Conversion of effector CD4+ T cells to a CD8+ MHC II-recognizing lineage.

CD4+ and CD8+ T cells are dichotomous lineages in adaptive immunity. While conventionally viewed as distinct fates that are fixed after thymic development, accumulating evidence indicates that these two populations can exhibit significant lineage plasticity, particularly upon TCR-mediated activation. We define a novel CD4-CD8αß+ MHC II-recognizing population generated by lineage conversion from effector CD4+ T cells. CD4-CD8αß+ effector T cells downregulated the expression of T helper cell-associated costimulatory molecules and increased the expression of cytotoxic T lymphocyte-associated cytotoxic molecules. This shift in functional potential corresponded with a CD8+-lineage skewed transcriptional profile. TCRß repertoire sequencing and in vivo genetic lineage tracing in acutely infected wild-type mice demonstrated that CD4-CD8αß+ effector T cells arise from fundamental lineage reprogramming of bona fide effector CD4+ T cells. Impairing autophagy via functional deletion of the initiating kinase Vps34 or the downstream enzyme Atg7 enhanced the generation of this cell population. These findings suggest that effector CD4+ T cells can exhibit a previously unreported degree of skewing towards the CD8+ T cell lineage, which may point towards a novel direction for HIV vaccine design.

Critical Role for Phosphatidylinositol-3 Kinase Vps34/PIK3C3 in ON-Bipolar Cells.

Purpose: Phosphatidylinositol-3-phosphate (PI(3)P), and Vps34, the type III phosphatidylinositol 3-kinase primarily responsible for its production, are important for function and survival of sensory neurons, where they have key roles in membrane processing events, such as autophagy, endosome processing, and fusion of membranes bearing ubiquitinated cargos with lysosomes. We examined their roles in the most abundant class of secondary neurons in the vertebrate retina, the ON-bipolar cells (ON-BCs). Methods: A conditional Vps34 knockout mouse line was generated by crossing Vps34 floxed mice with transgenic mice expressing Cre recombinase in ON-BCs. Structural changes in the retina were determined by immunofluorescence and electron microscopy, and bipolar cell function was determined by electroretinography. Results: Vps34 deletion led to selective death of ON-BCs, a thinning of the inner nuclear layer, and a progressive decline of electroretinogram b-wave amplitudes. There was no evidence for loss of other retinal neurons, or disruption of rod-horizontal cell contacts in the outer plexiform layer. Loss of Vps34 led to aberrant accumulation of membranes positive for autophagy markers LC3, p62, and ubiquitin, accumulation of endosomal membranes positive for Rab7, and accumulation of lysosomes. Similar effects were observed in Purkinje cells of the cerebellum, leading to severe and progressive ataxia. Conclusions: These results support an essential role for PI(3)P in fusion of autophagosomes with lysosomes and in late endosome maturation. The cell death resulting from Vps34 knockout suggests that these processes are essential for the health of ON-BCs.

VPS34 stimulation of p62 phosphorylation for cancer progression.

Vps34, a class III PtdIns3 lipid kinase involved in the control of both autophagic and endocytic systems, has been studied extensively in numerous fundamental cellular processes. Accumulating evidence indicates that Vps34 may also contribute to the development and progression of human cancers. However, the mechanism of Vps34 in tumorigenesis remains elusive. Here, we report an unanticipated role of Vps34 in the activation of p62 for cancer development. We identified that Vps34 is a transcriptional activator of p62 through competition of Nrf2 (nuclear factor erythroid 2-related factor 2) for Keap1 binding. Vps34 augments the association of PKC-δ with p62 for its phosphorylation at Serine 349, which leads to positive feedback on the Nrf2-dependent transcription of oncogenes. Additionally, we found that the expression of Vps34 is correlated with the tumorigenic activity of human breast cancer cells. Normally inactive in breast cancer, caspase 8 can cleave Vps34 at residue D285, which directly abolished its lipid kinase activity and dramatically altered cell invasion potential, colony formation, as well as tumorigenesis in orthotopic engraftments in mice. The cleavage at D285 blocks expression of LC3-II, Nrf2 and subsequently, p62, in addition to blocking tumor growth, indicating that the intact structure of Vps34 is essential for its activity. Moreover, either knockout of PKC-δ or knockdown of p62 by small interfering RNA in MCF-7 cells abrogates Vps34-dependent tumor growth. Data presented here suggested that Vps34 stimulates tumor development mainly through PKC-δ- activation of p62.

The Phosphatidylinositol 3-kinase Class III Complex Containing TcVps15 and TcVps34 Participates in Autophagy in Trypanosoma cruzi.

Autophagy is a degradative process by which eukaryotic cells digest their own components to provide aminoacids that may function as energy source under nutritional stress conditions. There is experimental evidence for autophagy in parasitic protists belonging to the family Trypanosomatidae. However, few proteins implicated in this process have been characterized so far in these parasites. Moreover, it has been shown that autophagy is involved in Trypanosoma cruzi differentiation and thus might have a role in pathogenicity. Here, we report the cloning and biochemical characterization of TcVps15. In addition, we demonstrate that TcVps15 interact with the PI3K TcVps34 and that both proteins associate with cellular membranes. Under nutritional stress conditions, TcVps15 and TcVps34 modify their subcellular distribution showing a partial co-localization in autophagosomes with TcAtg8.1 and using an active site TcVps15-mutated version (TcVps15-K219D-HA) we demonstrated that this relocalization depends on the TcVps15 catalytic activity. Overexpression of TcVps15-HA and TcVps15-K219D-HA also leads to increased accumulation of monodansylcadaverine (MDC) in autophagic vacuoles under nutritional stress conditions compared to wild-type cells. In addition, the MDC-specific activity shows to be significantly higher in TcVps15-HA overexpressing cells when compared with TcVps15-K219D-HA. Our results reveal for the first time a role of TcVps15 as a key regulator of TcVps34 enzymatic activity and implicate the TcVps15-Vps34 complex in autophagy in T. cruzi, exposing a new key pathway to explore novel chemotherapeutic targets.

Distinct Requirements for Vacuolar Protein Sorting 34 Downstream Effector Phosphatidylinositol 3-Phosphate 5-Kinase in Podocytes Versus Proximal Tubular Cells.

The mechanisms by which the glomerular filtration barrier prevents the loss of large macromolecules and simultaneously, maintains the filter remain poorly understood. Recent studies proposed that podocytes have an active role in both the endocytosis of filtered macromolecules and the maintenance of the filtration barrier. Deletion of a key endosomal trafficking regulator, the class 3 phosphatidylinositol (PtdIns) 3-kinase vacuolar protein sorting 34 (Vps34), in podocytes results in aberrant endosomal membrane morphology and podocyte dysfunction. We recently showed that the vacuolation phenotype in cultured Vps34-deficient podocytes is caused by the absence of a substrate for the Vps34 downstream effector PtdIns 3-phosphate 5-kinase (PIKfyve), which phosphorylates Vps34-generated PtdIns(3)P to produce PtdIns (3,5)P2. PIKfyve perturbation and PtdIns(3,5)P2 reduction result in massive membrane vacuolation along the endosomal system, but the cell-specific functions of PIKfyve in vivo remain unclear. We show here that the genetic deletion of PIKfyve in endocytically active proximal tubular cells resulted in the development of large cytoplasmic vacuoles caused by arrested endocytic traffic progression at a late-endosome stage. In contrast, deletion of PIKfyve in glomerular podocytes did not significantly alter the endosomal morphology, even in age 18-month-old mice. However, on culturing, the PIKfyve-deleted podocytes developed massive cytoplasmic vacuoles. In summary, these data suggest that glomerular podocytes and proximal tubules have different requirements for PIKfyve function, likely related to distinct in vivo needs for endocytic flux.

Autophagy in the lens.

The lens of the eye is a transparent tissue composed of lens fiber cells that differentiate from lens epithelial cells and degrade all cytoplasmic organelles during terminal differentiation. Autophagy is a major intracellular degradation system in which cytoplasmic proteins and organelles are degraded in the lysosome. Although autophagy is constitutively activated in the lens and has been proposed to be involved in lens organelle degradation, its precise role is not well understood. Recent genetic studies in mice have demonstrated that autophagy is critically important for intracellular quality control in the lens but can be dispensable for lens organelle degradation. Here, we review recent findings on the roles of autophagy and lysosomes in organelle degradation and intracellular quality control in the lens, and discuss their possible involvement in the development of human cataract.

Nuclear trafficking of EGFR by Vps34 represses Arf expression to promote lung tumor cell survival.

Epidermal growth factor receptor (EGFR) is a cell surface receptor that has an essential role in cell proliferation and survival, and overexpression of EGFR is a common feature of human cancers. In Non-small-cell lung cancer (NSCLC), activating mutations of EGFR have also been described. We recently showed that mutant EGFR-L858R inhibits the expression of the p14ARF tumor-suppressor protein to promote cell survival. In this study, we defined the molecular bases by which EGFR controls Arf expression. Using various lung tumor models, we showed that EGF stimulation inhibits Arf transcription by a mechanism involving the nuclear transport and recruitment of EGFR to the Arf promoter. We unraveled the vesicular trafficking protein Vps34 as a mediator of EGFR nuclear trafficking and showed that its neutralization prevents the accumulation of EGFR to the Arf promoter in response to ligand activation. Finally, in lung tumor cells that carry mutant EGFR-L858R, we demonstrated that inhibition of Vps34 using small interfering RNA restrains nuclear EGFR location and restores Arf expression leading to apoptosis. These findings identify the Arf tumor suppressor as a new transcriptional target of nuclear EGFR and highlight Vps34 as an important regulator of the nuclear EGFR/Arf survival pathway. As a whole, they provide a mechanistic explanation to the inverse correlation between nuclear expression of EGFR and overall survival in NSCLC patients.

Beclin 1 regulates growth factor receptor signaling in breast cancer.

Beclin 1 is a haploinsufficient tumor suppressor that is decreased in many human tumors. The function of beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, beclin 1 is a core component of the vacuolar protein sorting 34 (Vps34)/class III phosphatidylinositoI-3 kinase (PI3KC3) and Vps15/p150 complex that regulates multiple membrane-trafficking events. In the current study, we describe an alternative mechanism of action for beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P(-)) endosomes to PI3P(+) endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P(-)/APPL(+)-signaling-competent compartment. As a result, suppression of BECN1 sustains growth factor-stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of beclin 1 expression would enhance breast cancer progression.

Ironing out VPS34 inhibition.

The class III phosphoinositide 3-kinase VPS34 regulates autophagosome formation. Three groups have developed VPS34 inhibitors and shown their utility in investigating and defining autophagic processes.

Vps34 deficiency reveals the importance of endocytosis for podocyte homeostasis.

The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3-9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier.

Class III phosphatidylinositol 3-kinase and its catalytic product PtdIns3P in regulation of endocytic membrane traffic.

Endocytosis and subsequent membrane traffic through endosomes are cellular processes that are integral to eukaryotic evolution, and numerous human diseases are associated with their dysfunction. Consequently, it is important to untangle the molecular machineries that regulate membrane dynamics and protein flow in the endocytic pathway. Central in this context is class III phosphatidylinositol 3-kinase, an evolutionarily conserved enzyme complex that phosphorylates phosphatidylinositol into phosphatidylinositol 3-phosphate. Phosphatidylinositol 3-phosphate recruits specific effector proteins, most of which contain FYVE or PX domains, to promote endocytosis, endosome fusion, endosome motility and endosome maturation, as well as cargo sorting to lysosomes, the biosynthetic pathway or the plasma membrane. Here we review the functions of key phosphatidylinositol 3-phosphate effectors in regulation of endocytic membrane dynamics and protein sorting.

Mouse skeletal muscle fiber-type-specific macroautophagy and muscle wasting are regulated by a Fyn/STAT3/Vps34 signaling pathway.

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.

Role of autophagy in monokine induced by interferon γ (Mig) production during adenovirus-hepatitis B virus infection.

BACKGROUND/AIMS: The aim of this study was to investigate the effect of HBV-induced Mig and role of autophagy in the process. METHODOLOGY: Adxsi-1.3 x HBV plasmid was constructed and identified. The three cell lines (L02, HepG2, SMMC-7721) were infected with adenovirus-HBV. HBsAg and HBeAg were assessed by electrochemiluminescence immunoassay. HBV DNA, HBx, Beclin 1 and Mig expression were detected by quantitative real-Time PCR, western blotting and ELISA. The level of autophagy was evaluated by transmission electron microscope. RESULTS: Human fetal liver cells and hepatocellular carcinoma cells were successfully transfected with overlength HBV genome using an adenovirus vector (Ad-HBV). Ad-HBV induced Mig production and cell autophagy through up-regulation of Beclin 1 expression. We further demonstrated that the increased autophagy extent was in association with HBV-induced Mig expression. CONCLUSIONS: Autophagy may be a crucial intracellular mechanism of Mig induction in response to HBV infection. The results provide new insights into the pathogenesis of HBV.

The class III kinase Vps34 promotes T lymphocyte survival through regulating IL-7Rα surface expression.

IL-7Rα-mediated signals are essential for naive T lymphocyte survival. Recent studies show that IL-7Rα is internalized and either recycled to cell surface or degraded. However, how the intracellular process of IL-7Rα trafficking is regulated is unclear. In this paper, we show that Vps34, the class III PI3K, plays a critical role in proper IL-7Rα intracellular trafficking. Mice lacking Vps34 in T lymphocytes had a severely reduced T lymphocyte compartment. Vps34-deficient T lymphocytes exhibit increased death and reduced IL-7Rα surface expression, although three major forms of autophagy remain intact. Intracellular IL-7Rα in normal T lymphocytes at steady state is trafficked through either early endosome/multivesicular bodies to the late endosome-Golgi for surface expression or to the lysosome for degradation. However, Vps34-deficient T cells have mislocalized intracellular Eea1, HGF-regulated tyrosine kinase substrate, and Vps36 protein levels, the combined consequence of which is the inability to mobilize internalized IL-7Rα into the retromer pathway for surface display. Our studies reveal that Vps34, though dispensable for autophagy induction, is a critical regulator of naive T cell homeostasis, modulating IL-7Rα trafficking, signaling, and recycling.

The mammalian class 3 PI3K (PIK3C3) is required for early embryogenesis and cell proliferation.

The Pik3c3 gene encodes an 887 amino acid lipid kinase, phosphoinositide-3-kinase class 3 (PIK3C3). PIK3C3 is known to regulate various intracellular membrane trafficking events. However, little is known about its functions during early embryogenesis in mammals. To investigate the function of PIK3C3 in vivo, we generated Pik3c3 null mice. We show here that Pik3c3 heterozygous are normal and fertile. In contrast, Pik3c3 homozygous mutants are embryonic lethal and die between E7.5 and E8.5 of embryogenesis. Mutant embryos are poorly developed with no evidence of mesoderm formation, and suffer from severely reduced cell proliferations. Cell proliferation defect is also evident in vitro, where mutant blastocysts in culture fail to give rise to typical colonies formed by inner cell mass. Electron microscopic analysis revealed that epiblast cells in mutant embryos appear normal, whereas the visceral endoderm cells contain larger vesicles inside the lipid droplets. Finally, we provide evidence that mTOR signaling is drastically reduced in Pik3c3 null embryos, which could be a major contributor to the observed proliferation and embryogenesis defects.

Regulation of protein synthesis by amino acids in muscle of neonates.

The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed.