The control of phosphatidylinositol 3, 4, 5-trisphosphate [PtdIns(3,4,5)P3] level depends on the activities of both PI kinase and PtdIns(3,4,5)P3 phosphatases: 5-phosphatase like SHIP1 and SHIP2, and 3-phosphatase like PTEN. The ubiquitous SH2 domain containing inositol 5-phosphatase SHIP2 contains both a series of protein interacting domains and the ability to dephosphorylate PtdIns(3,4,5)P3. Previous reports obtained in SHIP2 deficient mice have shown that SHIP2 is involved in the control of insulin sensitivity and reducing weight gain on fatty diet. Since SHIP2 is a lipid phosphatase as well as a docking protein, the initial aim that emerged in the lab was to measure the inositol lipid levels in SHIP2 +/+ and deficient cells and compare the levels of 3-phosphoinositides PtdIns(3,4,5)P3 and PtdIns(3,4)P2. At first, we developed mouse embryonic fibroblasts (MEF) as a cellular model. Amongst various stimuli tested, surprisingly, only serum showed an obvious difference in terms of PtdIns(3,4,5)P3 level. This lipid was significantly up regulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P3 levels in heterozygous fibroblast cells was intermediate between the +/+ and -/- cells. Serum stimulated PI3K activity appeared to be comparable between +/+ and -/- cells. Moreover, PKB, but not MAP kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The up regulation of PKB activity in serum stimulated cells was totally reversed in the presence of the PI3K inhibitor LY-294002, in both +/+ and -/- cells. Reactive oxygen species (ROS) have emerged as physiological mediators of many cellular responses. H2O2 mimics some effects of insulin in a number of cell culture systems. It also inactivates tyrosine phosphatase activities including PTEN. In addition, in Swiss 3T3 fibroblasts, Gray et al reported that exposure of the cells to H2O2 resulted a huge increase in PtdIns(3,4)P2 level. The authors suspected that the effect was attributed to a inositol 5-phosphatase activity. We thus exposed our cells to H2O2 in order to address the question of the role of SHIP2 in response to oxidative stress. We worked on the same SHIP2 MEF model, stimulated by H2O2: at 15 min, PtdIns(3,4,5)P3 was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P2 could be detected at 15 or 120 min incubation of the cells with H2O2 (0.6 mM). PKB activity was upregulated in SHIP2 -/- cells in response to H2O2 and therefore follows the regulation of PtdIns(3,4,5)P3. As for serum, the PI3K activity appeared to be comparable between +/+ and -/- cells. The levels of PTEN and type I 4-phosphatase [an enzyme that acts on PtdIns(3,4)P2] remained unchanged between the two types of cells. SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role in the control of PtdIns(3,4,5)P3 level in response to H2O2: the decrease in PtdIns(3,4,5)P3, observed in SHIP2 expressing cells, was no longer seen in cells infected with a catalytic mutant of this enzyme.

Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.

Insulin signalling is an essential process in humans by which the level of plasma glucose is maintained within the physiologically healthy range. Insulin activates the phosphoinositide 3 kinase (PI3K) signalling pathway that generates the phospholipid messenger PtdIns(3,4,5)P3, which in turn enhances the activity of two important proteins, AKT and Rac1. This then leads to increase the presence of the glucose transporter 4 (GLUT4) at the plasma membrane that enhances the intake of glucose, particularly in skeletal muscle cells and adipocytes. Insulin signalling also triggers interconversion of several other phosphoinositides (PIs) which play pivotal roles in different steps of glucose regulation. PtdIns5P is an important PI that is robustly increased after insulin treatment in the skeletal muscle cell line, L6 myotubes. Many of PtdIns5P`s functions are not fully understood. To gain more knowledge of the role of PtdIns5P in insulin signalling in muscle cells, the PtdIns5P kinase phosphatidylinositol-5-phosphate 4-kinase a (PIP4K2a) was over-expressed in L6 myotubes as a way of removing PtdIns5P, and the consequences in insulin signalling were studied. Although PtdIns5P is converted by PIP4K2a to PtdIns(4,5)P2 which is a precursor of the potent PI PtdIns(3,4,5)P3, previous studies revealed that the increase in PtdIns(3,4,5)P3 induced by insulin in control cells is diminished in cells overexpressing PIP4K2a, for unknown reasons. Additionally, although the phosphorylation of the serine/threonine protein kinase AKT was not affected in these L6 cells, glucose uptake was attenuated. The current study investigates the possible causes of attenuating glucose uptake in PIP4K overexpressing myotubes by examining the small GTPase Rac1 which plays an important role in the cytoskeleton re-arrangement that is necessary for GLUT4 translocation. Furthermore, the possible roles of PI phosphatases that may cause the disturbance on the levels of PIs in response to insulin were evaluated. Additionally, the potential role of PtdIns5P in Rac1 activation in L6 myotubes was further investigated by delivering synthetic PtdIns5P using a carrier-based delivery approach. The results showed that the attenuation of glucose uptake documented in previous studies occurred as a result of a defect in the process of translocating GLUT4 from intracellular storage to the plasma membrane. Rac1 activity was significantly reduced in cells expressing PIP4K2a. Quantifying the level of PIs suggested that PIP4K2a expression increases the removal of PtdIns(3,4,5)P3 by the PI 5-phosphatase, SKIP. Silencing the expression of SKIP by siRNA restored the level of PtdIns(3,4,5)P3 but Rac1 activity and the attenuation GLUT4 translocation were not rescued possibly as a result of removing PtdIns5P by PIP4K2a. On the other hand, exogenous delivery of PtdIns5P in L6 myotubes activates both Rac1 and GLUT4 translocation in the absence of insulin. However, activating GLUT4 translocation by the exogenous PtdIns5P requires PI3K activity since redistribution of GLUT4 to the plasma membrane is inhibited by the PI3K inhibitor, wortmannin. Removing PtdIns5P reduces Rac1 activity and stimulates SKIP that inhibits PtdIns(3,4,5)P3 increase which attenuates GLUT4 translocation and hence glucose uptake. These results emphasise the critical role played by PtdIns5P which seems to serve as a regulator of insulin signalling, both directly and/or by regulating other enzymes involved in the metabolism of PIs.

