Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.     

Beclin 1 regulates neuronal transforming growth factor-β signaling by mediating recycling of the type I receptor ALK5

Background

Beclin 1 is a key regulator of multiple trafficking pathways, including autophagy and receptor recycling in yeast and microglia. Decreased beclin 1 levels in the CNS result in neurodegeneration, an effect attributed to impaired autophagy. However, neurons also rely heavily on trophic factors, and signaling through these pathways requires the proper trafficking of trophic factor receptors.

Results

We discovered that beclin 1 regulates signaling through the neuroprotective TGF-β pathway. Beclin 1 is required for recycling of the type I TGF-β receptor ALK5. We show that beclin 1 recruits the retromer to ALK5 and facilitates its localization to Rab11⁺ endosomes. Decreased levels of beclin 1, or its binding partners VPS34 and UVRAG, impair TGF-β signaling.

Conclusions

These findings identify beclin 1 as a positive regulator of a trophic signaling pathway via receptor recycling, and suggest that neuronal death induced by decreased beclin 1 levels may also be due to impaired trophic factor signaling.

     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

The G-Protein Rab5A Activates VPS34 Complex II,a Class III PI3K,by a Dual Regulatory Mechanism

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.     

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110Δkin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Two-layer regulation of PAQR3 on ATG14-linked class III PtdIns3K activation upon glucose starvation

As a central node of the macroautophagy/autophagy process, the BECN1/Beclin1-PIK3C3/VPS34 complex participates in different steps of autophagy by interacting with multiple molecules. The ATG14-associated PIK3C3 complex is involved in autophagy initiation, whereas the UVRAG-associated complex mainly modulates autophagosome maturation and endosome fusion. However, the molecular mechanism that coordinates the sequential execution of the autophagy program remains unknown. We have recently discovered that a Golgi-resident protein, PAQR3, regulates autophagy initiation as it preferentially facilitates the formation of the ATG14-linked PIK3C3 complex instead of the UVRAG-associated complex. Upon glucose starvation, AMPK directly phosphorylates T32 of PAQR3, which is crucial for the activation of the ATG14-associated class III PtdIns3K. Furthermore, Paqr3-deleted mice have a deficiency in exercise-induced autophagy as well as behavioral disorders. Thus, this work not only uncovers the regulatory mechanism of PAQR3 on autophagy initiation, but also provides a potential candidate therapeutic target for neurodegenerative diseases.     

Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.     

Atg14 and UVRAG: Mutually exclusive subunits of mammalian Beclin 1-PI3K complexes

Vps34, a Class III phosphatidylinositol 3-kinase (PI3-kinase), produces phosphatidylinositol 3 phosphate (PI3P) and functions in various membrane traffic pathways including endocytosis, multivesicular body formation and autophagy. In mammalian cells, Vps34 forms a complex with Beclin 1, but it remains unclear how this Vps34 complex exerts its specific function on each membrane trafficking pathway. We recently identified mammalian Atg14, a new binding partner of the Vps34-Beclin 1 complex, using a computational approach. The Atg14 complex consists of Vps34, Beclin 1 and p150, but lacks UVRAG, which was previously reported to bind the Vps34-Beclin 1 complex. Atg14 localizes to isolation membrane/phagophore during starvation and is essential for autophagosome formation. In contrast, UVRAG primarily localizes to late endosomes. Since UVRAG shows homology with yeast Vps38, we speculate that it could be a mammalian Vps38 ortholog. These findings indicate that the Vps34-Beclin 1 complex has at least two distinct functions, which can be promoted by its binding partners Atg14 and UVRAG.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.     

Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes

Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P(2) binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P(2) are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.     

Regulation of epidermal growth factor receptor endocytosis by wortmannin through activation of Rab5 rather than inhibition of phosphatidylinositol 3-kinase

The involvement of phosphatidylinositol 3-kinase (PI3K) in membrane trafficking in mammalian cells has largely come from experiments with wortmannin. This compound inhibits endosome fusion in vitro, possibly by inhibiting the production of phosphatidylinositol (PtdIns)-3-P, which co-regulates EEA1 with Rab5. However, the results from wortmannin inhibition experiments performed in vivo differ significantly. We have recently shown that wortmannin enlarges endosomes containing the epidermal growth factor receptor (EGFR) and enhances the lysosomal degradation of EGFR. In this report, we demonstrate that addition of the PI3K reaction products does not suppress wortmannin-induced enlargement of EGFR-containing endosomes and enhancement of EGFR degradation. Moreover, the effects of wortmannin on the intracellular trafficking of EGFR mimic those of the permanently activated Rab5 mutant, Rab5 Q79L, which stimulates endosome fusion. We also found that an inactive Rab5 mutant, Rab5 S34N, blocks wortmannin-induced endosome enlargement and that wortmannin stimulates the activation of Rab5. We further showed that wortmannin reduced the membrane association of p120 Ras GTPase-activating protein (GAP) and inhibited the interaction between Rab5 and p120 Ras GAP. We conclude that wortmannin alters intracellular trafficking of EGFR by activating Rab5 rather than by inhibiting PI3K.     

Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism

A marquee feature of the powerful human pathogen Mycobacterium tuberculosis is its macrophage parasitism. The intracellular survival of this microorganism rests upon its ability to arrest phagolysosome biogenesis, avoid direct cidal mechanisms in macrophages, and block efficient antigen processing and presentation. Mycobacteria prevent Rab conversion on their phagosomes and elaborate glycolipid and protein trafficking toxins that interfere with Rab effectors and regulation of specific organellar biogenesis in mammalian cells. One of the major Rab effectors affected in this process is the type III phosphatidylinositol 3-kinase hVPS34 and its enzymatic product phosphatidylinositol 3-phosphate (PI3P), a regulatory lipid earmarking organellar membranes for specific trafficking events. PI3P is also critical for the process of autophagy, recently recognized as an effector of innate and adaptive immunity. Induction of autophagy by physiological, pharmacological or immunological signals, including the major antituberculosis Th1 cytokine IFN-gamma and its downstream effector p47 GTPase LRG-47, can overcome mycobacterial phagosome maturation block and inhibit intracellular M. tuberculosis survival. This review summarizes the findings centred around the PI3P-nexus where the mycobacterial phagosome maturation block and execution stages of autophagy intersect.     

Autophagy regulation: RNF2 targets AMBRA1

The phosphatidylinositol 3-kinase complex I (PI3K complex I) is a crucial regulator of autophagy, which contains Beclin 1 (or ATG6), ATG14L, VPS34 (or the class III phosphatidylinositol 3-kinase and its adaptor VPS15) and AMBRA1, and controls autophagosome formation. In a paper recently published in Cell Research, Xia et al. report that during nutrient deprivation the ubiquitin E3 ligase RNF2 is recruited to the PI3K complex I, and ubiquitinates AMBRA1 to trigger its degradation and downregulate autophagy.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components¹. The ubiquitin-proteasome pathway is also critical during the process of autophagy. The formation of autophagosomes (double-membrane bound vacuoles that sequester cargo in bulk or in a selective manner before their delivery to the lysosomal compartment) depends on ubiquitination-like activity². The selective removal of cargo (e.g., protein aggregates, organelles) by autophagy is dependent in many cases on the ubiquitination of the cargo³. Recent studies also indicate that ubiquitination regulates the activity of autophagy proteins that comprise autophagosomes⁴.Autophagosome formation is dependent on evolutionarily conserved ATG (autophagy-related) proteins initially identified in yeast². These proteins function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via stages of initiation, elongation, and sealing. Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homolog of yeast ATG6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34² (Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P), is crucial to recruitment of ATG, which is required for the elongation and sealing of the autophagosomal membrane.Open in a separate windowFigure 1Role of RNF2 and WASH during autophagosome formation. Upon autophagy stimulation by nutrient deprivation, Beclin 1 forms a complex with VPS34 and its adaptor VPS15, ATG14L, and AMBRA1. In this complex, Beclin 1 is K63-polyubiquitylated (green circles) by the E3 ligase AMBRA1 to activate the production of PI3P by VPS34. The production of PI3P at the phagophore recruits WIPI proteins to trigger the ATG machinery to elongate and seal the membrane to form an autophagosome. After the induction of autophagy, WASH and RNF2 are recruited to downregulate the autophagy pathway by inhibiting the VPS34 activity. WASH negatively regulates autophagy through suppression of Beclin 1 K63-linked polyubiquitination whereas RNF2 is recruited to the complex via the Beclin 1 interactor WASH. RNF2 catalyzes K48-linked polyubiquitination (orange circles) of AMBRA1 to mediate its proteasomal degradation.Recently, Xia and colleagues⁵ demonstrate that RNF2 (also called Ring1B) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitination of AMBRA1. RNF2 is a member of the RING-domain ubiquitin E3 ligase family⁶. In a series of experiments involving two-hybrid screens with RNF2 as bait, Xia and colleagues⁵ showed that RNF2 interacts with AMBRA1 and that this interaction is enhanced upon autophagy stimulation in cells cultured in the absence of serum and amino acids. Deletion of RNF2 robustly stimulates autophagy in response to starvation whereas restoration of RNF2 in RNF2^−/− mouse embryo fibroblasts (MEFs) reduces autophagy. An RNF2 mutant with no E3 ligase activity does not impair autophagy in RNF2^−/− MEFs. The authors further showed that RNF2 downregulates autophagy by promoting the degradation of AMBRA1. RNF2 catalyzes the K48-linked polyubiquitination of AMBRA1 which mediates its proteasomal degradation. The crucial site for AMBRA1 K48-linked polyubiquitination is lysine 45, and a K45R AMBRA1 mutant is not sensitive to RNF2-mediated ubiquitination and is able to sustain VPS34 activity and autophagy. Beclin 1 exists in different complexes involved in different steps of the autophagic pathway². In the current study, the authors showed that RNF2 is associated with the PI3K complex 1 with Beclin 1 ATG14 and AMBRA1, a complex involved in early stage of autophagosome formation. Interestingly, in the absence of RNF2, the association of Beclin 1 with VPS34 in the PI3K complex 1 is enhanced.Recently, Fan''s group reported that AMBRA1-DDB1-CUL4A is the E3 ligase that mediates K63-linked ubiquitination of Beclin 1 to enhance its binding to VPS34 in response to starvation⁷. In the present study⁵, the group showed that the K63-linked ubiquitination of Beclin 1 is inhibited by RNF2. A screen identified WASH as an interactor of RNF2. WASH is part of a complex that promotes actin polymerization to facilitate endosomal protein sorting⁸ and has been recently shown to interact with Beclin 1 and to be associated with autophagosomal membrane⁷. This interaction impairs the AMBRA1-mediated K63 polyubiquitination of Beclin 1. WASH recruits RNF2 to AMBRA1 and the PI3K complex 1 in response to starvation. The absence of WASH abolishes the K48-linked polyubiquitination of AMBRA1. In addition, WASH overexpression partially impairs the interaction of AMBRA1 with Beclin1 to block its K63-linked polyubiquitination⁷. The study by Xia et al. reveals a novel layer of regulation: WASH recruits RNF2 to promote AMBRA1 degradation to impair Beclin 1 ubiquitination⁵. This work points to the complex role of ubiquitination in the regulation of the early stage of autophagy with a balance between activating K63-linked polyubiquitination of Beclin 1 and inhibitory K48-linked polyubiquitination of AMBRA1. These post-translational modifications depend on the activity of two E3 ligases, AMBRA1 and RNF2. This study also stresses the importance of AMBRA1 in stabilization of proteins engaged in the early stage of autophagosome formation via its E3 ligase activity with DDB1-CUL4A to enhance Beclin 1 association with VPS34⁷ and with TRAF6 to stabilize ULK1, which acts in a complex upstream of the PI3K complex 1 in autophagy⁹. Finally, the study by Xia et al. emphasizes that autophagy must be tightly regulated to avoid deleterious effects on cell homeostasis.The present study raises several questions regarding how and when WASH and RNF2 are recruited to downregulate autophagy in response to starvation. Recently it has been shown that WASH is a positive modulator of autophagosome biogenesis in mammalian cells through regulation of endosomal trafficking of ATG9A¹⁰. Altogether, these findings suggest that the role of WASH in autophagy is dependent on its subcellular localization and its partners in intracellular membranes.     

Binding Rubicon to cross the Rubicon

Beclin 1 is an antitumor protein, required for mammalian autophagy, but its precise molecular function is poorly understood. Mass spectrometry analysis reveals that two novel proteins, Atg14L and Rubicon, associate with Beclin 1, together with a known Beclin 1-binding protein, UVRAG. The interactions of Atg14L and UVRAG with the Beclin 1-Vps34 (class III PI3-kinase)-Vps15 core complex are mutually exclusive; Rubicon associates with a subpopulation of UVRAG-containing complexes. The Atg14L complex, which positively regulates autophagy at an early step, localizes to the phagophore/isolation membrane, autophagosome and endoplasmic reticulum. In contrast, the Rubicon-UVRAG complex localizes to the late endosome/lysosome and negatively regulates both autophagy at a later step and the endocytic pathway. Thus, the Beclin 1-Vps34-Vps15 complex functions in autophagy and the endocytic pathway, but its function in a given context depends on the identity of its interacting subunits.     

PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L‐associated PI3K activity

The Beclin1–VPS34 complex is recognized as a central node in regulating autophagy via interacting with diverse molecules such as ATG14L for autophagy initiation and UVRAG for autophagosome maturation. However, the underlying molecular mechanism that coordinates the timely activation of VPS34 complex is poorly understood. Here, we identify that PAQR3 governs the preferential formation and activation of ATG14L‐linked VPS34 complex for autophagy initiation via two levels of regulation. Firstly, PAQR3 functions as a scaffold protein that facilitates the formation of ATG14L‐ but not UVRAG‐linked VPS34 complex, leading to elevated capacity of PI(3)P generation ahead of starvation signals. Secondly, AMPK phosphorylates PAQR3 at threonine 32 and switches on PI(3)P production to initiate autophagosome formation swiftly after glucose starvation. Deletion of PAQR3 leads to reduction of exercise‐induced autophagy in mice, accompanied by a certain degree of disaggregation of ATG14L‐associated VPS34 complex. Together, this study uncovers that PAQR3 can not only enhance the capacity of pro‐autophagy class III PI3K due to its scaffold function, but also integrate AMPK signal to activation of ATG14L‐linked VPS34 complex upon glucose starvation.     

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.     

Rab39a Interacts with Phosphatidylinositol 3-Kinase and Negatively Regulates Autophagy Induced by Lipopolysaccharide Stimulation in Macrophages

Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS) stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K). Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34^th to 41^st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.     

The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila

Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.