Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. It was initially found that comparing to wild type cells, gsk3- cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.

Thesis advisor: Mary F. Roberts / Thesis advisor: Eranthie Weerapana / Small molecules are of interest both as metabolites in tumor cell biology and as potential therapeutics in the fight against cancer. In this work, small molecules in both roles have been examined. Modulation of tumor cell metabolism holds promise as a strategy to combat cancer, and both glucose and glutamine have been identified as critical fuels for tumor cell growth and proliferation. However, the reason for glutamine addiction is poorly understood. The differential metabolism of glutamine and glucose was therefore examined using ¹³C labeling and NMR-based metabolomics in the VM-M3 tumor cell line, which requires both glucose and glutamine for survival and proliferation. In the course of this study, a novel mammalian metabolite itaconic acid was identified. Itaconic acid was detected in extracts and tissue culture media from the murine macrophage-derived tumor cell lines VM-M3 and RAW 264.7 as well as in primary macrophages. Production and secretion of itaconic acid was increased upon stimulation. LC-MS and NMR based metabolomics studies show that this metabolite is synthesized by the decarboxylation of cis-aconitate from the TCA cycle, and provided evidence for a novel mammalian homologue of the enzyme cis-aconitic decarboxylase. D-3-deoxy diC₈PI is a small molecule of interest as a potential cancer therapeutic. This compound was designed to induce apoptosis in tumor cells by competitively binding to the Akt PH domain and preventing Akt translocation. However, high resolution ³¹P field-cycling studies show that both D-3-deoxy diC₈PI and an inactive analogue L-3,5-dideoxy diC₈PI bind to the same site on the PH domain, which is distinct from the binding site of the ligand diC₈PI(3,4,5)P₃. This makes the aforementioned mechanism of cytotoxicity unlikely. Aggregation of the PH domain in the presence of soluble headgroup IP₆ was also observed, which may be related to a physiological function of this protein and invalidates at least one other binding assay. Investigation into alterations in signaling pathways in the MCF-7 breast cancer cell line showed that D-3-deoxy diC₈PI activates the p38MAPK pathway which results in CREB hyperphosphorylation. However, activation of this pathway appears to be compensatory and unrelated to the mechanism of action. D-3-deoxy diC₈PI also decreases levels of cyclin D1 and cyclin D3, which regulate the progression of the cell cycle. These decreases appear to be occurring at the transcriptional level rather than due to increased proteasomal degradation. The loss of these two proteins does not cause apoptosis in MCF-7 cells, but siRNA knockdown of specifically cyclin D1 inhibits proliferation. This is consistent with the cell cycle arrest observed upon D-3-deoxy diC₈PI treatment in these cells. These findings do not conclusively elucidate the mechanism of cytotoxicity of D-3-deoxy diC₈PI, but provide a characterization of some of its effects in the MCF-7 cell line which may be useful for further studies. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Les phosphoinositides (PIs) sont des phospholipides membranaires qui jouent un rôle crucial dans le contrôle de l'organisation spatio-temporelle de nombreuses voies de signalisation intracellulaire, du réarrangement du cytosquelette d'actine et du trafic de vésicules. Dans la plaquette, le métabolisme des PIs est particulièrement actif et génère, par le jeu de kinases, phosphatases et phospholipases spécifiques, des seconds messagers indispensables à l'activation plaquettaire, notamment le phosphatidylinositol 3,4,5-trisphosphate (PIP3). La première partie de la thèse concerne l'étude des différentes espèces moléculaires (composition en acides gras) des 4 grandes classes de PIs (PI, PIP, PIP2 et PIP3) dans les plaquettes humaines et de souris au repos ou lors de leur activation. Cette analyse, jamais réalisée précédemment, a été possible grâce à une technique de spectrométrie de masse (LC-MS), basée sur la méthylation avec le TMS-diazomethane des groupements phosphates des PIs. Cette étude montre une augmentation rapide et transitoire de 2 espèces moléculaires majoritaires de PIP3 lors d'une stimulation plaquettaire avec une réactivité différente des plaquettes humaines et de souris en réponse aux agonistes plaquettaires (thrombine et CRP). En utilisant des modèles murins présentant une inactivation des PI3-kinases (PI3K) dans la lignée mégacaryocytaire et des inhibiteurs spécifiques de PI3K, j'ai montré que l'isoforme PI3Kß (p110ß) de classe I est très majoritairement responsable de la production des diverses espèces moléculaires de PI(3,4,5)P3 en réponse à la thrombine ou au CRP alors que la PI3Ka (p110a) est faiblement impliquée. Les résultats montrent également une grande variété d'espèces moléculaires de PI et seulement 2 espèces moléculaires prédominantes pour les PIP, PIP2 et PIP3, aussi bien chez l'homme que chez la souris malgré des régimes alimentaires très différents. Nous montrons des différences importantes dans le métabolisme des espèces moléculaires de PI, PIP et PIP2 dans les plaquettes humaines et de souris lors de la stimulation. Dans cette étude, nous avons identifié pour la première fois des espèces moléculaires minoritaire de PIP2 mais qui augmentent de façon importante lors de la stimulation plaquettaire. Ce travail permet de dresser la première cartographie des différentes espèces moléculaires de PIs présents dans les plaquettes humaines et de souris et les modifications induites par leur activation. La deuxième partie de la thèse montre pour la première fois une localisation atypique du phosphatidylinositol 3- monophosphate (PI3P), dans le feuillet externe de la membrane plasmique plaquettaire. Je démontre que ce lipide minoritaire (environ 10% de PIP), connu pour être intracellulaire et impliqué dans le trafic vésiculaire, est également présent à la surface des plaquettes au repos. Aucun autre PI n'a pu être détecté dans le feuillet externe de la membrane plasmique plaquettaire. Ce résultat a été obtenu en utilisant différentes sondes fluorescentes se liant spécifiquement au PI3P et leurs contrôles mutées. Nous montrons que le traitement des plaquettes avec des enzymes métabolisant spécifiquement le PI3P (MTM1 et ABH) réduit significativement ce pool de PI3P. Les plaquettes de souris déficientes en PI3K de classe II et III présentent une diminution du PI3P de surface. De manière intéressante, ce pool externe de PI3P permet l'endocytose des protéines circulantes liant le PI3P, in vitro, ex vivo et in vivo. Les sondes PI3P spécifiques internalisées dans la plaquette sont stockées dans les granules a puis sécrétées lors de l'activation plaquettaire. Cette étude montre que le PI3P se comporte comme un récepteur permettant l'endocytose de protéines plasmatiques spécifiques. / Phosphoinositides (PIs) are membrane phospholipids that play a crucial role in controlling the spatiotemporal organization of many intracellular signaling pathways, actin cytoskeleton rearrangement, and vesicle trafficking. In platelet, the metabolism of PIs is highly active and generates, by the interplay of specific kinases, phosphatases and phospholipases, second messengers essential for platelet activation, in particular phosphatidylinositol 3,4,5-trisphosphate (PIP3). The first part of the thesis concerns the study of the different molecular species (fatty-acyl composition) of 4 PIs classes (PI, PIP, PIP2 and PIP3) in resting and stimulated human and mouse platelets. This analysis, never realized previously, was possible thanks to a mass spectrometry (LC-MS) technique, based on methylation of PIs phosphates groups with TMS- diazomethane. This study shows a rapid and transient increase in the 2 major molecular species of PIP3 during platelet stimulation with a different reactivity of human and mice platelets according to the used agonists (thrombin and CRP). Using mice models with selective deletion of PI3-kinases (PI3K) in the megakaryocyte lineage and specific PI3K inhibitor, I showed that the class I PI3Kß (p110ß) is the major isoform responsible for the production of the various molecular species of PIP3 in response to thrombin or CRP whereas class I PI3Ka (p110a) is weakly involved. The results also show a large variety of molecular species of PI while only 2 predominant molecular species for PIP, PIP2 and PIP3, both in humans and mice platelets despite very different diet. We show a significant difference in terms of PI, PIP and PIP2 molecular species metabolism in human and mice platelets during stimulation. In this study, we identified for the first time the presence of low-abundance molecular species of PIP2 but which increase significantly during platelet stimulation. This work constitutes the first comprehensive analysis of PIs molecular species and the changes in their actual mass during platelet stimulation. The second part of the thesis shows for the first time an atypical localization of phosphatidylinositol 3-monophosphate (PI3P), in the outer leaflet of the platelet plasma membrane. I demonstrate that this minor lipid (about 10% of PIP), known to be intracellular and involved in vesicular trafficking, is also present at the surface of resting platelet. No other PIs could be detected in the outer leaflet of the platelet plasma membrane. This result was obtained using fluorescent probes binding specifically to PI3P and their mutated controls. Treatment of platelets with PI3P specific metabolizing enzymes (MTM1 and ABH) significantly reduced this particular pool of PI3P. Class II and III PI3K deficient mouse platelets showed a decrease in surface PI3P. Interestingly, this external pool of PI3P was able to mediate endocytosis of circulating PI3P- binding proteins, in vitro, ex vivo and in vivo. Internalized specific PI3P probes were stored into platelets a-granules and could then be secreted during platelets activation. This study shows that PI3P acts as a receptor allowing endocytosis of specific plasma proteins.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 87-94.

Ulm, Univ., Diss., 2008.

University, Diss., 2003--Jena.

Universiẗat, Diss., 2002--Jena.

Universiẗat, Diss., 2005--Jena.