Vps34 Lipid Kinase Research Articles

The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization

Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy.

Abstract LB317: TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance

Abstract Triple negative breast cancer (TNBC) carries a poorer prognosis than other breast cancer subtypes due to high metastatic incidence, lack of molecular drug targets, and rapid development of chemoresistance. Cancer-associated fibroblasts (CAFs) are key extrinsic drivers of chemoresistance in primary TNBC tumors, but it is unknown whether resident fibroblasts in distant metastatic tissues also influence TNBC drug sensitivity. Since the lung is a top metastatic site in TNBC, we developed a breast cancer cell (BCC) - primary human donor-derived lung fibroblast (LF) co-culture model to identify therapeutic vulnerabilities and mechanisms of fibroblast-mediated extrinsic chemoresistance in lung-metastatic TNBC. We characterized the growth and chemoresistance phenotypes of BCC co-cultured with primary LFs via bioluminescence and fluorescence imaging and evaluated their transcriptional and secretory profiles using bulk and scRNAseq and multiplex cytokine analysis. Co-culture of human BCC and primary LFs resulted in BCC molecular subtype-dependent changes in cell growth and drug resistance compared with conventional monoculture. Moreover, we also observed increased LF secretion of the immunomodulatory cytokines IL-6 and CCL2 in response to both BCC co-culture and BCC-conditioned media. Transcriptomic analysis of LFs sorted from co-culture identified upregulation of pro-inflammatory and pro-fibrotic iCAF and myCAF-like gene signatures, while co-cultured TNBC cells upregulated interferon response-mediated multi-therapy resistance genes and cellular dormancy signatures. We applied this model in a high throughput screen of 846 kinase inhibitor compounds, which identified reproducible differential BCC responses to kinase inhibitors between monoculture and co-culture conditions. When co-cultured with LFs, BCC were broadly more resistant to kinase inhibitor compounds but showed increased sensitivity to inhibitors of the lipid kinase VPS34, an important initiator of autophagy. We subsequently established that autophagy is increased in co-cultured TNBC cells and contributes to LF-mediated resistance to kinase inhibitors. Moreover, we determined that while paracrine signals from BCC can promote LF activation indicated by enhanced cytokine secretion, cell-cell contact is required for LF modulation of TNBC therapy responses in co-culture. Thus, we conclude that breast cancer cell co-culture with primary lung fibroblasts is a scalable in vitro model that can facilitate therapeutic discovery and mechanistic evaluation of extrinsic modulation of therapy responses in metastatic breast cancer, and further demonstrate that BCC can promote paracrine activation of co-cultured LFs in association with juxtacrine induction of BCC autophagy and extrinsic therapy resistance. Citation Format: Marika L. Klosowski, Kathryn Cronise, Claire Stratton, Qiong Zhou, Hector Esquer, Daniel LaBarbera, Daniel Regan. TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB317.

ULK1-mediated metabolic reprogramming regulates Vps34 lipid kinase activity by its lactylation.

Autophagy and glycolysis are highly conserved biological processes involved in both physiological and pathological cellular programs, but the interplay between these processes is poorly understood. Here, we show that the glycolytic enzyme lactate dehydrogenase A (LDHA) is activated upon UNC-51-like kinase 1 (ULK1) activation under nutrient deprivation. Specifically, ULK1 directly interacts with LDHA, phosphorylates serine-196 when nutrients are scarce and promotes lactate production. Lactate connects autophagy and glycolysis through Vps34 lactylation (at lysine-356 and lysine-781), which is mediated by the acyltransferase KAT5/TIP60. Vps34 lactylation enhances the association of Vps34 with Beclin1, Atg14L, and UVRAG, and then increases Vps34 lipid kinase activity. Vps34 lactylation promotes autophagic flux and endolysosomal trafficking. Vps34 lactylation in skeletal muscle during intense exercise maintains muscle cell homeostasis and correlates with cancer progress by inducing cell autophagy. Together, our findings describe autophagy regulation mechanism and then integrate cell autophagy and glycolysis.

LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models

Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. Here we show that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. Our results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.

VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney.

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased β-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection.

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

Endosomal phosphatidylinositol 3‐phosphate controls synaptic vesicle cycling and neurotransmission

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration.

Self-transducible LRS-UNE-L peptide enhances muscle regeneration.

BackgroundMuscle regeneration includes proliferation and differentiation of muscle satellite cells, which involves the mammalian target of rapamycin (mTOR). We identified the C‐terminal unique attached sequence motif (UNE) domain of leucyl‐tRNA synthetase (LRS‐UNE‐L) as an mTORC1 (mTOR complex1)‐activating domain that acts through Vps34 and phospholipase D1 (PLD1) when introduced in the form of a muscle‐enhancing peptide.Methods In vitro Vps34 lipid kinase assay, phosphatidylinositol 3‐phosphate (PI(3)P) measurement, in vivo PLD1 assay, and western blot assay were performed in HEK293 cells to test the effect of the LRS‐UNE‐L on the Vps34‐PLD1‐mTOR pathway. Adeno‐associated virus (AAV)‐LRS‐UNE‐L was transduced in C2C12 cells in vitro, in BaCl2‐injured tibialis anterior (TA) muscles, and in 18‐month‐old TA muscles to analyse its effect on myogenesis, muscle regeneration, and aged muscle, respectively. The muscle‐specific cell‐permeable peptide M12 was fused with LRS‐UNE‐L and tested for cell integration in C2C12 and HEK293 cells using FACS analysis and immunocytochemistry. Finally, M12‐LRS‐UNE‐L was introduced into BaCl2‐injured TA muscles of 15‐week‐old Pld1 +/+ or Pld1 −/− mice, and its effect was analysed by measurement of cross‐sectional area of regenerating muscle fibres.ResultsThe LRS‐UNE‐L expression restored amino acid‐induced S6K1 phosphorylation in LRS knockdown cells in a RagD GTPases‐independent manner (421%, P = 0.007 vs. LRS knockdown control cells). The LRS‐UNE‐L domain was directly bound to Vps34; this interaction was accompanied by increases in Vps34 activity (166%, P = 0.0352), PI(3)P levels (146%, P = 0.0039), and PLD1 activity (228%, P = 0.0294) compared with amino acid‐treated control cells, but it did not affect autophagic flux. AAV‐delivered LRS‐UNE‐L domain augmented S6K1 phosphorylation (174%, P = 0.0013), mRNA levels of myosin heavy chain (MHC) (122%, P = 0.0282) and insulin‐like growth factor 2 (IGF2) (146%, P = 0.008), and myogenic fusion (133%, P = 0.0479) in C2C12 myotubes. AAV‐LRS‐UNE‐L increased the size of regenerating muscle fibres in BaCl2‐injured TA muscles (124%, P = 0.0279) (n = 9–10), but it did not change the muscle fibre size of TA muscles in old mice. M12‐LRS‐UNE‐L was preferentially delivered into C2C12 cells compared with HEK293 cells and augmented regeneration of BaCl2‐injured TA muscles in a PLD1‐dependent manner (116%, P = 0.0022) (n = 6).ConclusionsOur results provide compelling evidence that M12‐LRS‐UNE‐L could be a muscle‐enhancing protein targeting mTOR.

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex. SummaryIn this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.

ULK1-Mediated Metabolic Reprogramming Regulates Vps34 Lipid Kinase Activity by Its Lactylation

ULK1-Mediated Metabolic Reprogramming Regulates Vps34 Lipid Kinase Activity by Its Lactylation