No description available.

Le Phosphatidylinositol(4,5)P2 (PtdIns(4,5)P2) est essentiel pour réguler divers processus cellulaires, y compris la signalisation cellulaire, le trafic intracellulaire et la cytocinèse. Le contrôle strict de son homéostasie est donc crucial et la dérégulation des kinases, des phosphatases et des phospholipases qui la contrôlent conduit à de multiples pathologies. Parmi elles, le syndrome de Lowe est une maladie rare et incurable causée par des mutations du gène OCRL qui code pour la PtdIns(4,5)P2 phosphatase OCRL1. La déplétion de dOCRL, l’orthologue d’OCRL1 chez la drosophile altère l’homéostasie du PtdIns(4,5)P2 avec (i) une accumulation anormale de PtdIns(4,5)P2 sur les endomembranes conduisant (ii) à des défauts de cytocinèse et à de la multinucléation. L’objectif de cette thèse était de comprendre comment le PtdIns(4,5)P2 est régulé sur les endomembranes. Dans les cellules de drosophile, nous avons découvert une fonction nouvelle et inattendue pour le suppresseur de tumeur PTEN, indépendante de son activité phosphatase. En effet, nous avons constaté que PTEN réduit les niveaux de PtdIns(4,5)P2 sur les endosomes grâce à l’action enzymatique de dPLCXD, une phospholipase C (PLC) atypique. Ainsi la voie de signalisation PTEN/dPLCXD peut compenser pour les défauts de cytocinèse dus à la perte de dOCRL. Enfin, nous avons identifié un activateur chimique des PLC qui restaure la perte fonctionnelle d’OCRL dans trois modèles de syndrome de Lowe distincts. Par la suite, nous avons étudié le rôle de la PTEN/PLCXD pendant l’autophagie, mécanisme d’autodigestion du matériel cellulaire. En effet, l’homéostasie du PtdIns(4,5)P2 lysosomale est essentielle pour l’étape autophagique de fusion des autophagosomes avec lysosomes. Nous avons observé que la déplétion de PLCXD et la surexpression d’un mutant catalytiquement inactif de PTEN altèrent l’autophagie chez les cellules de drosophile et de mammifère. Ces données suggèrent que la voie PTEN/PLCXD nouvellement identifiée régule le flux autophagique. Dans cette thèse, nous avons mis en lumière une nouvelle voie de signalisation PTEN/dPLCXD qui contrôle les niveaux de PtdIns(4,5)P2 sur les endolysosomes. Cette voie peut réguler l’autophagie et compenser la perte de dOCRL. Il s’agit d’une nouvelle fonction de PTEN indépendante de son activité phosphatase et c’est une première fonction biologique connue pour PLCXD. Cette découverte a conduit à l’identification d’une stratégie thérapeutique potentielle pour traiter les patients atteints du syndrome de Lowe. / Phosphatidylinositol(4,5)P2 (PtdIns(4,5)P2) is essential for various cellular processes, including cell signaling, intracellular traffic and cytokinesis. Therefore, strict control of its homeostasis is crucial. Indeed, the deregulation of the kinases, phosphatases and phospholipases which controls PtdIns(4,5)P2 leads to multiple pathologies. Among them, the Lowe syndrome is a rare and incurable disease caused by mutations in the OCRL gene which codes for PtdIns(4,5)P2 phosphatase OCRL1. Depletion of dOCRL, the orthologue of OCRL1 in drosophila, alters the homeostasis of PtdIns(4,5)P2 with (i) an abnormal accumulation of PtdIns(4,5)P2 on the endomembranes leading (ii) to cytokinesis defects and multinucleation. The objective of this thesis was to understand how PtdIns(4,5)P2 is regulated on endomembranes. In drosophila cells, we have discovered a new and unexpected function for the tumor suppressor PTEN independent of its phosphatase activity. Indeed, we have found that PTEN reduces the levels of PtdIns(4,5)P2 on endolysosomes thanks to the enzymatic action of dPLCXD, an atypical phospholipase C (PLC). Thus, the PTEN/dPLCXD signaling pathway can compensate for cytokinesis defects due to the loss of dOCRL. Finally, we identified a chemical activator of PLC that restores the functional loss of OCRL in three distinct Lowe syndrome models. Next, we studied the role of this newly identified PTEN/PLCXD pathway during autophagy, a self-digestion mechanism. Indeed, the homeostasis of lysosomal PtdIns(4,5)P2 is essential for the fusion of autophagosomes with lysosomes during autophagy. We have observed that depletion of PLCXD and overexpression of a catalytically inactive mutant of PTEN both alter autophagy in Drosophila and mammalian cells. These data suggest that this newly identified PTEN/PLCXD pathway regulates the autophagic flux. In this thesis, we have highlighted a new PTEN/dPLCXD signaling pathway which controls the levels of PtdIns(4,5)P2 on endolysosomes. This new PTEN function is independent of its phosphatase activity and the first biological function for PLCXD can regulate autophagy and compensate for the loss of dOCRL. This discovery led to the identification of a potential therapeutic strategy for treating patients with Lowe’s syndrome.

