eisenberg-Lerner
A.
Kimchi
A.
PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of DAPk
Cell Death Differ.
2012
, vol. 
19
 (pg. 
788
-
797
)
[PubMed]
67
Wang
R.C.
Wei
Y.
An
Z.
Zou
Z.
Xiao
G.
Bhagat
G.
White
M.
Reichelt
J.
Levine
B.
Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation
Science
2012
, vol. 
338
 (pg. 
956
-
959
)
[PubMed]
68
Wei
Y.
Zou
Z.
Becker
N.
Anderson
M.
Sumpter
R.
Xiao
G.
Kinch
L.
Koduru
P.
Christudass
C.S.
Veltri
R.W.
, et al. 
EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance
Cell
2013
, vol. 
154
 (pg. 
1269
-
1284
)
[PubMed]
69
Eskelinen
E.L.
Prescott
A.R.
Cooper
J.
Brachmann
S.M.
Wang
L.
Tang
X.
Backer
J.M.
Lucocq
J.M.
Inhibition of autophagy in mitotic animal cells
Traffic
2002
, vol. 
3
 (pg. 
878
-
893
)
[PubMed]
70
Furuya
T.
Kim
M.
Lipinski
M.
Li
J.
Kim
D.
Lu
T.
Shen
Y.
Rameh
L.
Yankner
B.
Tsai
L.H.
Yuan
J.
Negative regulation of Vps34 by Cdk mediated phosphorylation
Mol. Cell
2010
, vol. 
38
 (pg. 
500
-
511
)
[PubMed]
71
Veldhoen
R.A.
Banman
S.L.
Hemmerling
D.R.
Odsen
R.
Simmen
T.
Simmonds
A.J.
Underhill
D.A.
Goping
I.S.
The chemotherapeutic agent paclitaxel inhibits autophagy through two distinct mechanisms that regulate apoptosis
Oncogene
2013
, vol. 
32
 (pg. 
736
-
746
)
[PubMed]
72
Hirsch
D.S.
Shen
Y.
Dokmanovic
M.
Wu
W.J.
pp60c-Src phosphorylates and activates vacuolar protein sorting 34 to mediate cellular transformation
Cancer Res.
2010
, vol. 
70
 (pg. 
5974
-
5983
)
[PubMed]
73
Hirsch
D.S.
Shen
Y.
Dokmanovic
M.
Yu
J.
Mohan
N.
Elzarrad
M.K.
Wu
W.J.
Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1
Cell. Signal.
2014
, vol. 
26
 (pg. 
1258
-
1268
)
[PubMed]
74
Maejima
Y.
Kyoi
S.
Zhai
P.
Liu
T.
Li
H.
Ivessa
A.
Sciarretta
S.
Del Re
D.P.
Zablocki
D.K.
Hsu
C.P.
, et al. 
Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2
Nat. Med.
2013
, vol. 
19
 (pg. 
1478
-
1488
)
[PubMed]
75
Pattingre
S.
Tassa
A.
Qu
X.
Garuti
R.
Liang
X.H.
Mizushima
N.
Packer
M.
Schneider
M.D.
Levine
B.
Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy
Cell
2005
, vol. 
122
 (pg. 
927
-
939
)
[PubMed]
76
Li
X.
He
L.
Che
K.H.
Funderburk
S.F.
Pan
L.
Pan
N.
Zhang
M.
Yue
Z.
Zhao
Y.
Imperfect interface of Beclin1 coiled-coil domain regulates homodimer and heterodimer formation with Atg14L and UVRAG
Nat. Commun.
2012
, vol. 
3
 pg. 
662
 
[PubMed]
77
Hurley
J.H.
Schulman
B.A.
Atomistic autophagy: the structures of cellular self-digestion
Cell
2014
, vol. 
157
 (pg. 
300
-
311
)
[PubMed]
78
Noble
C.G.
Dong
J.M.
Manser
E.
Song
H.
Bcl-xL and UVRAG cause a monomer-dimer switch in Beclin1
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
26274
-
26282
)
[PubMed]
79
Oberstein
A.
Jeffrey
P.D.
Shi
Y.
Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
13123
-
13132
)
[PubMed]
80
Ku
B.
Woo
J.S.
Liang
C.
Lee
K.H.
Jung
J.U.
Oh
B.H.
An insight into the mechanistic role of Beclin 1 and its inhibition by prosurvival Bcl-2 family proteins
Autophagy
2008
, vol. 
4
 (pg. 
519
-
520
)
[PubMed]
81
Wei
Y.
Pattingre
S.
Sinha
S.
Bassik
M.
Levine
B.
JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy
Mol. Cell
2008
, vol. 
30
 (pg. 
678
-
688
)
[PubMed]
82
Chen
D.
Gao
F.
Li
B.
Wang
H.
Xu
Y.
Zhu
C.
Wang
G.
Parkin mono-ubiquitinates Bcl-2 and regulates autophagy
J. Biol. Chem.
2010
, vol. 
285
 (pg. 
38214
-
38223
)
[PubMed]
83
Chang
N.C.
Nguyen
M.
Germain
M.
Shore
G.C.
Antagonism of Beclin 1-dependent autophagy by BCL-2 at the endoplasmic reticulum requires NAF-1
EMBO J.
2010
, vol. 
29
 (pg. 
606
-
618
)
[PubMed]
84
Tang
D.
Kang
R.
Livesey
K.M.
Cheh
C.W.
Farkas
A.
Loughran
P.
Hoppe
G.
Bianchi
M.E.
Tracey
K.J.
Zeh
H.J.
3rd
Lotze
M.T.
Endogenous HMGB1 regulates autophagy
J. Cell Biol.
2010
, vol. 
190
 (pg. 
881
-
892
)
[PubMed]
85
Fimia
G.M.
Stoykova
A.
Romagnoli
A.
Giunta
L.
Di Bartolomeo
S.
Nardacci
R.
Corazzari
M.
Fuoco
C.
Ucar
A.
Schwartz
P.
, et al. 
Ambra1 regulates autophagy and development of the nervous system
Nature
2007
, vol. 
447
 (pg. 
1121
-
1125
)
[PubMed]
86
Ma
B.
Liu
B.
Cao
W.
Gao
C.
Qi
Z.
Ning
Y.
Chen
Y.G.
The Wnt signaling antagonist Dapper1 accelerates dishevelled2 degradation via promoting its ubiquitination and aggregate-induced autophagy
J. Biol. Chem.
2015
, vol. 
290
 (pg. 
12346
-
12354
)
[PubMed]
87
Xu
D.
Zhang
T.
Xiao
J.
Zhu
K.
Wei
R.
Wu
Z.
Meng
H.
Li
Y.
Yuan
J.
Modification of BECN1 by ISG15 plays a crucial role in autophagy regulation by type I IFN/interferon
Autophagy
2015
, vol. 
11
 (pg. 
617
-
628
)
[PubMed]
88
Jang
Y.H.
Choi
K.Y.
Min
D.S.
Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy
Cell Death Differ.
2014
, vol. 
21
 (pg. 
533
-
546
)
[PubMed]
89
Liu
F.T.
Yang
Y.J.
Wu
J.J.
Li
S.
Tang
Y.L.
Zhao
J.
Liu
Z.Y.
Xiao
B.G.
Zuo
J.
Liu
W.
Wang
J.
Fasudil, a Rho kinase inhibitor, promotes the autophagic degradation of A53T alpha-synuclein by activating the JNK 1/Bcl-2/beclin 1 pathway
Brain. Res.
2016
, vol. 
1632
 (pg. 
9
-
18
)
[PubMed]
90
Cinque
L.
Forrester
A.
Bartolomeo
R.
Svelto
M.
Venditti
R.
Montefusco
S.
Polishchuk
E.
Nusco
E.
Rossi
A.
Medina
D.L.
, et al. 
FGF signalling regulates bone growth through autophagy
Nature
2015
, vol. 
528
 (pg. 
272
-
275
)
[PubMed]
91
Bologna
C.
Buonincontri
R.
Serra
S.
Vaisitti
T.
Audrito
V.
Brusa
D.
Pagnani
A.
Coscia
M.
D'Arena
G.
Mereu
E.
, et al. 
SLAMF1 regulation of chemotaxis and autophagy determines CLL patient response
J. Clin. Invest.
2016
, vol. 
126
 (pg. 
181
-
194
)
[PubMed]
92
Kang
R.
Tang
D.
Schapiro
N.E.
Livesey
K.M.
Farkas
A.
Loughran
P.
Bierhaus
A.
Lotze
M.T.
Zeh
H.J.
The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival
Cell Death Differ.
2010
, vol. 
17
 (pg. 
666
-
676
)
[PubMed]
93
Ma
C.
Wang
N.
Detre
C.
Wang
G.
O'Keeffe
M.
Terhorst
C.
Receptor signaling lymphocyte-activation molecule family 1 (Slamf1) regulates membrane fusion and NADPH oxidase 2 (NOX2) activity by recruiting a Beclin-1/Vps34/ultraviolet radiation resistance-associated gene (UVRAG) complex
J. Biol. Chem.
2012
, vol. 
287
 (pg. 
18359
-
18365
)
[PubMed]
94
Liang
C.
Feng
P.
Ku
B.
Dotan
I.
Canaani
D.
Oh
B.H.
Jung
J.U.
Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG
Nat. Cell Biol.
2006
, vol. 
8
 (pg. 
688
-
699
)
[PubMed]
95
Munson
M.J.
Allen
G.F.
Toth
R.
Campbell
D.G.
Lucocq
J.M.
Ganley
I.G.
mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival
EMBO J.
2015
, vol. 
34
 (pg. 
2272
-
2290
)
[PubMed]
96
Hein
M.Y.
Hubner
N.C.
Poser
I.
Cox
J.
Nagaraj
N.
Toyoda
Y.
Gak
I.A.
Weisswange
I.
Mansfeld
J.
Buchholz
F.
, et al. 
A human interactome in three quantitative dimensions organized by stoichiometries and abundances
Cell
2015
, vol. 
163
 (pg. 
712
-
723
)
[PubMed]
97
Maiuri
M.C.
Le Toumelin
G.
Criollo
A.
Rain
J.C.
Gautier
F.
Juin
P.
Tasdemir
E.
Pierron
G.
Troulinaki
K.
Tavernarakis
N.
, et al. 
Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1
EMBO J.
2007
, vol. 
26
 (pg. 
2527
-
2539
)
[PubMed]
98
Adi-Harel
S.
Erlich
S.
Schmukler
E.
Cohen-Kedar
S.
Segev
O.
Mizrachy
L.
Hirsch
J.A.
Pinkas-Kramarski
R.
Beclin 1 self-association is independent of autophagy induction by amino acid deprivation and rapamycin treatment
J. Cell. Biochem.
2010
, vol. 
110
 (pg. 
1262
-
1271
)
[PubMed]
99
Maiuri
M.C.
Criollo
A.
Tasdemir
E.
Vicencio
J.M.
Tajeddine
N.
Hickman
J.A.
Geneste
O.
Kroemer
G.
BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L)
Autophagy
2007
, vol. 
3
 (pg. 
374
-
376
)
[PubMed]
100
Tian
S.
Lin
J.
Jun Zhou
J.
Wang
X.
Li
Y.
Ren
X.
Yu
W.
Zhong
W.
Xiao
J.
Sheng
F.
, et al. 
Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound, Z18
Autophagy
2010
, vol. 
6
 (pg. 
1032
-
1041
)
[PubMed]
101
Jin
J.
Arias
E.E.
Chen
J.
Harper
J.W.
Walter
J.C.
A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1
Mol. Cell
2006
, vol. 
23
 (pg. 
709
-
721
)
[PubMed]
102
Nazio
F.
Strappazzon
F.
Antonioli
M.
Bielli
P.
Cianfanelli
V.
Bordi
M.
Gretzmeier
C.
Dengjel
J.
Piacentini
M.
Fimia
G.M.
Cecconi
F.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6
Nat. Cell Biol.
2013
, vol. 
15
 (pg. 
406
-
416
)
[PubMed]
103
Strappazzon
F.
Vietri-Rudan
M.
Campello
S.
Nazio
F.
Florenzano
F.
Fimia
G.M.
Piacentini
M.
Levine
B.
Cecconi
F.
Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy
EMBO J.
2011
, vol. 
30
 (pg. 
1195
-
1208
)
[PubMed]
104
Di Bartolomeo
S.
Corazzari
M.
Nazio
F.
Oliverio
S.
Lisi
G.
Antonioli
M.
Pagliarini
V.
Matteoni
S.
Fuoco
C.
Giunta
L.
, et al. 
The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy
J. Cell Biol.
2010
, vol. 
191
 (pg. 
155
-
168
)
[PubMed]
105
Pampliega
O.
Orhon
I.
Patel
B.
Sridhar
S.
Diaz-Carretero
A.
Beau
I.
Codogno
P.
Satir
B.H.
Satir
P.
Cuervo
A.M.
Functional interaction between autophagy and ciliogenesis
Nature
2013
, vol. 
502
 (pg. 
194
-
200
)
[PubMed]
106
Takahashi
Y.
Coppola
D.
Matsushita
N.
Cualing
H.D.
Sun
M.
Sato
Y.
Liang
C.
Jung
J.U.
Cheng
J.Q.
Mul
J.J.
, et al. 
Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis
Nat. Cell Biol.
2007
, vol. 
9
 (pg. 
1142
-
1151
)
[PubMed]
107
Farsad
K.
Ringstad
N.
Takei
K.
Floyd
S.R.
Rose
K.
De Camilli
P.
Generation of high curvature membranes mediated by direct endophilin bilayer interactions
J. Cell Biol.
2001
, vol. 
155
 (pg. 
193
-
200
)
[PubMed]
108
Takahashi
Y.
Meyerkord
C.L.
Wang
H.G.
Bif-1/endophilin B1: a candidate for crescent driving force in autophagy
Cell Death Differ.
2009
, vol. 
16
 (pg. 
947
-
955
)
[PubMed]
109
Mari
M.
Macia
E.
Le Marchand-Brustel
Y.
Cormont
M.
Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
42501
-
42508
)
[PubMed]
110
Recacha
R.
Boulet
A.
Jollivet
F.
Monier
S.
Houdusse
A.
Goud
B.
Khan
A.R.
Structural basis for recruitment of Rab6-interacting protein 1 to Golgi via a RUN domain
Structure
2009
, vol. 
17
 (pg. 
21
-
30
)
[PubMed]
111
Niso-Santano
M.
Criollo
A.
Malik
S.A.
Michaud
M.
Morselli
E.
Marino
G.
Lachkar
S.
Galluzzi
L.
Maiuri
M.C.
Kroemer
G.
Direct molecular interactions between Beclin 1 and the canonical NFkappaB activation pathway
Autophagy
2012
, vol. 
8
 (pg. 
268
-
270
)
[PubMed]
112
Criollo
A.
Niso-Santano
M.
Malik
S.A.
Michaud
M.
Morselli
E.
Marino
G.
Lachkar
S.
Arkhipenko
A.V.
Harper
F.
Pierron
G.
, et al. 
Inhibition of autophagy by TAB2 and TAB3
EMBO J.
2011
, vol. 
30
 (pg. 
4908
-
4920
)
[PubMed]
113
Liu
K.
Jian
Y.
Sun
X.
Yang
C.
Gao
Z.
Zhang
Z.
Liu
X.
Li
Y.
Xu
J.
Jing
Y.
, et al. 
Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion
J. Cell Biol.
2016
, vol. 
212
 (pg. 
181
-
198
)
[PubMed]
114
Shi
C.S.
Kehrl
J.H.
TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy
Sci. Signal.
2010
, vol. 
3
 pg. 
ra42
 
[PubMed]
115
Sun
T.
Li
X.
Zhang
P.
Chen
W.D.
Zhang
H.L.
Li
D.D.
Deng
R.
Qian
X.J.
Jiao
L.
Ji
J.
, et al. 
Acetylation of Beclin 1 inhibits autophagosome maturation and promotes tumour growth
Nat. Commun.
2015
, vol. 
6
 pg. 
7215
 
[PubMed]
116
Obara
K.
Sekito
T.
Ohsumi
Y.
Assortment of phosphatidylinositol 3-kinase complexes—Atg14p directs association of complex I to the pre-autophagosomal structure in Saccharomyces cerevisiae
Mol. Biol. Cell
2006
, vol. 
17
 (pg. 
1527
-
1539
)
[PubMed]
117
Matsunaga
K.
Morita
E.
Saitoh
T.
Akira
S.
Ktistakis
N.T.
Izumi
T.
Noda
T.
Yoshimori
T.
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L
J. Cell Biol.
2010
, vol. 
190
 (pg. 
511
-
521
)
[PubMed]
118
Jao
C.C.
Ragusa
M.J.
Stanley
R.E.
Hurley
J.H.
A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy
Proc. Natl. Acad. Sci. U.S.A.
2013
, vol. 
110
 (pg. 
5486
-
5491
)
[PubMed]
119
Luo
S.
Garcia-Arencibia
M.
Zhao
R.
Puri
C.
Toh
P.P.
Sadiq
O.
Rubinsztein
D.C.
Bim inhibits autophagy by recruiting Beclin 1 to microtubules
Mol. Cell
2012
, vol. 
47
 (pg. 
359
-
370
)
[PubMed]
120
Ravikumar
B.
Moreau
K.
Jahreiss
L.
Puri
C.
Rubinsztein
D.C.
Plasma membrane contributes to the formation of pre-autophagosomal structures
Nat. Cell Biol.
2010
, vol. 
12
 (pg. 
747
-
757
)
[PubMed]
121
Bejarano
E.
Yuste
A.
Patel
B.
Stout
R.F.
Jr.
Spray
D.C.
Cuervo
A.M.
Connexins modulate autophagosome biogenesis
Nat. Cell Biol.
2014
, vol. 
16
 (pg. 
401
-
414
)
[PubMed]
122
Nah
J.
Pyo
J.O.
Jung
S.
Yoo
S.M.
Kam
T.I.
Chang
J.
Han
J.
Soo
A.A.S.
Onodera
T.
Jung
Y.K.
BECN1/Beclin 1 is recruited into lipid rafts by prion to activate autophagy in response to amyloid beta 42
Autophagy
2013
, vol. 
9
 (pg. 
2009
-
2021
)
[PubMed]
123
Liang
J.
Xu
Z.X.
Ding
Z.
Lu
Y.
Yu
Q.
Werle
K.D.
Zhou
G.
Park
Y.Y.
Peng
G.
Gambello
M.J.
Mills
G.B.
Myristoylation confers noncanonical AMPK functions in autophagy selectivity and mitochondrial surveillance
Nat. Commun.
2015
, vol. 
6
 pg. 
7926
 
[PubMed]
124
Bodemann
B.O.
Orvedahl
A.
Cheng
T.
Ram
R.R.
Ou
Y.H.
Formstecher
E.
Maiti
M.
Hazelett
C.C.
Wauson
E.M.
Balakireva
M.
, et al. 
RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly
Cell
2011
, vol. 
144
 (pg. 
253
-
267
)
[PubMed]
125
He
B.
Guo
W.
The exocyst complex in polarized exocytosis
Curr. Opin. Cell Biol.
2009
, vol. 
21
 (pg. 
537
-
542
)
[PubMed]
126
Moskalenko
S.
Tong
C.
Rosse
C.
Mirey
G.
Formstecher
E.
Daviet
L.
Camonis
J.
White
M.A.
Ral GTPases regulate exocyst assembly through dual subunit interactions
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
51743
-
51748
)
[PubMed]
127
Joffre
C.
Dupont
N.
Hoa
L.
Gomez
V.
Pardo
R.
Goncalves-Pimentel
C.
Achard
P.
Bettoun
A.
Meunier
B.
Bauvy
C.
, et al. 
The pro-apoptotic STK38 kinase is a new Beclin1 partner positively regulating autophagy
Curr. Biol.
2015
, vol. 
25
 (pg. 
2479
-
2492
)
[PubMed]
128
Platta
H.W.
Abrahamsen
H.
Thoresen
S.B.
Stenmark
H.
Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1
Biochem. J.
2012
, vol. 
441
 (pg. 
399
-
406
)
[PubMed]
129
Liu
J.
Xia
H.
Kim
M.
Xu
L.
Li
Y.
Zhang
L.
Cai
Y.
Norberg
H.V.
Zhang
T.
Furuya
T.
, et al. 
Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13
Cell
2011
, vol. 
147
 (pg. 
223
-
234
)
[PubMed]
130
Xia
P.
Wang
S.
Huang
G.
Du
Y.
Zhu
P.
Li
M.
Fan
Z.
RNF2 is recruited by WASH to ubiquitinate AMBRA1 leading to downregulation of autophagy
Cell Res.
2014
, vol. 
24
 (pg. 
943
-
958
)
[PubMed]
131
Liu
C.C.
Lin
Y.C.
Chen
Y.H.
Chen
C.M.
Pang
L.Y.
Chen
H.A.
Wu
P.R.
Lin
M.Y.
Jiang
S.T.
Tsai
T.F.
Chen
R.H.
Cul3-KLHL20 ubiquitin ligase governs the turnover of ULK1 and VPS34 complexes to control autophagy termination
Mol. Cell
2016
, vol. 
61
 (pg. 
84
-
97
)
[PubMed]
132
Zhang
T.
Dong
K.
Liang
W.
Xu
D.
Xia
H.
Geng
J.
Najafov
A.
Liu
M.
Li
Y.
Han
X.
, et al. 
G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L
eLife
2015
, vol. 
4
 pg. 
e06734
 
[PubMed]
133
Xiao
J.
Zhang
T.
Xu
D.
Wang
H.
Cai
Y.
Jin
T.
Liu
M.
Jin
M.
Wu
K.
Yuan
J.
FBXL20-mediated Vps34 ubiquitination as a p53 controlled checkpoint in regulating autophagy and receptor degradation
Genes. Dev.
2015
, vol. 
29
 (pg. 
184
-
196
)
[PubMed]
134
Luo
S.
Rubinsztein
D.C.
Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL
Cell Death Differ.
2010
, vol. 
17
 (pg. 
268
-
277
)
[PubMed]
135
Levine
B.
Liu
R.
Dong
X.
Zhong
Q.
Beclin orthologs: integrative hubs of cell signaling, membrane trafficking, and physiology
Trends Cell Biol.
2015
, vol. 
25
 (pg. 
533
-
544
)
[PubMed]
136
Feng
Y.
He
D.
Yao
Z.
Klionsky
D.J.
The machinery of macroautophagy
Cell Res.
2014
, vol. 
24
 (pg. 
24
-
41
)
[PubMed]
137
Klionsky
D.J.
Codogno
P.
The mechanism and physiological function of macroautophagy
J. Innate Immun.
2013
, vol. 
5
 (pg. 
427
-
433
)
[PubMed]
138
Schneider
J.L.
Cuervo
A.M.
Autophagy and human disease: emerging themes
Curr. Opin. Genet. Dev.
2014
, vol. 
26
 (pg. 
16
-
23
)
[PubMed]
139
Schink
K.O.
Raiborg
C.
Stenmark
H.
Phosphatidylinositol 3-phosphate, a lipid that regulates membrane dynamics, protein sorting and cell signalling
BioEssays
2013
, vol. 
35
 (pg. 
900
-
912
)
[PubMed]
140
Raiborg
C.
Schink
K.O.
Stenmark
H.
Class III phosphatidylinositol 3-kinase and its catalytic product PtdIns3P in regulation of endocytic membrane traffic
FEBS J.
2013
, vol. 
280
 (pg. 
2730
-
2742
)
[PubMed]
141
Bago
R.
Malik
N.
Munson
M.J.
Prescott
A.R.
Davies
P.
Sommer
E.
Shpiro
N.
Ward
R.
Cross
D.
Ganley
I.G.
Alessi
D.R.
Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase
Biochem. J.
2014
, vol. 
463
 (pg. 
413
-
427
)
[PubMed]
142
Pasquier
B.
El-Ahmad
Y.
Filoche-Romme
B.
Dureuil
C.
Fassy
F.
Abecassis
P.Y.
Mathieu
M.
Bertrand
T.
Benard
T.
Barriere
C.
, et al. 
Discovery of (2S)-8-[(3R)-3-methylmorpholin-4-yl]-1-(3-methyl-2-oxobutyl)-2-(trifluoromethyl)- 3,4-dihydro-2H-pyrimido[1,2-a]pyrimidin-6-one: a novel potent and selective inhibitor of Vps34 for the treatment of solid tumors
J. Med. Chem.
2015
, vol. 
58
 (pg. 
376
-
400
)
[PubMed]
143
Dowdle
W.E.
Nyfeler
B.
Nagel
J.
Elling
R.A.
Liu
S.
Triantafellow
E.
Menon
S.
Wang
Z.
Honda
A.
Pardee
G.
, et al. 
Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo
Nat. Cell Biol.
2014
, vol. 
16
 (pg. 
1069
-
1079
)
[PubMed]
144
Ronan
B.
Flamand
O.
Vescovi
L.
Dureuil
C.
Durand
L.
Fassy
F.
Bachelot
M.F.
Lamberton
A.
Mathieu
M.
Bertrand
T.
, et al. 
A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy
Nat. Chem. Biol.
2014
, vol. 
10
 (pg. 
1013
-
1019
)
[PubMed]
145
Juhasz
G.
Hill
J.H.
Yan
Y.
Sass
M.
Baehrecke
E.H.
Backer
J.M.
Neufeld
T.P.
The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila
J. Cell Biol.
2008
, vol. 
181
 (pg. 
655
-
666
)
[PubMed]
146
Jaber
N.
Dou
Z.
Chen
J.S.
Catanzaro
J.
Jiang
Y.P.
Ballou
L.M.
Selinger
E.
Ouyang
X.
Lin
R.Z.
Zhang
J.
Zong
W.X.
Class III PI3K Vps34 plays an essential role in autophagy and in heart and liver function
Proc. Natl. Acad. Sci. U.S.A.
2012
, vol. 
109
 (pg. 
2003
-
2008
)
[PubMed]
147
MacKenzie
M.G.
Hamilton
D.L.
Murray
J.T.
Taylor
P.M.
Baar
K.
mVps34 is activated following high-resistance contractions
J. Physiol.
2009
, vol. 
587
 (pg. 
253
-
260
)
[PubMed]
148
Fetalvero
K.M.
Yu
Y.
Goetschkes
M.
Liang
G.
Valdez
R.A.
Gould
T.
Triantafellow
E.
Bergling
S.
Loureiro
J.
Eash
J.
, et al. 
Defective autophagy and mTORC1 signaling in myotubularin null mice
Mol. Cell. Biol.
2013
, vol. 
33
 (pg. 
98
-
110
)
[PubMed]
149
Bridges
D.
Ma
J.T.
Park
S.
Inoki
K.
Weisman
L.S.
Saltiel
A.R.
Phosphatidylinositol 3,5-bisphosphate plays a role in the activation and subcellular localization of mechanistic target of rapamycin 1
Mol. Biol. Cell
2012
, vol. 
23
 (pg. 
2955
-
2962
)
[PubMed]
150
Fang
Y.
Vilella-Bach
M.
Bachmann
R.
Flanigan
A.
Chen
J.
Phosphatidic acid-mediated mitogenic activation of mTOR signaling
Science
2001
, vol. 
294
 (pg. 
1942
-
1945
)
[PubMed]
151
Chen
Y.
Zheng
Y.
Foster
D.A.
Phospholipase D confers rapamycin resistance in human breast cancer cells
Oncogene
2003
, vol. 
22
 (pg. 
3937
-
3942
)
[PubMed]
152
Yoon
M.S.
Du
G.
Backer
J.M.
Frohman
M.A.
Chen
J.
Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway
J. Cell Biol.
2011
, vol. 
195
 (pg. 
435
-
447
)
[PubMed]
153
Xu
L.
Salloum
D.
Medlin
P.S.
Saqcena
M.
Yellen
P.
Perrella
B.
Foster
D.A.
Phospholipase D mediates nutrient input to mammalian target of rapamycin complex 1 (mTORC1)
J. Biol. Chem.
2011
, vol. 
286
 (pg. 
25477
-
25486
)
[PubMed]
154
Bar-Peled
L.
Sabatini
D.M.
Regulation of mTORC1 by amino acids
Trends Cell Biol.
2014
, vol. 
24
 (pg. 
400
-
406
)
[PubMed]
155
Toschi
A.
Lee
E.
Xu
L.
Garcia
A.
Gadir
N.
Foster
D.A.
Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin
Mol. Cell. Biol.
2009
, vol. 
29
 (pg. 
1411
-
1420
)
[PubMed]
156
Yoon
M.S.
Chen
J.
Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis
Mol. Biol. Cell
2013
, vol. 
24
 (pg. 
3754
-
3763
)
[PubMed]
157
Dall'Armi
C.
Hurtado-Lorenzo
A.
Tian
H.
Morel
E.
Nezu
A.
Chan
R.B.
Yu
W.H.
Robinson
K.S.
Yeku
O.
Small
S.A.
, et al. 
The phospholipase D1 pathway modulates macroautophagy
Nat. Commun.
2010
, vol. 
1
 pg. 
142
 
[PubMed]
158
Sagona
A.P.
Nezis
I.P.
Pedersen
N.M.
Liestol
K.
Poulton
J.
Rusten
T.E.
Skotheim
R.I.
Raiborg
C.
Stenmark
H.
PtdIns(3)P controls cytokinesis through KIF13A-mediated recruitment of FYVE-CENT to the midbody
Nat. Cell Biol.
2010
, vol. 
12
 (pg. 
362
-
371
)
[PubMed]
159
Nielsen
E.
Severin
F.
Backer
J.M.
Hyman
A.A.
Zerial
M.
Rab5 regulates motility of early endosomes on microtubules
Nat. Cell Biol.
1999
, vol. 
1
 (pg. 
376
-
382
)
[PubMed]
160
Carlton
J.G.
Martin-Serrano
J.
Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery
Science
2007
, vol. 
316
 (pg. 
1908
-
1912
)
[PubMed]
161
Slagsvold
T.
Aasland
R.
Hirano
S.
Bache
K.G.
Raiborg
C.
Trambaiolo
D.
Wakatsuki
S.
Stenmark
H.
Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
19600
-
19606
)
[PubMed]
162
Kuo
T.C.
Chen
C.T.
Baron
D.
Onder
T.T.
Loewer
S.
Almeida
S.
Weismann
C.M.
Xu
P.
Houghton
J.M.
Gao
F.B.
, et al. 
Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity
Nat. Cell Biol.
2011
, vol. 
13
 (pg. 
1214
-
1223
)
[PubMed]
163
Boularan
C.
Kamenyeva
O.
Cho
H.
Kehrl
J.H.
Resistance to inhibitors of cholinesterase (Ric)-8A and Galphai contribute to cytokinesis abscission by controlling vacuolar protein-sorting (Vps)34 activity
PLoS One
2014
, vol. 
9
 pg. 
e86680
 
[PubMed]
164
Xu
N.
Gao
X.Q.
Zhao
X.Y.
Zhu
D.Z.
Zhou
L.Z.
Zhang
X.S.
Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation
Plant Mol. Biol.
2011
, vol. 
77
 (pg. 
251
-
260
)
[PubMed]
165
Lee
Y.
Kim
E.S.
Choi
Y.
Hwang
I.
Staiger
C.J.
Chung
Y.Y.
Lee
Y.
The Arabidopsis phosphatidylinositol 3-kinase is important for pollen development
Plant Physiol.
2008
, vol. 
147
 (pg. 
1886
-
1897
)
[PubMed]
166
Bohdanowicz
M.
Grinstein
S.
Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis
Physiol. Rev.
2013
, vol. 
93
 (pg. 
69
-
106
)
[PubMed]
167
Vieira
O.V.
Botelho
R.J.
Rameh
L.
Brachmann
S.M.
Matsuo
T.
Davidson
H.W.
Schreiber
A.
Backer
J.M.
Cantley
L.C.
Grinstein
S.
Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation
J. Cell Biol.
2001
, vol. 
155
 (pg. 
19
-
25
)
[PubMed]
168
Henry
R.M.
Hoppe
A.D.
Joshi
N.
Swanson
J.A.
The uniformity of phagosome maturation in macrophages
J. Cell Biol.
2004
, vol. 
164
 (pg. 
185
-
194
)
[PubMed]
169
Feliciano
W.D.
Yoshida
S.
Straight
S.W.
Swanson
J.A.
Coordination of the Rab5 cycle on macropinosomes
Traffic
2011
, vol. 
12
 (pg. 
1911
-
1922
)
[PubMed]
170
Welliver
T.P.
Swanson
J.A.
A growth factor signaling cascade confined to circular ruffles in macrophages
Biol. Open
2012
, vol. 
1
 (pg. 
754
-
760
)
[PubMed]
171
Kinchen
J.M.
Doukoumetzidis
K.
Almendinger
J.
Stergiou
L.
Tosello-Trampont
A.
Sifri
C.D.
Hengartner
M.O.
Ravichandran
K.S.
A pathway for phagosome maturation during engulfment of apoptotic cells
Nat. Cell Biol.
2008
, vol. 
10
 (pg. 
556
-
566
)
[PubMed]
172
Bohdanowicz
M.
Cosio
G.
Backer
J.M.
Grinstein
S.
Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes
J. Cell Biol.
2010
, vol. 
191
 (pg. 
999
-
1012
)
[PubMed]
173
Anderson
K.E.
Boyle
K.B.
Davidson
K.
Chessa
T.A.
Kulkarni
S.
Jarvis
G.E.
Sindrilaru
A.
Scharffetter-Kochanek
K.
Rausch
O.
Stephens
L.R.
Hawkins
P.T.
CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks
Blood
2008
, vol. 
112
 (pg. 
5202
-
5211
)
[PubMed]
174
Kanai
F.
Liu
H.
Field
S.J.
Akbary
H.
Matsuo
T.
Brown
G.E.
Cantley
L.C.
Yaffe
M.B.
The PX domains of p47phox and p40phox bind to lipid products of PI(3)K
Nat. Cell Biol.
2001
, vol. 
3
 (pg. 
675
-
678
)
[PubMed]
175
Yang
C.S.
Lee
J.S.
Rodgers
M.
Min
C.K.
Lee
J.Y.
Kim
H.J.
Lee
K.H.
Kim
C.J.
Oh
B.
Zandi
E.
, et al. 
Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation
Cell Host Microbe
2012
, vol. 
11
 (pg. 
264
-
276
)
[PubMed]
176
Martinez
J.
Malireddi
R.K.
Lu
Q.
Cunha
L.D.
Pelletier
S.
Gingras
S.
Orchard
R.
Guan
J.L.
Tan
H.
Peng
J.
, et al. 
Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins
Nat. Cell Biol.
2015
, vol. 
17
 (pg. 
893
-
906
)
[PubMed]
177
Martinez
J.
Almendinger
J.
Oberst
A.
Ness
R.
Dillon
C.P.
Fitzgerald
P.
Hengartner
M.O.
Green
D.R.
Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells
Proc. Natl. Acad. Sci. U.S.A.
2011
, vol. 
108
 (pg. 
17396
-
17401
)
[PubMed]
178
Florey
O.
Kim
S.E.
Sandoval
C.P.
Haynes
C.M.
Overholtzer
M.
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes
Nat. Cell Biol.
2011
, vol. 
13
 (pg. 
1335
-
1343
)
[PubMed]
179
Lu
N.
Shen
Q.
Mahoney
T.R.
Neukomm
L.J.
Wang
Y.
Zhou
Z.
Two PI 3-kinases and one PI 3-phosphatase together establish the cyclic waves of phagosomal PtdIns(3)P critical for the degradation of apoptotic cells
PLoS Biol.
2012
, vol. 
10
 pg. 
e1001245
 
[PubMed]
180
Braccini
L.
Ciraolo
E.
Campa
C.C.
Perino
A.
Longo
D.L.
Tibolla
G.
Pregnolato
M.
Cao
Y.
Tassone
B.
Damilano
F.
, et al. 
PI3K-C2gamma is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling
Nat. Commun.
2015
, vol. 
6
 pg. 
7400
 
[PubMed]
181
Shin
H.W.
Hayashi
M.
Christoforidis
S.
Lacas-Gervais
S.
Hoepfner
S.
Wenk
M.R.
Modregger
J.
Uttenweiler-Joseph
S.
Wilm
M.
Nystuen
A.
, et al. 
An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway
J. Cell Biol.
2005
, vol. 
170
 (pg. 
607
-
618
)
[PubMed]
182
Berger
S.B.
Romero
X.
Ma
C.
Wang
G.
Faubion
W.A.
Liao
G.
Compeer
E.
Keszei
M.
Rameh
L.
Wang
N.
, et al. 
SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages
Nat. Immunol.
2010
, vol. 
11
 (pg. 
920
-
927
)
[PubMed]
183
Tatsuo
H.
Ono
N.
Tanaka
K.
Yanagi
Y.
SLAM (CDw150) is a cellular receptor for measles virus
Nature
2000
, vol. 
406
 (pg. 
893
-
897
)
[PubMed]
184
Joubert
P.E.
Meiffren
G.
Gregoire
I.P.
Pontini
G.
Richetta
C.
Flacher
M.
Azocar
O.
Vidalain
P.O.
Vidal
M.
Lotteau
V.
, et al. 
Autophagy induction by the pathogen receptor CD46
Cell Host Microbe
2009
, vol. 
6
 (pg. 
354
-
366
)
[PubMed]
185
Dehring
D.A.
Adler
A.S.
Hosseini
A.
Hicke
L.
A C-terminal sequence in the guanine nucleotide exchange factor Sec 7 mediates Golgi association and interaction with the Rsp5 ubiquitin ligase
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
34188
-
34196
)
[PubMed]
186
Cruz-Garcia
D.
Curwin
A.J.
Popoff
J.F.
Bruns
C.
Duran
J.M.
Malhotra
V.
Remodeling of secretory compartments creates CUPS during nutrient starvation
J. Cell Biol.
2014
, vol. 
207
 (pg. 
695
-
703
)
[PubMed]
187
Yu
L.
McPhee
C.K.
Zheng
L.
Mardones
G.A.
Rong
Y.
Peng
J.
Mi
N.
Zhao
Y.
Liu
Z.
Wan
F.
, et al. 
Termination of autophagy and reformation of lysosomes regulated by mTOR
Nature
2010
, vol. 
465
 (pg. 
942
-
946
)
[PubMed]
188
Lorenzo
D.N.
Badea
A.
Davis
J.
Hostettler
J.
He
J.
Zhong
G.
Zhuang
X.
Bennett
V.
A PIK3C3-ankyrin-B-dynactin pathway promotes axonal growth and multiorganelle transport
J. Cell Biol.
2014
, vol. 
207
 (pg. 
735
-
752
)
[PubMed]
189
Miaczynska
M.
Christoforidis
S.
Giner
A.
Shevchenko
A.
Uttenweiler-Joseph
S.
Habermann
B.
Wilm
M.
Parton
R.G.
Zerial
M.
APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment
Cell
2004
, vol. 
116
 (pg. 
445
-
456
)
[PubMed]
190
Schenck
A.
Goto-Silva
L.
Collinet
C.
Rhinn
M.
Giner
A.
Habermann
B.
Brand
M.
Zerial
M.
The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development
Cell
2008
, vol. 
133
 (pg. 
486
-
497
)
[PubMed]
191
Kalaidzidis
I.
Miaczynska
M.
Brewinska-Olchowik
M.
Hupalowska
A.
Ferguson
C.
Parton
R.G.
Kalaidzidis
Y.
Zerial
M.
APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments
J. Cell Biol.
2015
, vol. 
211
 (pg. 
123
-
144
)
[PubMed]
192
Di Fiore
P.P.
von Zastrow
M.
Endocytosis, signaling, and beyond
Cold Spring Harb. Perspect. Biol.
2014
, vol. 
6
 pg. 
a016865
 
[PubMed]
193
Rohatgi
R.A.
Janusis
J.
Leonard
D.
Bellve
K.D.
Fogarty
K.E.
Baehrecke
E.H.
Corvera
S.
Shaw
L.M.
Beclin 1 regulates growth factor receptor signaling in breast cancer
Oncogene
2015
, vol. 
34
 (pg. 
5352
-
5362
)
[PubMed]
194
Yue
Z.
Jin
S.
Yang
C.
Levine
A.J.
Heintz
N.
Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor
Proc. Natl. Acad. Sci. U.S.A.
2003
, vol. 
100
 (pg. 
15077
-
15082
)
[PubMed]
195
Nemazanyy
I.
Montagnac
G.
Russell
R.C.
Morzyglod
L.
Burnol
A.F.
Guan
K.L.
Pende
M.
Panasyuk
G.
Class III PI3K regulates organismal glucose homeostasis by providing negative feedback on hepatic insulin signalling
Nat. Commun.
2015
, vol. 
6
 pg. 
8283
 
[PubMed]
196
Dayde
D.
Guerard
M.
Perron
P.
Hatat
A.S.
Barrial
C.
Eymin
B.
Gazzeri
S.
Nuclear trafficking of EGFR by Vps34 represses Arf expression to promote lung tumor cell survival
Oncogene
2015
 
doi:10.1038/onc.2015.480
[PubMed]
197
Vasudevan
K.M.
Barbie
D.A.
Davies
M.A.
Rabinovsky
R.
McNear
C.J.
Kim
J.J.
Hennessy
B.T.
Tseng
H.
Pochanard
P.
Kim
S.Y.
, et al. 
AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer
Cancer Cell
2009
, vol. 
16
 (pg. 
21
-
32
)
[PubMed]
198
Virbasius
J.V.
Song
X.
Pomerleau
D.P.
Zhan
Y.
Zhou
G.W.
Czech
M.P.
Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate
Proc. Natl. Acad. Sci. U.S.A.
2001
, vol. 
98
 (pg. 
12908
-
12913
)
[PubMed]
199
Naguib
A.
Bencze
G.
Cho
H.
Zheng
W.
Tocilj
A.
Elkayam
E.
Faehnle
C.R.
Jaber
N.
Pratt
C.P.
Chen
M.
, et al. 
PTEN functions by recruitment to cytoplasmic vesicles
Mol. Cell
2015
, vol. 
58
 (pg. 
255
-
268
)
[PubMed]
200
Farkas
T.
Daugaard
M.
Jaattela
M.
Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy
J. Biol. Chem.
2011
, vol. 
286
 (pg. 
38904
-
38912
)
[PubMed]
201
Hu
G.
Hacham
M.
Waterman
S.R.
Panepinto
J.
Shin
S.
Liu
X.
Gibbons
J.
Valyi-Nagy
T.
Obara
K.
Jaffe
H.A.
, et al. 
PI3K signaling of autophagy is required for starvation tolerance and virulence of Cryptococcus neoformans
J. Clin. Invest.
2008
, vol. 
118
 (pg. 
1186
-
1197
)
[PubMed]
202
Jezewski
S.
von der Heide
M.
Poltermann
S.
Hartl
A.
Kunkel
W.
Zipfel
P.F.
Eck
R.
Role of the Vps34p-interacting protein Ade5,7p in hyphal growth and virulence of Candida albicans
Microbiology
2007
, vol. 
153
 (pg. 
2351
-
2362
)
[PubMed]
203
Mallo
G.V.
Espina
M.
Smith
A.C.
Terebiznik
M.R.
Aleman
A.
Finlay
B.B.
Rameh
L.E.
Grinstein
S.
Brumell
J.H.
SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34
J. Cell Biol.
2008
, vol. 
182
 (pg. 
741
-
752
)
[PubMed]
204
Chen
Y.
Johnson
J.
Macdonald
P.
Wu
B.
Mueller
J.D.
Observing protein interactions and their stoichiometry in living cells by brightness analysis of fluorescence fluctuation experiments
Methods Enzymol.
2010
, vol. 
472
 (pg. 
345
-
363
)
[PubMed]
205
Zi
Z.
Song
Z.
Zhang
S.
Ye
Y.
Li
C.
Xu
M.
Zou
Y.
He
L.
Zhu
H.
Rubicon deficiency enhances cardiac autophagy and protects mice from lipopolysaccharide-induced lethality and reduction in stroke volume
J. Cardiovasc. Pharmacol.
2015
, vol. 
65
 (pg. 
252
-
261
)
[PubMed]
206
Nemazanyy
I.
Blaauw
B.
Paolini
C.
Caillaud
C.
Protasi
F.
Mueller
A.
Proikas-Cezanne
T.
Russell
R.C.
Guan
K.L.
Nishino
I.
, et al. 
Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease
EMBO Mol. Med.
2013
, vol. 
5
 (pg. 
870
-
890
)
[PubMed]
207
Reifler
A.
Li
X.
Archambeau
A.J.
McDade
J.R.
Sabha
N.
Michele
D.E.
Dowling
J.J.
Conditional knockout of pik3c3 causes a murine muscular dystrophy
Am. J. Pathol.
2014
, vol. 
184
 (pg. 
1819
-
1830
)
[PubMed]
208
Bechtel
W.
Helmstadter
M.
Balica
J.
Hartleben
B.
Schell
C.
Huber
T.B.
The class III phosphatidylinositol 3-kinase PIK3C3/VPS34 regulates endocytosis and autophagosome-autolysosome formation in podocytes
Autophagy
2013
, vol. 
9
 (pg. 
1097
-
1099
)
[PubMed]
209
Wang
L.
Budolfson
K.
Wang
F.
Pik3c3 deletion in pyramidal neurons results in loss of synapses, extensive gliosis and progressive neurodegeneration
Neuroscience
2011
, vol. 
172
 (pg. 
427
-
442
)
[PubMed]
210
Zhou
X.
Wang
L.
Hasegawa
H.
Amin
P.
Han
B.X.
Kaneko
S.
He
Y.
Wang
F.
Deletion of PIK3C3/Vps34 in sensory neurons causes rapid neurodegeneration by disrupting the endosomal but not the autophagic pathway
Proc. Natl. Acad. Sci. U.S.A.
2010
, vol. 
107
 (pg. 
9424
-
9429
)
[PubMed]
211
Morel
E.
Chamoun
Z.
Lasiecka
Z.M.
Chan
R.B.
Williamson
R.L.
Vetanovetz
C.
Dall'Armi
C.
Simoes
S.
Point Du Jour
K.S.
McCabe
B.D.
, et al. 
Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system
Nat. Commun.
2013
, vol. 
4
 pg. 
2250
 
[PubMed]
212
Issman-Zecharya
N.
Schuldiner
O.
The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation
Dev. Cell
2014
, vol. 
31
 (pg. 
461
-
473
)
[PubMed]
213
McLeod
I.X.
Zhou
X.
Li
Q.J.
Wang
F.
He
Y.W.
The class III kinase Vps34 promotes T lymphocyte survival through regulating IL-7Ralpha surface expression
J. Immunol.
2011
, vol. 
187
 (pg. 
5051
-
5061
)
[PubMed]
214
Willinger
T.
Flavell
R.A.
Canonical autophagy dependent on the class III phosphoinositide-3 kinase Vps34 is required for naive T-cell homeostasis
Proc. Natl. Acad. Sci. U.S.A.
2012
, vol. 
109
 (pg. 
8670
-
8675
)
[PubMed]
215
Parekh
V.V.
Wu
L.
Boyd
K.L.
Williams
J.A.
Gaddy
J.A.
Olivares-Villagomez
D.
Cover
T.L.
Zong
W.X.
Zhang
J.
Van Kaer
L.
Impaired autophagy, defective T cell homeostasis, and a wasting syndrome in mice with a T cell-specific deletion of Vps34
J. Immunol.
2013
, vol. 
190
 (pg. 
5086
-
5101
)
[PubMed]
216
Anding
A.L.
Baehrecke
E.H.
Vps15 is required for stress induced and developmentally triggered autophagy and salivary gland protein secretion in Drosophila
Cell Death Differ.
2015
, vol. 
22
 (pg. 
457
-
464
)
[PubMed]
217
Folkes
A.J.
Ahmadi
K.
Alderton
W.K.
Alix
S.
Baker
S.J.
Box
G.
Chuckowree
I.S.
Clarke
P.A.
Depledge
P.
Eccles
S.A.
, et al. 
The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-t hieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer
J. Med. Chem.
2008
, vol. 
51
 (pg. 
5522
-
5532
)
[PubMed]