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

Abstract A01: Vps34 promotes macropinocytosis in Tsc2-deficient cells

Abstract Purpose: The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is constitutively active in many human cancers and in tuberous sclerosis complex (TSC). mTORC1 hyperactivation drives extensive metabolic reprogramming via several interconnected mechanisms, including glucose and glutamine utilization, nucleic acid synthesis, and lipid synthesis. We recently found that treatment with chloroquine (CQ), a lysosomal/autophagy inhibitor, upregulates the uptake and endosomal processing of cholesterol selectively in TSC2-deficient cells, creating a TSC2-specific dependence on exogenous cholesterol. This dependence on exogenous nutrients and the mechanisms of nutrient uptake may be therapeutically targetable. Our central hypothesis is that Tsc2-deficient cells depend on the uptake of exogenous nutrients via macropinocytosis to maintain cellular metabolic homeostasis and proliferation. Methods and Results: To investigate the role of macropinocytosis-mediated nutrient uptake in Tsc2-deficient cells, we measured the uptake of dextran, a polysaccharide taken up via macropinocytosis. Tsc2-deficient cells showed a striking increase in both dextran (3.6-fold, p<0.001) and exogenous protein uptake (~2-fold, p<0.001) relative to Tsc2-expressing cells. Interestingly, mTOR inhibition using Torin1 decreased dextran uptake (3-fold, p<0.001). Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of dextran in Tsc2-deficient cells (~50%, p<0.0001). Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms (25%, p<0.001), suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Additionally, exogenous protein activates mTORC1 signaling of Tsc2-deficient cells grown in amino acid-free media. Treatment with ethylisopropylamiloride (EIPA) resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p<0.0001). Genetic inhibition of autophagy (Atg5-/- MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p<0.0001). Finally, pharmacologic inhibition of lysosomal function with CQ and macropinocytosis with SAR405 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Conclusions: These data suggest that macropinocytosis is upregulated in Tsc2-deficient cells via a Vps34-dependent mechanism to support tumorigenesis. Macropinocytosis-mediated nutrient uptake represents an unexplored opportunity for therapeutic intervention in TSC and may also be relevant to other diseases characterized by mTORC1 hyperactivation. Citation Format: Charilaos Filippakis, Amine Belaid, Brian Siroky, Constance Wu, Nicola Alesi, Thomas Alesi, Julie Nijmeh, Hilaire Colletta Lam, Ellizabeth Henske. Vps34 promotes macropinocytosis in Tsc2-deficient cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A01.

Vps34 derived phosphatidylinositol 3‐monophosphate modulates megakaryocyte maturation and proplatelet production through late endosomes/lysosomes

BackgroundDevelopment of platelet precursor cells, megakaryocytes (MKs), implies an increase in their size; formation of the elaborate demarcation membrane system (DMS); and extension of branched cytoplasmic structures, proplatelets, that will release platelets. The membrane source(s) for MK expansion and proplatelet formation have remained elusive. ObjectiveWe hypothesized that traffic of membranes regulated by phosphatidylinositol 3‐monophosphate (PI3P) contributes to MK maturation and proplatelet formation. ResultsIn immature MKs, PI3P produced by the lipid kinase Vps34 is confined to perinuclear early endosomes (EE), while in mature MKs PI3P shifts to late endosomes and lysosomes (LE/Lys). PI3P partially colocalized with the plasma membrane marker phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) and with LE/Lys in mature MKs, suggests that PI3P‐containing LE/Lys membranes contribute to MK expansion and proplatelet formation. Consistently, we found that sequestration of PI3P, specific pharmacological inhibition of Vps34‐mediated PI3P production, or depletion of PI3P by PI3‐phosphatase (MTM1)‐mediated hydrolysis potently blocked proplatelet formation. Moreover, Vps34 inhibition led to the intracellular accumulation of enlarged LE/Lys, and decreased expression of surface LE/Lys markers. Inhibiting Vps34 at earlier MK stages caused aberrant DMS development. Finally, inhibition of LE/Lys membrane fusion by a dominant negative mutant of the small GTPase Rab7 or pharmacological inhibition of PI3P conversion into PI(3,5)P2 led to enlarged LE/Lys, reduced surface levels of LE/Lys markers, and decreased proplatelet formation. ConclusionOur results suggest that PI3P‐positive LE/Lys contribute to the membrane growth and proplatelet formation in MKs by their translocation to the cell periphery and fusion with the plasma membrane.

Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes.

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

Beclin 1-ATG14L Protein-Protein Interaction Inhibitor Selectively Inhibits Autophagy through Disruption of VPS34 Complex I.

Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.

Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

ULK1 O-GlcNAcylation Is Crucial for Activating VPS34 via ATG14L during Autophagy Initiation

Unc-51-like-kinase 1 (ULK1) is a target of both the mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), whose role is to facilitate the initiation of autophagy in response to starvation. Upon glucose starvation, dissociation of mTOR from ULK1 and phosphorylation by AMPK leads to the activation of ULK1 activity. Here, we provide evidence that ULK1 is the attachment of O-linked N-acetylglucosamine (O-GlcNAcylated) on the threonine 754 site by O-linked N-acetylglucosamine transferase (OGT) upon glucose starvation. ULK1 O-GlcNAcylation occurs after dephosphorylationof adjacent mTOR-dependent phosphorylation on the serine 757 site by protein phosphatase 1(PP1) and phosphorylation by AMPK. ULK1 O-GlcNAcylation is crucial for binding and phosphorylation ofATG14L, allowing the activation of lipid kinase VPS34 and leading to the production of phosphatidylinositol-(3)-phosphate (PI(3)P), which is required for phagophore formation and initiation of autophagy. Our findings provide insights into the crosstalk between dephosphorylation and O-GlcNAcylation during autophagy and specify a molecular framework for potential therapeutic intervention in autophagy-related diseases.

Vps34-mediated macropinocytosis in Tuberous Sclerosis Complex 2-deficient cells supports tumorigenesis

Tuberous Sclerosis Complex (TSC), a rare genetic disorder with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivation, is characterized by multi-organ hamartomatous benign tumors including brain, skin, kidney, and lung (Lymphangioleiomyomatosis). mTORC1 hyperactivation drives metabolic reprogramming including glucose and glutamine utilization, protein, nucleic acid and lipid synthesis. To investigate the mechanisms of exogenous nutrients uptake in Tsc2-deficient cells, we measured dextran uptake, a polysaccharide internalized via macropinocytosis. Tsc2-deficient cells showed a striking increase in dextran uptake (3-fold, p < 0.0001) relative to Tsc2-expressing cells, which was decreased (3-fold, p < 0.0001) with mTOR inhibitor, Torin1. Pharmacologic and genetic inhibition of the lipid kinase Vps34 markedly abrogated uptake of Dextran in Tsc2-deficient cells. Macropinocytosis was further increased in Tsc2-deficient cells that lack autophagic mechanisms, suggesting that autophagy inhibition leads to dependence on exogenous nutrient uptake in Tsc2-deficient cells. Treatment with a macropinocytosis inhibitor, ethylisopropylamiloride (EIPA), resulted in selective growth inhibition of Atg5-deficient, Tsc2-deficient cells (50%, p < 0.0001). Genetic inhibition of autophagy (Atg5−/− MEFs) sensitized cells with Tsc2 downregulation to the Vps34 inhibitor, SAR405, resulting in growth inhibition (75%, p < 0.0001). Finally, genetic downregulation of Vps34 inhibited tumor growth and increased tumor latency in an in vivo xenograft model of TSC. Our findings show that macropinocytosis is upregulated with Tsc2-deficiency via a Vps34-dependent mechanism to support their anabolic state. The dependence of Tsc2-deficient cells on exogenous nutrients may provide novel approaches for the treatment of TSC.

Regulation of the Tumor-Suppressor BECLIN 1 by Distinct Ubiquitination Cascades.

Autophagy contributes to cellular homeostasis through the degradation of various intracellular targets such as proteins, organelles and microbes. This relates autophagy to various diseases such as infections, neurodegenerative diseases and cancer. A central component of the autophagy machinery is the class III phosphatidylinositol 3-kinase (PI3K-III) complex, which generates the signaling lipid phosphatidylinositol 3-phosphate (PtdIns3P). The catalytic subunit of this complex is the lipid-kinase VPS34, which associates with the membrane-targeting factor VPS15 as well as the multivalent adaptor protein BECLIN 1. A growing list of regulatory proteins binds to BECLIN 1 and modulates the activity of the PI3K-III complex. Here we discuss the regulation of BECLIN 1 by several different types of ubiquitination, resulting in distinct polyubiquitin chain linkages catalyzed by a set of E3 ligases. This contribution is part of the Special Issue “Ubiquitin System”.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

AbstractTo uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.