No description available.

The control of phosphatidylinositol 3, 4, 5-trisphosphate [PtdIns(3,4,5)P3] level depends on the activities of both PI kinase and PtdIns(3,4,5)P3 phosphatases: 5-phosphatase like SHIP1 and SHIP2, and 3-phosphatase like PTEN. The ubiquitous SH2 domain containing inositol 5-phosphatase SHIP2 contains both a series of protein interacting domains and the ability to dephosphorylate PtdIns(3,4,5)P3. Previous reports obtained in SHIP2 deficient mice have shown that SHIP2 is involved in the control of insulin sensitivity and reducing weight gain on fatty diet. <p><p>Since SHIP2 is a lipid phosphatase as well as a docking protein, the initial aim that emerged in the lab was to measure the inositol lipid levels in SHIP2 +/+ and deficient cells and compare the levels of 3-phosphoinositides PtdIns(3,4,5)P3 and PtdIns(3,4)P2. At first, we developed mouse embryonic fibroblasts (MEF) as a cellular model. Amongst various stimuli tested, surprisingly, only serum showed an obvious difference in terms of PtdIns(3,4,5)P3 level. This lipid was significantly up regulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P3 levels in heterozygous fibroblast cells was intermediate between the +/+ and -/- cells. Serum stimulated PI3K activity appeared to be comparable between +/+ and -/- cells. Moreover, PKB, but not MAP kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The up regulation of PKB activity in serum stimulated cells was totally reversed in the presence of the PI3K inhibitor LY-294002, in both +/+ and -/- cells.<p><p>Reactive oxygen species (ROS) have emerged as physiological mediators of many cellular responses. H2O2 mimics some effects of insulin in a number of cell culture systems. It also inactivates tyrosine phosphatase activities including PTEN. In addition, in Swiss 3T3 fibroblasts, Gray et al reported that exposure of the cells to H2O2 resulted a huge increase in PtdIns(3,4)P2 level. The authors suspected that the effect was attributed to a inositol 5-phosphatase activity. We thus exposed our cells to H2O2 in order to address the question of the role of SHIP2 in response to oxidative stress.<p><p>We worked on the same SHIP2 MEF model, stimulated by H2O2: at 15 min, PtdIns(3,4,5)P3 was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P2 could be detected at 15 or 120 min incubation of the cells with H2O2 (0.6 mM). PKB activity was upregulated in SHIP2 -/- cells in response to H2O2 and therefore follows the regulation of PtdIns(3,4,5)P3. As for serum, the PI3K activity appeared to be comparable between +/+ and -/- cells. The levels of PTEN and type I 4-phosphatase [an enzyme that acts on PtdIns(3,4)P2] remained unchanged between the two types of cells. SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role in the control of PtdIns(3,4,5)P3 level in response to H2O2: the decrease in PtdIns(3,4,5)P3, observed in SHIP2 expressing cells, was no longer seen in cells infected with a catalytic mutant of this enzyme. <p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished