VMP1 is a new player in the regulation of the autophagy-specific phosphatidylinositol 3-kinase complex activation

We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells.     

Chromaffin granule-associated phosphatidylinositol 4-kinase activity is required for stimulated secretion.

Permeabilized bovine adrenal chromaffin cells have been used to characterize the MgATP requirement of processes preceding exocytosis. Incubation of primary cultures with the membrane-permeable phenylarsine oxide (PAO) at 20 microM inhibited the phosphorylation of phosphatidylinositol (PtdIns) and completely blocked secretion. This block could be reversed by addition of 2,3-dimercaptopropanol to the permeabilized cells. Simultaneous addition of [gamma32P]ATP and 2,3-dimercaptopropanol permitted a comparison between recovery of secretion and phosphorylation of intracellular components. Recovery of secretion closely correlated with phosphorylation of PtdIns and PtdIns4P. Subcellular fractionation of permeabilized cells after recovery of secretion revealed that the majority of newly phosphorylated PtdIns4P was localized on the chromaffin granules. In accordance with these results, PtdIns 4-kinase activity was found in protein extracts of permeabilized cells as well as associated with purified chromaffin granules, sensitive in both cases to PAO. Additionally, PtdIns 4-kinase activity in these two assays was inhibited by quercetin. In permeabilized cells, quercetin decreased the levels of labeled PtdIns4P and Ptdlns(4,5)P2 and inhibited secretion. Our data suggest that a chromaffin granule-associated PtdIns 4-kinase acts in the priming of exocytosis.     

A nanodomain-anchored scaffolding complex is required for the function and localization of phosphatidylinositol 4-kinase alpha in plants

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.

PI4Kα1 is targeted to plasma membrane nanodomains by a lipid-anchored heterotetrameric complex essential for plant cell survival, including gametophytic, embryonic, and post-embryonic development.     

Atg27 is required for autophagy-dependent cycling of Atg9

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.     

A late, prolonged activation of the phosphatidylinositol 3-kinase pathway is required for T cell proliferation

Activation of the phosphatidylinositol-3 kinase (PI 3-K) pathway is associated with the proliferation of many cell types, including T lymphocytes. However, recent studies in cell lines stably expressing deletion mutants of IL-2R that fail to activate PI 3-K have questioned the requirement for this pathway in cell cycle regulation. In this study with IL-2 and IL-7, we show in primary T cells that, unlike IL-2, IL-7 fails to induce the early activation of PI 3-K seen within minutes and normally associated with cytokine signaling. However, kinetic experiments showed that both of these T cell growth factors induce a distinct and sustained phase of PI 3-K activity several hours after stimulation. This delayed activation correlates with cell cycle induction and from studies using inhibitors of PI 3-K signaling, we show that this later phase, unlike the early activation within minutes, is required for cell cycle induction. The data presented here will have major implications for our understanding of the mechanism of T cell proliferation as well as the regulation of PI 3-K activity.     

The Arabidopsis phosphatidylinositol 3-kinase is important for pollen development

Phosphatidylinositol 3-kinase has been reported to be important for normal plant growth. To characterize the role of the enzyme further, we attempted to isolate Arabidopsis (Arabidopsis thaliana) plants that do not express the gene, but we could not recover homozygous mutant plants. The progeny of VPS34/vps34 heterozygous plants, harboring a T-DNA insertion, showed a segregation ratio of 1:1:0 for wild-type, heterozygous, and homozygous mutant plants, indicating a gametophytic defect. Genetic transmission analysis showed that the abnormal segregation ratio was due to failure to transmit the mutant allele through the male gametophyte. Microscopic observation revealed that 2-fold higher proportions of pollen grains in heterozygous plants than wild-type plants were dead or showed reduced numbers of nuclei. Many mature pollen grains from the heterozygous plants contained large vacuoles even until the mature pollen stage, whereas pollen from wild-type plants contained many small vacuoles beginning from the vacuolated pollen stage, which indicated that vacuoles in many of the heterozygous mutant pollen did not undergo normal fission after the first mitotic division. Taken together, our results suggest that phosphatidylinositol 3-kinase is essential for vacuole reorganization and nuclear division during pollen development.     

p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.     

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.     

Preconditioning enhanced glucose uptake is mediated by p38 MAP kinase not by phosphatidylinositol 3-kinase

Ischemia is reported to stimulate glucose uptake, but the signaling pathways involved are poorly understood. Modulation of glucose transport could be important for the cardioprotective effects of brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning. Previous work indicates that preconditioning reduces production of acid and lactate during subsequent sustained ischemia, consistent with decreased glucose utilization. However, there are also data that preconditioning enhances glucose uptake. The present study examines whether preconditioning alters glucose transport and whether this is mediated by either phosphatidylinositol 3-kinase (PI3K) or p38 MAP kinase. Langendorff-perfused rat hearts were preconditioned with 4 cycles of 5 min of ischemia and 5 min of reperfusion, with glucose as substrate. During the last reflow, glucose was replaced with 5 mM acetate and 5 mM 2-deoxyglucose (2DG), and hexose transport was measured from the rate of production of 2-deoxyglucose 6-phosphate (2DG6P), using (31)P nuclear magnetic resonance. Preconditioning stimulated 2DG uptake; after 15 min of perfusion with 2DG, 2DG6P levels were 165% of initial ATP in preconditioned hearts compared with 96% in control hearts (p < 0.05). Wortmannin, an inhibitor of PI3K, did not block the preconditioning induced stimulation of 2DG6P production, but perfusion with SB202190, an inhibitor of p38 MAP kinase, did attenuate 2DG6P accumulation (111% of initial ATP, p < 0. 05 compared with preconditioned hearts). SB202190 had no effect on 2DG6P accumulation in nonpreconditioned hearts. Preconditioning stimulation of translocation of GLUT4 to the plasma membrane was not inhibited by wortmannin. The data demonstrate that ischemic preconditioning increases hexose transport and that this is mediated by p38 MAP kinase and is PI3K-independent.     

Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption

Aldosterone, a steroid hormone, regulates renalNa⁺ reabsorption and, therefore,plays an important role in the maintenance of salt and water balance.In a model renal epithelial cell line (A6) we have found thatphosphoinositide 3-kinase (PI 3-kinase) activity is required foraldosterone-stimulated Na⁺reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na⁺ transport. Further, one of theproducts of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, isincreased in response to aldosterone in intact A6 monolayers. Thisincrease occurs just before the manifestation of the functional effectof the hormone and is also inhibited by LY-294002. With the use ofblocker-induced noise analysis, it has been demonstrated thatinhibition of phosphoinositide formation causes an inhibition ofNa⁺ entry in both control andaldosterone-pretreated cultures by reducing the number of openfunctional epithelial Na⁺ channels(ENaCs) in the apical membrane of the A6 cells. These novelobservations indicate that phosphoinositides are required for ENaCexpression and suggest a mechanism for aldosterone regulation ofchannel function.

     

A direct interaction between oncogenic Ha-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependent transformation of epithelial cells

Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.     

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17- Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.     

The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.     

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.     

The recruitment of phosphatidylinositol 3-kinase to the E-cadherin-catenin complex at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and human keratinocyte differentiation

Calcium induces epidermal keratinocyte differentiation, but the mechanism is not completely understood. We have previously demonstrated that calcium-induced human keratinocyte differentiation requires an intracellular calcium rise caused by phosphatidylinositol 3-kinase (PI3K)-dependent activation of phospholipase C-gamma1. In this study we sought to identify the upstream signaling pathway necessary for calcium activation of PI3K and its subsequent activation of phospholipase C-gamma1. We found that calcium induces the recruitment of PI3K to the E-cadherin-catenin complex at the plasma membrane of human keratinocytes. Knocking-down E-cadherin, beta-catenin, or p120-catenin expression blocked calcium activation of PI3K and phospholipase C-gamma1 and calcium-induced keratinocyte differentiation. However, knocking-down gamma-catenin expression had no effect. Calcium-induced PI3K recruitment to E-cadherin stabilized by p120-catenin at the plasma membrane requires beta-catenin but not gamma-catenin. These data indicate that the recruitment of PI3K to the E-cadherin/beta-catenin/p120-catenin complex via beta-catenin at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and, ultimately, keratinocyte differentiation.     

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.