The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))-kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34(-/-) animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation.     

The class IA phosphatidylinositol 3-kinase p110-beta subunit is a positive regulator of autophagy

Autophagy is an evolutionarily conserved cell renewal process that depends on phosphatidylinositol 3-phosphate (PtdIns(3)P). In metazoans, autophagy is inhibited by PtdIns(3,4,5)P(3), the product of class IA PI3Ks, which mediates the activation of the Akt-TOR kinase cascade. However, the precise function of class IA PI3Ks in autophagy remains undetermined. Class IA PI3Ks are heterodimeric proteins consisting of an 85-kD regulatory subunit and a 110-kD catalytic subunit. Here we show that the class IA p110-β catalytic subunit is a positive regulator of autophagy. Genetic deletion of p110-β results in impaired autophagy in mouse embryonic fibroblasts, liver, and heart. p110-β does not promote autophagy by affecting the Akt-TOR pathway. Rather, it associates with the autophagy-promoting Vps34-Vps15-Beclin 1-Atg14L complex and facilitates the generation of cellular PtdIns(3)P. Our results unveil a previously unknown function for p110-β as a positive regulator of autophagy in multicellular organisms.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Δvps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Δvps15 larvae. Fluorescence microscopy showed that Δvps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates.. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.     

The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates

Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Deltavps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.     

Elevated RalA activity in the hippocampus of PI3Kγ knock-out mice lacking NMDAR-dependent long-term depression

Phosphoinositide 3-kinases (PI3Ks) play key roles in synaptic plasticity and cognitive functions in the brain. We recently found that genetic deletion of PI3Kγ, the only known member of class IB PI3Ks, results in impaired N-methyl-_D-aspartate receptor-dependent long-term depression (NMDAR-LTD) in the hippocampus. The activity of RalA, a small GTP-binding protein, increases following NMDAR-LTD inducing stimuli, and this increase in RalA activity is essential for inducing NMDAR-LTD. We found that RalA activity increased significantly in PI3Kγ knockout mice. Furthermore, NMDAR-LTDinducing stimuli did not increase RalA activity in PI3Kγ knockout mice. These results suggest that constitutively increased RalA activity occludes further increases in RalA activity during induction of LTD, causing impaired NMDARLTD. We propose that PI3Kγ regulates the activity of RalA, which is one of the molecular mechanisms inducing NMDARdependent LTD. [BMB Reports 2013; 46(2): 103-106]     

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

The Class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first described as a component of the vacuolar sorting system in Saccharomyces cerevisiae and is the sole PI3K in yeast. The homologue in mammalian cells, hVps34, has been studied extensively in the context of endocytic sorting. However, hVps34 also plays an important role in the ability of cells to respond to changes in nutrient conditions. Recent studies have shown that mammalian hVps34 is required for the activation of the mTOR (mammalian target of rapamycin)/S6K1 (S6 kinase 1) pathway, which regulates protein synthesis in response to nutrient availability. In both yeast and mammalian cells, Class III PI3Ks are also required for the induction of autophagy during nutrient deprivation. Finally, mammalian hVps34 is itself regulated by nutrients. Thus Class III PI3Ks are implicated in the regulation of both autophagy and, through the mTOR pathway, protein synthesis, and thus contribute to the integration of cellular responses to changing nutritional status.     

Vps34 and PLD1 take center stage in nutrient signaling: their dual roles in regulating autophagy

Autophagy is a critical pathway leading to lysosomal degradation of cellular components in response to changes in nutrient availability. Autophagy includes the biogenesis of autophagosomes and their sequential maturation through fusion with endo-lysosomes. The class III PI3 kinase Vps34 and its product phosphatidylinositol-3-phosphate (PI(3)P) play a critical role in this process, and enable the amino acid-mediated activation of mammalian target of rapamycin (mTOR), a suppressor of autophagy. Recent studies have shown that phospholipase PLD1, a downstream regulator of Vps34, is also closely involved in both mTOR activation and autophagy. This mini review summarizes recent findings in the regulation of Vps34 and PLD1 and highlights the role of these lipid-metabolizing enzymes in both mTOR activation and autophagy.     

Elevating PI3P drives select downstream membrane trafficking pathways

Phosphoinositide signaling lipids are essential for several cellular processes. The requirement for a phosphoinositide is conventionally studied by depleting the corresponding lipid kinase. However, there are very few reports on the impact of elevating phosphoinositides. That phosphoinositides are dynamically elevated in response to stimuli suggests that, in addition to being required, phosphoinositides drive downstream pathways. To test this hypothesis, we elevated the levels of phosphatidylinositol-3-phosphate (PI3P) by generating hyperactive alleles of the yeast phosphatidylinositol 3-kinase, Vps34. We find that hyperactive Vps34 drives certain pathways, including phosphatidylinositol-3,5-bisphosphate synthesis and retrograde transport from the vacuole. This demonstrates that PI3P is rate limiting in some pathways. Interestingly, hyperactive Vps34 does not affect endosomal sorting complexes required for transport (ESCRT) function. Thus, elevating PI3P does not always increase the rate of PI3P-dependent pathways. Elevating PI3P can also delay a pathway. Elevating PI3P slowed late steps in autophagy, in part by delaying the disassembly of autophagy proteins from mature autophagosomes as well as delaying fusion of autophagosomes with the vacuole. This latter defect is likely due to a more general defect in vacuole fusion, as assessed by changes in vacuole morphology. These studies suggest that stimulus-induced elevation of phosphoinositides provides a way for these stimuli to selectively regulate downstream processes.     

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.Key words: PIK3C3/Vps34, ATG7, sensory neurons, neurodegeneration, autophagy, abnormal endosomePIK3C3 (commonly known as Vps34) is the class III phosphatidylinositol 3-kinase (PtdIns3K) that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PtdIns3P). It is the only PtdIns3K that is conserved from lower eukaryotes to mammals, and represents the most ancient form of PtdIns3Ks. Studies in invertebrate organisms as well as mammalian cell lines show that PIK3C3/Vps34 regulates multiple aspects of both the endocytic and the autophagic pathways. On one hand, PIK3C3 is important for the progression of early endosome to late endosome, and the biogenesis of multivesicular bodies. On the other hand, PIK3C3 is critical for the initiation of autophagosome formation. A chemical inhibitor of PIK3C3, 3-MA, has been commonly used as a specific inhibitor for autophagy. The distinct functions of PIK3C3 are thought to be carried out by at least two different PIK3C3 complexes. In yeast, complex I (Vps34, Vps15, Atg6 and Atg14) is involved in autophagy, whereas complex II (Vps34, Vps15, Atg6 and Vps38) functions in the vacuolar protein sorting process. In mammals, the homologue of complex I (PIK3C3, p150, Beclin 1 and Atg14L) activates autophagy, whereas the homologue of complex II (PIK3C3, p150, Beclin 1 and UVRAG/Vps38) regulates endocytic trafficking.To characterize the in vivo function of PIK3C3 in mammals, we generated a conditional allele of the Pik3c3 gene in mouse and specifically deleted it in postmitotic sensory neurons (Pik3c3-cKO mouse). We focused our analyses on sensory neurons because Pik3c3 is most abundantly expressed in these neurons. Detailed analyses of the sensory ganglia in the knockout mice reveal rapid but differential neurodegenerations of different types of sensory neurons within a few days after birth. Large-diameter myelinated mechanosensory and proprioceptive neurons undergo fast degeneration, whereas mutant small-diameter unmyelinated nociceptive neurons degenerate slower and survive longer.Interestingly, the large-diameter Pik3c3-deleted neurons rapidly accumulate ubiquitin-positive aggregates as well as numerous enlarged vesicles, which are likely abnormal endosomes. The accumulation of enlarged vesicles not only sequesters the cellular membrane source, but also could create trafficking jams that block the transport of prosurvival signals and/or material and organelles, and thus may underlie the rapid demise of large neurons. By contrast, the small-diameter Pik3c3-deleted neurons contain a limited number of vacuoles but gradually build up lysosome- like organelles. The marked increase of lysosomes seems to be more tolerable by neurons, but the mechanism underlying this phenotype is unclear. It could represent a protective and homeostatic response of neurons challenged with stress and insults to their endomembrane system. Alternatively, since sorting of many lysosomal proteins requires PtdIns3P, this phenotype may also result from a build-up of nonfunctional lysosomes as was the case in cathepsin B and L knockout mice. It is also unclear why two types of sensory neurons respond differently to a universal insult. One speculative explanation is that the large-diameter neurons are constantly activated under normal physiological conditions by touch and body movement and thus they contain more active endocytic and membrane trafficking processes; whereas small-diameter pain-sensing neurons are normally not activated and have less endocytic events. These differences might allow the two types of neurons to respond differently to PIK3C3 deletion.We further show that the fast and differential degeneration phenotypes in the Pik3c3-cKO mice are caused primarily by a disruption in the endosomal but not the autophagic pathway. This is validated by comparing the neuronal phenotypes of Pik3c3-cKO mice with those of Atg7-cKO mice, in which the autophagy-specific gene Atg7 is deleted using the same sensory neuron-specific cre driver. Disrupting autophagy leads to a slow degeneration of all types of sensory neurons over a period of several months, and formation of very large intracellular inclusion bodies in all sensory neurons. No increase of lysosomes or accumulation of enlarged vesicles is observed. The completely distinct phenotypes observed in Atg7-cKO versus Pik3c3-cKO mice suggest that inactivation of PIK3C3 primarily disrupts the endosomal pathway rather than inhibiting autophagy (at least in neurons). It calls into attention that care needs to be taken to interpret the results of using PIK3C3 inhibitors such as 3-MA as autophagy-specific inhibitors.The most surprising finding is the existence and activation of a noncanonical, PIK3C3-independent macroautophagy pathway in small-diameter Pik3c3-mutant neurons. Although PIK3C3 is traditionally viewed as indispensable for autophagy initiation, several recent studies suggest a possible PIK3C3-independent autophagy pathway in various cell lines and in Drosophila. We show that this noncanonical autophagy pathway can occur in sensory neurons in vivo using three different assays: crossing Pik3c3-cKO mice to the GFP-LC3 reporter line, western blot analyses of LC3 isoforms, and performing autophagy flux experiments. Interestingly, analyses of Pik3c3/Atg7 double-mutant neurons indicate that this alternative autophagosome initiation pathway still requires ATG7 and hence the conventional conjugation systems. Therefore, this non-canonical autophagy is distinct from the newly reported ATG5/ATG7-independent but PIK3C3-dependent autophagy. We speculate that activation of this PIK3C3-independent autophagy in small-diameter mutant neurons is part of the reason for their longer survival period.The molecular mechanism underlying the PIK3C3-independent autophagosome formation is unknown. It is possible that PtdIns3P can be generated at a low level on the membrane of pre-autophagosomes/phagophores by salvage pathways using other lipid kinases or phosphatases. Alternatively, other mechanisms may direct the formation of the crescent-shaped double membrane structures. For instance, asymmetric insertion into the membrane of proteins with amphipathic helices can induce membrane curvature; BAR domain-containing proteins can also detect and facilitate the formation of curved membrane structures. Thus, these types of proteins might potentially be recruited to nucleate the formation of pre-autophagosomes in the absence of PIK3C3. Finally, the role of this PIK3C3-independent autophagy under normal physiological conditions in vivo needs to be explored.     

Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity

Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.     

Finding a fitting shoe for Cinderella: Searching for an autophagy inhibitor

Vps34 is the ancestral phosphatidylinositol 3-kinase (PtdIns3K) isoform and is essential for endosomal trafficking of proteins to the vacuole/lysosome, autophagy and phagocytosis. Vps34-containing complexes associate with specific cellular compartments to produce PtdIns(3)P. Understanding the roles of Vps34 has been hampered by the lack of potent, specific inhibitors. To boost development of Vps34 inhibitors, we determined the crystal structures of Vps34 alone and in complexes with multitargeted PtdIns3K inhibitors. These structures provided a first glimpse into the uniquely constricted ATP-binding site of Vps34 and enabled us to model Vps34 regulation. We showed that the substrate-binding “activation” loop and the flexibly attached amphipathic C-terminal helix are crucial for catalysis on membranes. The C-terminal helix also suppresses ATP hydrolysis in the absence of membranes. We propose that membrane binding shifts the C-terminal helix to orient the enzyme for catalysis, and the Vps15 regulatory subunit, which binds to this and the preceding helix, may facilitate this process. This C-terminal region may also represent a target for specific, non-ATP-competitive PtdIns3K inhibitors.Key words: Vps34, PI 3-kinase, structure, inhibitor, enzyme, autophagy, Vps15, PtdIns3P, phosphoinositidePtdIns3Ks phosphorylate their lipid substrates at the 3-hydroxyl position of the inositol headgroup. Vps34 is the primordial PtdIns3K present in all eukaryotes and the only PtdIns3K in fungi and plants. This Cinderella of the PtdIns3Ks is responsible for much of a cell''s cleaning and self-feeding: It is essential for multivesicular body formation, autophagy and phagocytosis. It associates with endosomes, omegasomes and phagosomes producing PtdIns(3)P, the most abundant 3-phosphoinositide in resting mammalian cells, which is essential for recruiting a range of complexes to intracellular membranes, including the autophagy machinery, ESCRTs, the retromer, motor proteins and components necessary for abscission in cytokinesis. In cells, Vps34 is at the core of larger complexes that also contain two regulatory proteins, Vps15 and Beclin 1, which bind directly to Vps34. The N-terminally myristoylated putative Ser/Thr protein kinase p150/Vps15 increases the lipid kinase activity of Vps34 and facilitates its translocation to endosomal membranes and the phagophore assembly site (PAS) or phagophore (Fig. 1A).Open in a separate windowFigure 1(A) Domain organization of Vps34, its regulatory subunit Vps15 and the adaptor proteins required for autophagy induction in mammalian cells, Beclin 1 and Atg14L/Barkor (Beclin1-associated autophagy-related key regulator). (B) Structure of Drosophila Vps34 helical (green) and catalytic (red/yellow) domains. A PtdIns substrate molecule has been modeled between the activation loop (magenta) and the catalytic loop (black) and ATP was modeled based on the p110γ/ATP structure (PDB ID 1E8X). The C2 domain (cyan) was also modeled from the p110γ/ATP structure. The enzyme is oriented so that the C2 domain and C-terminal helix interact with the membrane. Two regulatory proteins bind directly to Vps34: Vps15 binds to helices kα11 and kα12 (orange), and Beclin 1 binds to the C2 domain. Both Vps15 and Beclin 1 stimulate Vps34 activity. (C) A schematic representation of the Vps34 domains and the putative change in conformation of the kα12 helix. In solution (right), the helix is closed and interacts with residues in the substrate-binding and catalytic loops to exclude water. At the membrane (left), the kα12 helix undergoes a conformational change and interacts with the membrane, enabling productive substrate binding and catalysis.We have determined the structure of the catalytic core of Vps34 (PDB ID 2X6H) (Fig. 1B), which consists of a helical solenoid domain forming an extensive interface with a bilobal catalytic domain. The catalytic domain reveals key features that are important for the catalytic mechanism of all PtdIns3Ks: A phosphate-binding loop (P-loop) that interacts with the phosphates of ATP, a substrate-binding loop or “activation” loop that recognizes the PtdIns substrate, and a catalytic loop that is required for the transfer of the ATP γ-phosphate to the 3-hydroxyl of PtdIns. For the first time in any PtdIns3K structure, all three of these elements are completely ordered. The C-terminal helix (kα12) was previously shown to be required for Vps34 catalytic activity. However, the molecular basis for its function was unknown. The Vps34 structure suggests that the C-terminal helix closely associates with the substrate-binding loop and catalytic loop in the closed conformation. Site-specific mutagenesis guided by the crystal structure provides key insights into mechanisms of enzymatic regulation of Vps34 by this C-terminal helix. Deletion of the last 10 residues or point mutations within this helix, dramatically impairs lipid kinase activity in the presence of substrate lipids, but increases basal ATPase activity in the absence of substrate. These results suggest that in the closed form of the enzyme, the amphipathic C-terminal helix acts as a lid on the catalytic site to suppress activity in the absence of substrate lipid. Hydrophobic residues in this helix are also important for membrane interaction. Enzymatic activity and membrane binding measurements are consistent with a model whereby the C-terminal helix shifts to facilitate membrane interaction and orientation of the enzyme on the membrane interface for optimal catalysis (Fig. 1C). The amphipathic character of the C-terminal region is conserved in all of the PtdIns3Ks, and it probably represents a common regulatory element in the entire family of enzymes. This may also extend to the PtdIns3Krelated enzymes such as TOR where the equivalent region has been denoted as the “FATC” domain, which also associates with membranes.Early reports showed that methylated adenosine derivatives can inhibit autophagy. It was later demonstrated that the autophagy inhibitor 3-methyladenine (3-MA) inhibits PtdIns3Ks and that other general PtdIns3K inhibitors, such as wortmannin also inhibit autophagy. Although 3-MA shows some limited Vps34 preference in vitro, with an IC₅₀ of 25 µM for Vps34 as compared with 60 µM for PtdIns3Kγ it is typically employed in cellular studies at a concentration of 10 mM, which can inhibit all PtdIns3Ks. Specific, potent inhibitors of Vps34 are acutely needed. All current PtdIns3K inhibitors are ATP-competitive, i.e., they target the ATP-binding site that is conserved among various PtdIns3K isotypes. The Vps34 structure suggests that the lack of potent Vps34 inhibitors could be accounted for by the uniquely constricted conformation of the Vps34 ATP-binding site in comparison with other PtdIns3Ks. Our structures of Vps34 in complexes with 3-MA and multitargeted PtdIns3K inhibitors (PIK-90, PIK-93 and PI-103) have provided insight into how this enzyme might be specifically inhibited. The slight preference for Vps34 inhibition by 3-MA probably arises from a hydrophobic ring specific to Vps34, which encircles the 3-methyl group of 3-MA. The insights arising from these structures have enabled us to develop a first generation of inhibitors with improved potency and Vps34 selectivity, e.g., the compound PT210 that has an IC₅₀ of 0.45 µM as compared with 4.5 µM for PtdIns3Kγ. Further development of inhibitors guided by structures could lead to a new generation of improved inhibitors with applications as chemical tools to investigate PtdIns(3) P-dependendent pathways and as therapeutic agents.     

Vps15 is required for stress induced and developmentally triggered autophagy and salivary gland protein secretion in Drosophila

Autophagy is a catabolic process used to deliver cellular material to the lysosome for degradation. The core Vps34/class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of Atg6, Vps15, and Vps34, is highly conserved throughout evolution, critical for recruiting autophagy-related proteins to the preautophagosomal structure and for other vesicular trafficking processes, including vacuolar protein sorting. Atg6 and Vps34 have been well characterized, but the Vps15 kinase remains poorly characterized with most studies focusing on nutrient deprivation-induced autophagy. Here, we investigate the function of Vps15 in different cellular contexts and find that it is necessary for both stress-induced and developmentally programmed autophagy in various tissues in Drosophila melanogaster. Vps15 is required for autophagy that is induced by multiple forms of stress, including nutrient deprivation, hypoxia, and oxidative stress. Furthermore, autophagy that is triggered by physiological stimuli during development in the fat body, intestine, and salivary gland also require the function of Vps15. In addition, we show that Vps15 is necessary for efficient salivary gland protein secretion. These data illustrate the broad importance of Vps15 in multiple forms of autophagy in different animal cells, and also highlight the pleiotropic function of this kinase in multiple vesicle-trafficking pathways.Autophagy is an evolutionarily conserved process in which cytoplasmic proteins or organelles are packaged into lysosomes for degradation. This process can be initiated by a variety of stimuli, such as high levels of starvation or stress, to provide nutrients to the cells or to clear the cell of damaged organelles or protein aggregates.¹ In some circumstances, autophagy can promote an alternative form of cell death, such as in the clearance of larval tissues in Drosophila melanogaster.² As defects in autophagy have been implicated in several physiological and pathological conditions, such as cancer, neurodegenerative diseases, and aging,^3,4 it is important to obtain a complete understanding of the molecular mechanisms controlling autophagy.The induction of autophagy is regulated by the Atg1/Ulk1 complex, and this complex is regulated by mechanistic target of rapamycin (mTOR).⁵ Vesicle nucleation is controlled by the class III phosphoinositide 3-kinase (PI3K) complex that generates phosphatidylinositol 3-phosphate (PI3P).⁶ This conserved complex consists of vacuolar protein sorting 34 (Vps34; also known as Pik3c3), Atg6/Becn1 (also known as Vps30 in yeast), and the serine-threonine kinase Vps15/ird1 (p150 in mammals; also known as Pik3r4).^7,8 Localized production of PI3P by Vps34 can act to recruit proteins containing PX or FYVE domains to membrane compartments, such as the autophagosome isolation membrane.⁹ Vps34 is also required more broadly for several vesicular trafficking processes such as the sorting of hydrolytic enzymes to the yeast vacuole and mammalian lysosome, and endocytic trafficking.^{10, 11, 12} There is mounting evidence demonstrating the pleiotropic function of the PI3K/Vps34 complex, but this has not been well studied in the context of autophagy under different physiological and cell contexts in animals.Of the three core PI3K complex proteins, Vps15 remains an understudied kinase, and its function has not been rigorously investigated in multicellular organisms in vivo. Most of the focus on the role of this complex in autophagy regulation has been on nutrient deprivation-initiated autophagy. Indeed, previous studies determined Vps15 to be necessary for starvation-induced autophagy in the Drosophila larval fat body.^13,14 However, its role in hormone-regulated autophagy, a process that occurs in the intestine,¹⁵ salivary glands,¹⁶ and fat body¹⁷ of developing Drosophila, as well as its role in other stress-induced conditions have not yet been examined. In order to address the role of Vps15 in these and other processes regulated by autophagy, we utilized Vps15 knockdown as well as a previously described null mutant¹⁴ to examine its role in a multicellular organism in vivo. We found that Vps15 is required not only for stress-induced autophagy in multiple tissues, but it is also a broad regulator of developmentally programmed autophagy in Drosophila. In addition, Vps15 is necessary for efficient protein secretion, as indicated by its role in the secretion of glue proteins from the Drosophila salivary gland. Together, these results highlight the importance of Vps15 in multiple processes in vivo.     

The beta identity of class I PtdIns3K: A positive role of p110β in autophagy revealed

Autophagy is critically controlled by phosphatidylinositol 3-kinases (PtdIns3Ks). The common understanding for mammalian autophagy is that class I PtdIns3Ks inhibit autophagy by activating the Akt-TOR kinase cascade, whereas the class III PtdIns3K (Vps34) promotes autophagy by generating the phospholipid PtdIns(3)P. However, direct genetic evidence for a role of class I PtdIns3Ks in autophagy has been lacking. Using mice with a conditional deletion of the class I PtdIns3K catalytic subunit isoform p110α or p110β, we revealed an unexpected function of p110β as a positive regulator of autophagy.     

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase,two proteins involved in Lafora disease

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.     

A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens

Selective autophagy of bacterial pathogens represents a host innate immune mechanism. Selective autophagy has been characterized on the basis of distinct cargo receptors but the mechanisms by which different cargo receptors are targeted for autophagic degradation remain unclear. In this study we identified a highly conserved Tectonin domain-containing protein, Tecpr1, as an Atg5 binding partner that colocalized with Atg5 at Shigella-containing phagophores. Tecpr1 activity is necessary for efficient autophagic targeting of bacteria, but has no effect on rapamycin- or starvation-induced canonical autophagy. Tecpr1 interacts with WIPI-2, a yeast Atg18 homolog and PI(3)P-interacting protein required for phagophore formation, and they colocalize to phagophores. Although Tecpr1-deficient mice appear normal, Tecpr1-deficient MEFs were defective for selective autophagy and supported increased intracellular multiplication of Shigella. Further, depolarized mitochondria and misfolded protein aggregates accumulated in the Tecpr1-knockout MEFs. Thus, we identify a Tecpr1-dependent pathway as important in targeting bacterial pathogens for selective autophagy.     

AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.     

Class II phosphoinositide 3-kinase C2alpha: what we learned so far

More than fifteen years after the first identification of a class II isoform of phosphoinositide 3-kinase (PI3K) in Drosophila melanoǵaster this subfamily remains the most enigmatic among all PI3Ks. What are the functions of these enzymes? What are their mechanisms of activation? Which downstream effectors are specifically regulated by these isoforms? Are class I and class II PI3Ks redundant or do they control different intracellular processes? And, more important, do class II PI3Ks have a role in human diseases? The recent increased interest on class II PI3Ks has started providing some answers to these questions but still a lot needs to be done to completely uncover the contribution of these enzymes to physiological processes and possibly to pathological conditions. Here we will summarise the recent findings on the alpha isoform of mammalian class II PI3Ks (PI3K-C2α ) and we will discuss the potential involvement of this enzyme in human diseases.